MicroRNAome Genome:
A Treasure for Cancer Diagnosis and Therapy
Ioana Berindan-Neagoe,PhD1;Paloma del C.Monroig,BS2;Barbara Pasculli,MS3;George A.Calin,MD,PhD4
The interplay between abnormalities in genes coding for proteins and noncoding microRNAs(miRNAs)has been among the most exciting yet unexpected discoveries in oncology over the last decade.The complexity of this network has redefined cancer research as miRNAs,produced from what was once considered“genomic trash,”have shown to be crucial for cancer initiation, progression,and dissemination.Naturally occurring miRNAs are very short transcripts that never produce a protein or amino acid chain,but act by regulating protein expression during cellular processes such as growth,development,and differentiation at the transcriptional,posttranscriptional,and/or translational level.In this review article,miRNAs are presented as ubiquitous players involved in all cancer hallmarks.The authors also describe the most used methods to detect their expression,which have revealed
the identity of hundreds of miRNAs dysregulated in cancer cells or tumor microenvironment cells.Furthermore,the role of miRNAs
as hormones and as reliable cancer biomarkers and predictors of treatment response is discussed.Along with this,the authors explore current strategies in designing miRNA-targeting therapeutics,as well as the associated challenges that research envisions
to overcome.Finally,a new wave in molecular oncology translational research is introduced:the study of long noncoding RNAs.
CA Cancer J Clin2014;64:311-336.V C2014American Cancer Society.
Keywords:microRNA,diagnosis,therapy,cancer.
Introduction
MicroRNAs Are Strangers in the Genomic Galaxy
The central dogma of molecular biology is an explanation of the?ow of genetic information within a biological system and
it is summarized in the fact that“DNA makes RNA,which encodes protein.”However,over the past few years,DNA seg-ments have been shown to produce RNA transcripts that do not encode proteins.These transcripts were named noncoding RNAs(ncRNAs),and they were considered to be part of a“dark”unexplored segment of the human genome.MicroRNAs (miRNAs)are a class of small ncRNAs19to25nucleotides(nt)in length that can regulate gene expression by various mechanisms that have still not been fully investigated.They represent the most explored side of the“dark”matter of the genome,1-3and the full complement of known(cloned)miRNAs present in a genome is named microRNAome(for a glos-sary of terms,see Table1).
Initially,the DNA segments containing miRNA-coding genes are transcribed by an RNA polymerase II or III(RNA
Pol II and III)to initiate their biogenesis.The primary transcript(pri-miRNAs)can be hundreds or thousands of nt long,
but it is further processed,forming a$100-nt precursor transcript that folds upon itself(Fig.1).4-6The precursor sequence
is then exported to the cytoplasm,where it undergoes a series of catalytic steps prior to achieving maturation.In the cyto-plasm,mature single-stranded miRNAs are integrated into a number of proteins that compose the RNA-induced silencing complex(RISC),and thereafter they interact by sequence complementarity with the messenger RNA(mRNA)(Fig.1).4-6,7
1Department of Functional Genomics,The Oncology Institute,Research Center for Functional Genomics,Biomedicine and Translational Medicine, Department of Immunology,Iuliu Hatieganu University of Medicine and Pharmacy,Cluj-Napoca,Romania;2Department of Experimental Therapeutics,
The University of Texas MD Anderson Cancer Center,Houston,TX,University of Puerto Rico School of Medicine,San Juan,Puerto Rico;3Visiting PhD Student,Department of Experimental Therapeutics,The University of Texas MD Anderson Cancer Center,Houston TX;PhD Student,Department of Bio-sciences,Biotechnology and Biopharmaceutics,University of Bari,Bari,Italy;4Department of Experimental Therapeutics,Department of Leukemia,Cen-
ter for RNA Interference and Non-Coding RNAs,The University of Texas MD Anderson Cancer Center,Houston,TX.
Corresponding author:George A.Calin,MD,PhD,Department of Experimental Therapeutics,The University of Texas MD Anderson Cancer Center,1881East
Rd,Unit1950,3SCR4.3424,Houston,TX77030;gcalin@https://www.doczj.com/doc/355952503.html,
DISCLOSURES:George A.Calin,MD,PhD,is The Alan M.Gewirtz Leukemia&Lymphoma Society Scholar.Work in Dr.Calin’s laboratory is supported in part by National Institutes of Health/National Cancer Institute grants1UH2TR00943-01and1R01CA182905-01;Developmental Research Awards in prostate cancer, multiple myeloma,leukemia(P50CA100632),and head and neck(P50CA097007)Specialized Programs of Research Excellence(SPORE)grants;a Sister Institu-
tion Network Fund(SINF)University of Texas MD Anderson Cancer Center DKFZ grant in chronic lymphocytic leukemia(CLL);a SINF grant in colon cancer;a Kidney Cancer Pilot Project;The University of Texas MD Anderson Cancer Center Duncan Family Institute for Cancer Prevention and Risk Assessment;The Blanton-Davis Ovarian Cancer-2013Sprint for Life Research Award;the Laura and John Arnold Foundation;the RGK Foundation and the Estate of C.G.Johnson,
Jr;and by the CLL Global Research Foundation.Dr.Berindan-Neagoe’s work was financed by a POSCCE709/2010grant.
doi:10.3322/caac.21244.Available online at https://www.doczj.com/doc/355952503.html,
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As part of the new ncRNA-based dogma,miRNAs have been known to bind to mRNA at the30-untranslated region(UTR)and cause the downregulation of protein-coding genes(named targets)in the cytoplasm.They do so by inhibiting the ability of the ribosome to“translate”the mRNA.Alternatively,miRNAs can increase mRNA deg-radation,also reducing the possibility of the mRNA being translated into proteins.8The level of complementarity between miRNA and the target mRNA may determine the mechanism by which the translation of mRNA to pro-tein is blocked.Perfect or near-perfect complementarity has been found to induce mRNA degradation by RISC, and partial complementarity has been found to repress mRNA translation by blocking ribosomal access to the mRNA.9Due to the fact that each miRNA has hundreds or thousands of mRNA targets,a broad segment of the protein-coding genome is under their control.The regula-tion of miRNA-coding genes is of critical interest because they might be involved in any type of pathophysiological process and/or pathway,such as B-cell survival(miR-15a and miR-16-1),B-cell lineage fate(miR-181),brain patterning(miR-430),pancreatic cell insulin secretion (miR-375),adipocyte development(miR-145),cell prolifer-ation control(miR-125b and let-7),or cell survival(let-7 family).10
The understanding of the mechanisms of action of miRNAs has signi?cantly expanded in the last few years, with discoveries demonstrating unexpected complexities of their regulative manner,such as promoter binding,protein binding,or direct interaction with other ncRNAs(Fig.2). miRNAs can be relocalized in the nucleus;for example, human miR-29b has been found predominantly in the nucleus,demonstrating that despite their small size,speci?c miRNAs contain speci?c nucleotide sequences that control their subcellular localization.11This localization supports the already proven hypothesis that miRNAs can alternatively regulate transcriptional processes at a DNA level.For exam-ple,human miR-373binds to the E-cadherin(CDH1)pro-moter,thereby inducing its expression.12Futhermore, miRNAs target other genetic regions at the mRNA level besides the30-UTRs,such as50-UTR and coding regions.13-15Finally,besides mRNAs,miRNAs can target
TABLE1.Glossary of Terms Used in the MicroRNA World
ASO:An antisense oligonucleotide is a single-stranded,chemically modified DNA-like molecule that is17to22nucleotides in length and designed to be
complementary to a selected messenger RNA(mRNA)or noncoding RNA and thereby specifically inhibits expression of that gene.
Exome sequencing(targeted exome capture):An efficient strategy to selectively sequence the coding regions of the genome.
Messenger RNA(mRNA):The form of RNA that mediates the transfer of genetic information from the DNA in the cell nucleus to ribosomes in the cytoplasm,in which it serves as a template for protein synthesis.
MicroRNAome:The full complement of known(cloned)microRNAs(miRNAs)present in a genome.Due to multiple cloning efforts,it is growing constantly and by May2014contains2578mature human miRNAs(release20of miRBase available at https://www.doczj.com/doc/355952503.html,/index.shtml).
Noncoding RNAs(ncRNAs):Any RNA molecule that is not translated into a protein.
Oncogenic miRNA:A miRNA that when expressed at higher levels than normal initiates or favors the development of a tumor.
Open reading frame(ORF):A section of an mRNA that begins with an initiation(methionine ATG)codon and ends with a nonsense codon.ORFs all have the potential to encode a protein or polypeptide;however,many may not actually do so.
Pol II:RNA polymerase II(also called RNAP II)catalyzes the transcription of DNA to synthesize precursors of miRNAs and most small nuclear RNAs.
Pol III:RNA polymerase III(also called RNAP III)transcribes DNA to synthesize ribosomals RNA,transfer RNAs,and other small RNAs.The genes transcribed by RNA Pol III fall in the category of“housekeeping”genes whose expression is required by all cell types and most environmental conditions.
Pseudogene:A copy of a gene that usually lacks introns and other essential DNA sequences necessary for function.A pseudogene has been mutated into an inactive form over the course of evolution,but contains the majority of interactor sites with miRNAs as the original functional gene.
Sense/antisense:Refers to the strand of a nucleic acid that directly specifies the product or refers to the strand of a double-stranded molecule that does not directly encode the product but is complementary to it(antisense).
Single nucleotide polymorphism(SNP):A variation at a single position in a DNA sequence among individuals.If a SNP occurs within a gene,then the gene has more than one allele.
The Cancer Genome Atlas(TCGA):A project started in2005to catalogue genetic alterations responsible for cancer,using gene expression profiling,copy number variation profiling,single nucleotide polymorphism genotyping,genome-wide DNA methylation profiling,miRNA profiling,and exome sequencing.
Transcription:The process by which RNA is synthesized from a DNA template.
Translation:The process of ribosome-mediated production of a protein by which the primary structure of the protein is determined by the codon nucleotide sequence of an mRNA.
Tumor suppressor miRNA:A miRNA that when expressed normally blocks the initiation or development of a tumor;at lower levels of expression,this braking effect disappears and the tumor can develop.The same miRNA can behave as an oncogene in one type of tissue and as a tumor suppressor in another type.
Untranslated region(UTR):5’UTR is the portion of an mRNA from the5’end to the position of the first codon used in translation.The3’UTR is the portion of an mRNA from the3’end of the mRNA to the position of the last codon used in translation.
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different types of ncRNAs,some of them being highly conserved among species such as the ultraconserved genes 4or poorly conserved such as the pseudogenes (see Table 1for the de?nition of terms).5
In the past few years,miRNAs have also been found to favor protein expression (in addition to downregulating it).MiR-369-3p was shown to interact with adenylate uridy-late-rich elements in the tumor necrosis factor-alpha (TNF-alpha)mRNA,consequently recruiting proteins that
increased the process of translation during cell cycle arrest.6Similarly,miR-328was revealed to increase translation by interacting with hnRNPE2,a translational regulator,lead-ing to the release of CCAAT/enhancer-binding protein alpha mRNA.Conversely,it decreased the translation of PIM1kinase by binding speci?cally to its mRNA.Both mechanisms are active during the acute transformation of the chronic phase of chronic myeloid leukemia (CML)and are independent of patients’response to imatinib.Together,these data reveal that miRNAs possess the ability to control the fate of protein-coding genes by attaching through base pairing complementary to mRNA sequences or by interfering with regulatory proteins directly.16
MiRNAs also work as secreted molecules that trigger a receptor-mediated response in a different cell or tissue.They have the ability to be released into the extracellular environment within exosomes (cell-derived vesicles origi-nated by the inward budding in the plasma membrane gen-erating multivesicular bodies)that are present in many and perhaps all biological ?uids.In this way,they can act as “hormones.”17For example,it has been shown that macro-phages in?uence breast cancer cell invasion through the exosome-mediated delivery of oncogenic miR-223;more-over,pretreatment of mice with tumor-derived exosomes accelerates lung metastasis formation.18
Similarly,exosomes have been shown to modulate tumor microenvironments by releasing miRNAs in a coordinated manner.In this regard,exosomes derived from hypoxic cul-tures of a leukemic cell line in vitro were found to carry and release miR-210among other angiogenic miRNAs,increasing tube formation by endothelial cells.19Along this line,it was also shown that miR-21and miR-29a can be transported through exosomes and can act as direct agonists of Toll-like receptors (TLRs).By binding as ligands to receptors of the TLR family in immune cells,these miRNAs were shown to trigger a TLR-mediated prometastatic in?ammatory response (such as secretion of interleukins)that could favor tumor growth and metastasis.20,21Under-standing such newly recognized ways in which the miRNAs are working is important for both scientists and physicians as novel therapeutic approaches can be designed,such as blocking of miR-328interaction with hnRNP E2in patients with CML or blocking the miR-21and miR-29a agonistic effects on TLRs in patients with metastatic diseases.
MiRNAs as Ubiquitous Players in Cancer
MiRNA alterations have been identi?ed in many human diseases such as autoimmune and cardiac disorders,schizo-phrenia,and cancer (where they have been found to be highly dysregulated).22MiRNAs have been found to be differentially expressed between all types of analyzed human tumors and normal tissues,including benign
and
FIGURE 1.MicroRNA Processing.RNA polymerase II is responsible for the
initial transcription of the microRNA (miRNA)gene into a long,capped,and polyadenylated (poly-A)precursor,called primary miRNA (pri-miRNA).4,5A double-stranded RNA-specific ribonuclease,Drosha,in conjunction with its binding partner DGCR8(DiGeorge syndrome critical region gene 8or Pasha),further processes pri-miRNA into a 70-to 100-nucleotide hairpin RNA precursor (pre-miRNA).6Pre-miRNA is translocated from the nucleus to the cytoplasm by Exportin-5/RanGTP,and cleaved into an 18-to 24-nucleotide duplex by a ribonucleoprotein complex composed of ribonucle-ase III (Dicer)and TRBP (human immunodeficiency virus-1transactivating response RNA-binding protein).Finally,the duplex interacts with the RNA-induced silencing complex (RISC),which includes proteins of the Argonaute family (Ago1to Ago4in humans).One strand of the miRNA duplex remains stably associated with RISC and becomes the mature miRNA,which pri-marily,but not exclusively,guides the RISC complex to the 30-untranslated region of target messenger RNAs (mRNA).The other strand named “star”has also been found to be functional.Although the interaction between miRNA and mRNA usually results in translation inhibition,some cleavage of target mRNAs has also been observed.
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malignant tumors 23such as leukemias,lymphomas,lung cancers,breast cancers,colorectal cancers (CRCs),papillary thyroid carcinomas,glioblastomas and other brain tumors,hepatocellular carcinomas,pancreatic tumors,cervical cancers,prostate cancers,kidney and bladder cancers,or pituitary adenomas.23,24
MiRNAs can act as oncogenes and/or tumor suppressors.MiRNAs have been proven to work as oncogenes (Table 1),such as miR-21or miR-155,both of which are among the most frequently overexpressed miRNAs in human cancers.23For example,in a transgenic miR-155mouse model,the rodents initially exhibited a preleukemic pre-B-cell prolifera-tion evident in the spleen and bone marrow,followed by frank B-cell malignancy.25Overexpression of miR-21in mice similarly leads to a pre-B-cell malignant lymphoid-like phenotype,demonstrating that this gene is also a genuine oncogene.When miR-21was inactivated in vivo,the tumors regressed completely within a few days,partly as a result of enhanced apoptosis.26
MiRNAs can act also as tumor suppressors,such as the miR-15a/16-1cluster,which is frequently deleted in chronic lymphocytic leukemia (CLL)27and prostate cancers.28The induced deletion of these miRNAs in a knockout mouse model causes the development of indolent B-cell,autono-mous,clonal lymphoproliferative disorders,recapitulating the spectrum of CLL-associated phenotypes observed in humans.The miR-15a/16-1-deletion has been demonstrated to accelerate the proliferation of both human
and mouse B cells by modulating the expression of genes controlling cell cycle progression.29
In some instances,the same miRNA can have dual activ-ities,thereby acting as an oncogene in one speci?c cell type and as a tumor suppressor in another.For example,miR-221is overexpressed in liver cancers,in which it targets the PTEN tumor suppressor,and in this way it promoted liver tumorigenicity in a miR-221mouse transgenic model.30Similarly,in CRC,this same miRNA promotes cell invasion and metastasis by targeting RECK (which normally inhibits invasion and metastasis).31However,in other tumor types such as gastrointestinal stromal tumors,miR-221is downre-gulated and the consequent derepression of c-KIT and ETV1(target oncogenes)promotes this malignancy.32
The genetic basis for abnormal miRNA expression in cancer cells is complex.The widespread disruption of miRNA expression in malignant cells is only beginning to be understood,and a variety of abnormalities could contrib-ute to the miRNAome expression pro?le in each tumor.Transcription factors are involved in the regulation of spe-ci?c miRNAs,such as the miR-34family and miR-15/16cluster modulated by TP53,33,34the miR-17-92cluster regulated by MYC,34,35or miR-210regulated by hypoxia-inducible factor-1alpha (HIF-1a ).36The aberrant expres-sion causes abnormal levels of mature and/or precursor miRNAs in comparison with the corresponding levels in normal tissues.37At least 3different mechanisms (that could act independently or together)have been described
to
FIGURE 2.Mechanisms of Action of MicroRNAs.MicroRNAs can work through a variety of mechanisms,including (A)direct binding by complementarity to
multiple regions at the messenger RNA (mRNA)including the 30-untranslated region (UTR),50UTR,and coding regions and decreasing protein expression (majority of miRNAs);(B)positive regulation of translation (miR-369-3p)through the increased recruitment of processing proteins;(C)direct interaction with promoter sequences (miR-373);(D)direct agonism of receptors such as Toll-like receptors (miR-21,miR-29a,and let-7);and (E)direct interaction with pro-tein and enhancing protein expression through a decoy mechanism (miR-328).For gene abbreviations please see https://www.doczj.com/doc/355952503.html,/gene.
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cause this:the location of miRNA-coding genes at cancer-associated genomic regions frequently deleted or ampli-?ed38;epigenetic regulation of miRNA expression by methylation(which adds methyl groups to the cytosine or adenine DNA nucleotides,thereby suppressing genetic expression)or histone modi?cation(which are proteins that package and order the DNA)39;and?nally,abnormal-ities in miRNA processing genes or proteins(required for miRNA biogenesis and maturation).40,41
Germline and Somatic Mutations in miRNAs Variations in the sequences of miRNAs located in the mature,precursor,or primary transcript may contribute to cancer predisposition and initiation.42For example,germ-line(passed from parental germ cell)or somatic(acquired by the somatic cell)mutations of some miRNA genes were found in patients with CLL.In the initial analyses of sequence variations in miRNAs,it was reported that in2 patients diagnosed with CLL,a nucleotide substitution (C for T)was associated with lower levels of mature miR-16(a tumor suppressor miRNA).43This mutation proved to affect the SRp20site,which is an RNA-splicing protein implicated in processing the primary miR-16transcript, and this was hypothesized to be contributing to the devel-opment of CLL.44Findings such as this point out that some patients with CLL may have a genetic predisposition toward this type of cancer.Furthermore,a mouse model also supported the role of certain miRNAs in the pathoge-nesis of CLL.Similarly,these mice harbor a point muta-tion(one nt)adjacent to miR-16,which results in its reduced overall expression.45
On a separate note,single nucleotide polymorphisms (SNP,see Table1for de?nition)in the protein-coding mRNAs targeted by miRNAs have shown to in?uence can-cer risk as well.For example,let-7,a tumor suppressor miRNA known to target the mRNA of the KRAS onco-gene,has been proven to be unable to bind to a KRAS SNP variant found to be disproportionately enriched in non-small cell lung carcinoma(NSCLC)(present in18%-20% of cases).46This same KRAS variant was also associated with an increased risk of developing epithelial ovarian can-cer,a?nding that was consistent among3independent cohorts and2case-control studies.46Moreover,it was pres-ent in61%of patients with a history of hereditary breast and ovarian cancer who were previously genetically uninfor-mative(BRCA1-2negative).46This suggests that the SNP variant may be a new independent cancer risk biomarker for hereditary breast and ovarian cancer families.To date,most studies of miRNA binding-site SNPs have followed the case-control study design,and therefore they are centered on cancer risks.Consequently,the majority of our knowl-edge to date centers on cancer risk.For more information regarding miRNA binding-site SNPs considered to be can-
cer risk biomarkers,see the recent review by Preskill and Weidhaas.46
MiRNAs as Hormones
A large amount of data have accumulated over the last few years describing the participation of miRNAs and micro-environment cells in malignant processes.47,48Similar to hormones,miRNAs are released by a donor cell as“free”molecules or in secreted vesicles,and delivered from bodily
?uids into receptor cells located in other areas.17For exam-ple,the tumor suppressor miR-143was found to be released by normal epithelial prostate cells,inducing growth inhibition of prostate cancer cells in vitro and in vivo.49Similarly,intercellular transfer of miR-142and
miR-223from immune cells to malignant cells(hepatocel-lular carcinoma cells)inhibited proliferation and destabi-lized microtubule regulation in vitro.50In addition,in a breast cancer study,miR-210was shown to be released from tumor cells to endothelial cells,thereby promoting angiogenesis and metastasis through an exosomal-mediated transfer.51MiR-210was also proven to be traf-
?cked through exosomes in a leukemia cell line(under hypoxic conditions).52Along with other oncogenic miR-NAs,the delivery of miR-210resulted in increased angio-genesis through the promotion of tube formation by endothelial cells in vitro.19
MiRNAs Are Involved in All Cancer Hallmarks
The hallmarks of cancer comprise the biological capabilities acquired during the stepwise process of developing human tumors(Fig.3).MiRNAs have the potential to in?uence these hallmarks,and the recognition of the applicability of these interactions will increasingly in?uence the develop-ment of new therapeutic alternatives for patients with can-cer.53,54Herein are some representative examples of miRNAs that act as regulators of tumor biology;more detailed information on the roles of miRNAs in cancer can
be found in several reviews.22,24,40,55,56
Self-Sufficiency in Growth Signals
Activation of the RAS oncogene is a common event that allows tumor cells to escape growth factor dependency and become“oncogene addicted.”All3RAS genes(K-RAS, NRAS,and H-RAS)have been proven to be directly modu-lated by the tumor suppressor miRNA family of let-7’s in a posttranscriptional manner.57Alternatively,let-7also tar-gets high mobility group A2(HMGA2),a pleiotropic tran-scription factor.The characteristic downregulation of let-7 accompanying tumor development results in an increased expression of the RAS oncogenes along with their down-stream effects.It also derepresses HMGA2,thereby
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facilitating
anchorage-independent growth in cell cul-
tures,58as well as cell proliferation and differentiation.59 Evidently,both of these mechanisms have signi?cant rele-vance regarding tumorigenesis and cancer development. Furthermore,reconstituting the levels of let-7in an experi-mental approach demonstrated that this precursor miRNA family can inhibit cell proliferation,resulting in tumor regression as shown in lung cancer models in mice.60 Insensitivity to Antigrowth Signals
E2F is a group of genes that encode a family of transcrip-tion factors that tightly regulate cell cycle progression and DNA synthesis.Three of these,E2F1,E2F2,and E2F3a, are known as the cell cycle“activators,“and they represent attractive factors to target in cancer because they contrib-ute to uncontrolled cell growth.Several miRNAs have been demonstrated to have the potential to modulate the translation of the mRNAs of these transcription factors. For example,miR-20a,miR-17-5p,miR-93,and miR-106b have been shown to negatively regulate E2F1.61,62 Moreover,the miR-17-92cluster has been shown to decrease the levels of E2F1-3.63Therefore,in this sense,it is likely that the downregulation of these miRNAs in dif-ferent types of cancer could favor a proliferative transcrip-tional network contributing to tumorigenesis.Thus, reconstituting the basal levels of expression of these miRNAs(in E2Fs-dependent tumors)could serve as a future therapeutic alternative with clinical relevance.Some of these miRNAs are part of positive or negative feedback mechanisms,and thus the end result of modulating their levels still remains to be explored.
Finally,in separate studies,the transcription factor FOXO1(tumor suppressor that controls proliferation and regulates apoptosis)was found to be decreased in classic Hodgkin lymphoma.In classic Hodgkin lymphoma cell lines,the levels of FOXO1were proven to be repressed by 3upregulated microRNAs:miR-96,miR-182,and miR-183.64This repression highly increased proliferation,and at the same time inhibited apoptosis in vitro.
Evasion From Apoptosis
Apoptosis is a physiological self-destructive cellular mecha-nism that leads to the removal of unwanted cells.MiR-25 was identi?ed as being elevated in cholangiocarcinoma cell lines as well as patient samples,and its increased levels were shown to contribute to the evasion of TNF-related apoptosis. In experiments that reduced levels of miR-25(in vitro),cells in culture were sensitized to apoptotic death,thereby impli-cating the miRNA in the control of tumor cell apoptosis.65 The cancer-associated genomic region1p36(frequently lost or rearranged in many types of leukemias)contains a crucial tumor suppressor,miR-34a.In neuroblastoma,loss of miR-34a synergizes with MYCN oncogene ampli?cation, and miR-34a has been shown to be a MYCN-negative regulator.66In addition,miR-34a is known to induce cell cycle arrest and subsequent caspase-dependent
FIGURE3.Examples of MicroRNAs Involved in the Cancer Hallmarks.The8biological capabilities acquired during the multistep development of human tumors include sustaining proliferative signaling,evading growth suppressors,resisting cell death,enabling replicative immortality,inducing angiogenesis,activating invasion and metastasis,reprogramming of energy metabolism,and evading immune destruction.For each,one representative example of a microRNA is presented.
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apoptosis by repressing the antiapoptotic protein Bcl266and the transcription inducer of cell cycle progression E2F3.67 The overexpression of this same miRNA exhibits a tran-scriptome expression similar to that observed with p53(a widely known tumor suppressor)induction,being highly enriched for genes regulating apoptosis,cell cycle progres-sion,DNA repair,and angiogenesis.68-70This,along with other observations,points to the fact that the loss of this miR-34a sidesteps typical apoptotic pathways.Recently, another miRNA was also found to in?uence pathways lead-ing to apoptotic evasion.In a gastric cancer study,a novel mechanism whereby miR-185directly targets apoptosis repressor with caspase recruitment domain(ARC)was revealed.The role of this miRNA was studied in vitro and further validated in a gastric tumor xenograft model.71 Limitless Replicative Potential
Cellular senescence is a physiological withdrawal from the cell cycle in response to a variety of stressful stimuli and involves telomerase(an enzyme that prevents the loss of important DNA from chromosome ends)deregulation. MiRNAs have been linked to premature senescence,and their relevance has been addressed through the generation of a genetic miRNA screening library.The miR-29and miR-30 miRNA families are upregulated during induced replicative senescence and their high levels in?uence the repression of the MYBL2oncogene inhibiting cellular DNA synthesis.72 Moreover,miR-373and miR-372were identi?ed as being capable of allowing proliferation and tumorigenesis of pri-mary human cells harboring oncogenic RAS and wild-type TP53(a functional tumor suppressor).73These miRNAs neu-tralized p53-mediated cyclin-dependent kinase inhibition, possibly through direct inhibition of the expression of a tumor suppressor called LATS2.This evidence de?nitely implicates both of the miRNAs as being oncogenic,particularly in the development of human testicular germ cell tumors. Angiogenesis
Tumor cells activate an“angiogenic switch,”producing high amounts of proangiogenic factors that promote neo-vascularization.The most important one,vascular endothe-lial growth factor(VEGF),is highly expressed in most tumors,both solid and hematologic,and has been proven to be induced by hypoxia.During tumor progression, hypoxia has been found to contribute to the modulation of miRNA expression,in part by a direct transcriptional acti-vation of speci?c miRNAs(such as miR-26,miR-107,and miR-210)triggered by HIF-1.36These miRNAs have dual functions:on the one hand,they participate in angiogenesis but conversely,they potentiate the ability of cells to engage in antiapoptotic mechanisms to sustain survival.For example,miR-27a restrains the zinc?nger gene ZBTB10,a negative regulator of the Speci?cities Protein(SP)tran-scription factors,and through this repression it induces a
SP-dependent transcription of genes related to both survival
and angiogenesis(ie,survivin,VEGF,and VEGF receptor [VEGFR]).74Furthermore,miR-210,through direct modu-lation of the tyrosine kinase receptor ligand Ephrin A,repre-sents a component of the circuitry controlling endothelial
cell chemotaxis and tubulogenesis.75In addition,the down-regulation of miR-18a has also been recently linked to angiogenesis.MiR-18a is known to inhibit the phosphoryla-
tion of2substrates of the mammalian target of rapamycin pathway.This results in an inactivation of the pathway and a consequent downregulation of factors that stimulate blood vessel production,such as HIF-1a and VEGF.76
Invasion and Metastasis
The process of metastasis begins with the acquisition of invasive properties that allow cells to detach from the pri-mary tumor,enter the blood or lymphatic vasculature,and spread to distant organs.Upregulation of miR-10b has been demonstrated to promote invasion and metastasis by targeting HOXD10,a homeobox transcription factor that promotes or maintains a differentiated phenotype in epi-thelial cells.77,78Recently,a new miRNA that is involved in
the migration and metastases of lung cancer cells was iden-
ti?ed.79In lung cancer-positive transgenic mice,researchers found that miR-136,miR-376a,and miR-31were promi-nently overexpressed.Among these,the antagonization of
miR-31suppressed tumor growth,suggesting causation. This same group also identi?ed LATS2(a tumor suppres-
sor protein important for cytoskeleton function)and PPP2R2A(a phosphatase that catalyzes the removal of phosphate groups and is therefore considered an “inactivator protein”)mRNAs as targets of miR-31in mouse and human lung cancer models.In the attempt to identify miRNAs associated with lymph node metastasis in tissue samples from patients with lung adenocarcinoma, genome-wide next-generation miRNA sequencing and a training validation approach identi?ed and validated miR-
31upregulation as being signi?cantly associated with the presence of lymph node metastasis as well as poor patient survival.In addition,miR-31was shown to be able to mod-ulate the migratory,invasive,and proliferative behavior of lung adenocarcinoma cell lines in culture by stimulating the oncogenic ERK1/2signaling pathway.80
Reprogramming Energy Metabolism
Malignant cells tailor metabolic pathways to meet their energy requirements.81Glutamine and glucose are the2 major nutrients that fuel cellular metabolism and the path-ways using these nutrients are often altered in malignant tumors cancer.82Various studies have exposed the?ne
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interplay between metabolic pathways orchestrated by protein-coding genes and by miRNAs.For example,gluta-mine metabolism(glutaminolysis)was shown to be modu-lated by the MYC oncogene via miR-23a/b in prostate cancer and also in B-cell lymphoma.83In addition,it can also be modulated by a p65-mediated activation that down-regulates miR-23a levels.84Finally,glycolysis has been pro-ven to be modulated by a series of different miRNAs, including miR-378-star85and miR-143.86
Evading Immune Destruction
Signal transducer and activator of transcription3(STAT3) regulates a key pathway mediating immunosuppression in the tumor microenvironment.Recently,the role of miRNAs in tumor-mediated immunosuppression began to be discov-ered.MiR-124was found to be strongly downregulated in all grades and pathologic types of gliomas in comparison with normal brain tissue,and it was identi?ed as an impor-tant modulator of STAT3signaling.87Upregulation of miR-124in glioma cancer stem cells has been shown to inhibit the STAT3pathway.This resulted in the reversed induction of forkhead box P3regulatory T cells(Treg)and it also reversed the glioma cancer stem cells-mediated immunosuppression of T-cell proliferation.Furthermore, the systemic administration of miR-124by intratumoral or intravenous injection demonstrated to have antiglioma ther-apeutic effects in engineered murine glioblastoma models. The resulting effects indicated that miR-124depends on the presence of a T cell-mediated antitumor immune response.87 Methods to Detect the Expression of miRNAs Multiple platforms are available to identify and quantify miRNAs.An ideal method using miRNAs as biomarkers for human disease should be easy to perform and would not require expensive reagents or equipment;furthermore,it would be speci?c enough to detect only the miRNA of inter-est without detecting closely related miRNAs.It should also be sensitive enough to provide a quantitative expression anal-ysis,even with low amounts of starting material from small clinical samples,and it would need to possess the ability to process multiple samples in parallel.88,89The gold standard meeting these requirements for miRNA detection in clinical laboratories is quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR).MiRNA microarrays are more expensive than qRT-PCR and usually are used for the discovery step of biomarker identi?cation(Table2).90-96All these techniques,however,use miRNAs extracted from a patient tumor sample that includes malignant cells as well as nonneoplastic stromal and in?ammatory cells.If the detection aims to speci?cally analyze miRNA in malignant cells,it is advisable to use cell sorting with?ow cytometry for hematologic malignancies or laser capture microdissection for solid tumors.Another option is to perform in situ hybridiza-tion(ISH)97because the miRNA of interest can be detected within the different types of cells that compose the tumor (malignant vs microenvironment),and this approach provides additional information regarding the subcellular localization of the miRNA(Table2).90-96The newest method to dis-cover and measure miRNA expression is next-generation RNA sequencing.This technique is highly sensitive,highly speci?c,can be used for high-throughput analysis,and also for“de novo”discovering of miRNAs.RNA sequencing gen-erates a massive amount of complex data that need to be ana-lyzed by a well-trained bioinformatician.For this reason,as well as the high costs of a single RNA sequencing sample, this method is not yet appropriate for diagnostic purposes, but it can still be considered an alternative to investigate dis-eases such as cancer.
Understanding the Reproducibility of
Profiling Data
There are some technical challenges that make it dif?cult to compare results from similar pro?ling platforms used in different places.For example,the different primer design for measurements by qRT-PCR and microarrays is an important factor.98,99Another factor is the use of different protocols for sample preparation:some studies use an enriched small RNA fraction whereas others use total RNA.100Furthermore,technical and biological variability has been assessed by a range of different normalization processes,mostly based on the expression of reference genes.Speci?cally for cell-free miRNA analysis,no known extracellular reference RNA is currently suitable for a proper normalization.98Commonly used reference genes such as U6small nuclear RNA(RNU6B)and5S ribosomal RNA were found to be less stably expressed than others or degraded in serum samples.101In addition,the signi?cant differences in the choice of reference genes to use represent a major obstacle in comparing expression levels between normal tissue and tumors.Finally,the assessment of sample quantity and quality is more challenging for miRNAs than for mRNAs of protein-coding genes,for which the sizes and relative abundance of ribosomal RNAs can be used to check RNA integrity.
MicroRNAs:The Mix of Cancer Biomarkers and Hormones
Characteristics of a Good Biomarker
The measurement of miRNAs in bodily?uids including plasma and serum may represent a gold mine of noninvasive biomarkers for cancer.17,102An“ideal”biomarker should have a unique expression pro?le in the diseased compared with healthy tissues and should show highly increased or decreased expression levels in the diseased organ or tissue
318CA:A Cancer Journal for Clinicians
compared with nondiseased material.Moreover,the bio-marker should be reliable in detecting disease initiation or development before clinical symptoms appear with the goals of enabling early detection,persistence of minimal residual disease,or relapse after treatment.Furthermore, ideal biomarkers should be accessible through noninvasive methods;should have a long half-life in clinical samples; and should be rapidly detectable by simple,accurate,and inexpensive methods.103MiRNAs are very stable;even in bodily?uids such as plasma,serum,urine,and saliva,17 their expression is speci?c to tissues or organs and their expression level can be easily measured by methods such as quantitative PCR and miRNA microarrays.98Consistently, it has been shown that serum miRNAs remain stable after being subjected to severe conditions that would normally degrade most mRNAs,such as very low or high pH levels, boiling,extended storage,and10freeze-thaw cycles.These features make them very suitable as biomarkers.
MiRNA pro?ling of tumor versus normal tissues shows signi?cant changes and de?nes common altered miRNAs in human cancers.MiRNA pro?ling by various methods has allowed for the identi?cation of signatures associated with the diagnosis,staging,disease progression, prognosis,and response to treatment of human tumors (Table3).80,104-119MiRNA expression pro?les now allow for the classi?cation of human cancers.23To date,every type of tumor analyzed by miRNA pro?ling has shown signi?cantly different miRNA pro?les(for mature and/or precursor miRNAs)compared with normal cells from the same tissue type(Table3).80,104-119For example,it was shown that pro?ling a few hundred miRNAs has a signi?-cantly better predictive power of diagnosing a cancer of unknown primary site(CUP)compared with pro?ling sev-eral tens of thousands of mRNAs.120
MiRNAs in Bodily Fluids as Biomarkers of Therapeutic Response,Residual Disease,and Overall Survival in Patients With Cancer
Clinical studies have demonstrated the potential of using miRNAs as predictors of a cancer’s sensitivity to radiother-apy and anticancer agents(Table3).80,104-119For example, serum miR-21levels were found to be higher in patients with hormone-refractory prostate cancer whose disease was resistant to docetaxel-based chemotherapy when compared with those with chemosensitive disease.121In a separate study regarding lung adenocarcinoma and squamous cell carcinoma,11serum miRNAs proved to be able to separate patients into those with longer survival versus those with shorter survival.Furthermore,the levels of4of these miR-NAs were signi?cantly associated with overall survival(OS) and this same signature was consistently found to be an independent predictor of OS.122Likewise,upregulated and downregulated miRNAs have been detected in sensitive or resistant cell lines,in order to predict patient response to anticancer agents.One of these studies identi?ed a miRNA chemosensitivity pro?le from a set of59human cancer cell lines derived from diverse tissues(NCI-60cell lines). Downregulation of miR-34,miR-17,and let-7a was corre-lated with sensitivity to drugs commonly used as cancer treatments,such as5-?uorouacil,doxorubicin,and
TABLE2.Established Profiling Methods to Quantify miRNAs
ADVANTAGES LIMITATIONS REFERENCES
qRT-PCR l Semi-high throughput
l Good quantification
l Amplification enables
superior sensitivity(fM)l Difficult to distinguish single
nucleotide differences
l Not for discovery of ncRNAs
90,91
Microarray l Very high throughput
l Good ratio between cost and
generated information
l Easy to perform data analyses
l Results are often validated
with qRT-PCR l In some cases fair specificity
l Medium sensitivity(pM)
l Sensitivity and specificity can be
improved by LNA modification of the probes
l Limited for quantification
l Not useful for discovery of ncRNAs
92,93
In situ hybridization l Locate miRNA in tissue and cell
compartments
l miRNA and target identification
on the same slide l Low throughput
l Invasive sample collection
l Limited sensitivity
l Very limited quantification
94,95
RNA sequencing l High throughput due to barcoding
l High sensitivity( l High specificity l Can be used for discovery of novel ncRNAs l Large amount of complex data that need to be analyzed l High cost 92,96 fM indicates femtomole;pM,picomole;LNA,locked nucleic acids;miRNA,microRNA;ncRNA,noncoding RNA;qRT-PCR,quantitative reverse transcriptase-poly-merase chain reaction. VOLUME64_NUMBER5_SEPTEMBER/OCTOBER2014319 TABLE3.Examples of Malignant Cells or Body Fluid MicroRNAs Profiles With Clinical Significance for Patients With Cancer CANCER TYPE NO.OF PATIENTS miRNA ROLE REFERENCE Multiple cancers1809patients:7different types of cancers:breast cancer,primary head and neck squamous cell carcinoma, renal cancer,soft tissue sarcoma, pediatric osteosarcoma,bladder cancer,and glioblastoma miR-210Predictive effect on survival of patients with studied cancer types as indexed by disease-free survival, progression-free survival,and relapse-free survival 104 Multiple cancers174patients and39controls:50 breast cancers,30gastric cancers, 31lung cancers,31esophageal cancers,and31colorectal cancers miR-21Potential broad-spectrum serum- based diagnostic marker for the detection of solid tumors 105 Breast cancer120patients and40controls6circulating miRNAs: miR-10b,miR-17,miR-34a, miR-93,miR-155,and miR-373Known to be relevant for tumor development and progression;serum concentrations of deregulated miRNAs may be linked to a particular biology of breast cancer favoring progression and metastatic spread 106 Colon cancer102patients and54controls miR-141Highly correlated with TNM stage in patients with colorectal cancer; elevated levels associated with liver metastasis in patients with colorectal cancer 107 Colon cancer100patients with colorectal cancer, 37with adenomas,and59controls miR-29a Association with TNM stage in patients with colorectal cancer 108 Colon cancer103patients with colorectal cancer and37controls miR-221Potential noninvasive molecular marker for the diagnosis of colorectal cancer 109 Colon cancer Phase1:12patients with stage I and stage IV colorectal cancer Phase2:182patient with colorectal cancer and24controls Phase3:156matched tumor tissues from the phase2colorectal cancer cohort plus an independent set of20 matched primary colorectal cancers with corresponding liver metastases miR-200c Independent predictor of lymph node metastasis and tumor recurrence, emerging as an independent prognostic marker for colorectal cancer 110 Colon cancer Exosome-enriched serum samples from88patients with primary colorectal cancer and11healthy controls and29paired samples from patients after tumor resection let-7a,miR-21,miR-23a,miR-150, miR-223,miR-1229,and miR-1246 The serum exosomal levels of7 miRNAs were significantly higher in patients with primary colorectal cancer,even those with early-stage disease,compared with healthy con- trols,and were significantly downre- gulated after surgical resection of tumors 111 Gastric cancer124patients with noncardia gastric cancer and36patients with cardia adenocarcinoma and160controls miR-16,miR-25,miR-92a, miR-451,and miR-486-5p Detection of the early-stage gastric cancer 112 Gastric cancer104patients and65controls miR-18a Diagnostic power(high sensitivity and specificity) 113 HCC101patients,89controls,and48 patients with hepatitis B miR-21,miR-122,and miR-223Elevated in patients with HCC or chronic hepatitis,with strong potential to serve as novel biomarkers for liver injury but not specifically for HCC 114 Lung cancer Training set of64patients with adenocarcinoma;The Cancer Genome Atlas data set:223patients with adenocarcinoma miR-31Predictor of survival in a multivariate Cox regression model even when checking for cancer staging; exploratory in silico analysis indicated that low expression of miR- 31is associated with excellent sur- vival for patients with T2N0disease 80 320CA:A Cancer Journal for Clinicians cyclophosphamide,respectively.123These?ndings suggest that several miRNA-based models are ef?cient diagnostic tools,and they can furthermore be considered as useful when predicting patterns of resistance/sensitivity to drugs used as cancer treatment. MiRNA Profiling Represents a New and Useful Clinical Tool The identi?cation of noninvasive biomarkers still represents the most promising strategy for the early detection and accu-rate diagnosis of many malignancies.Due to their tissue-speci?c pro?les and stability in bodily?uids,miRNAs rank among the top candidates of biomarkers.Because of the exponential increase in the number of publications related to miRNA pro?ling in human tumors,we will highlight the signi?cance of recent studies reporting miRNA signatures published mainly within the last3years.92Many in-depth reviews cover publications from the last decade,including pro?ling topics in multiple types of cancers96,124such as lung cancers,125breast cancers,103gastroesophageal cancers,126 ovarian cancers,127,128hepatocellular carcinomas,129CLL,130 or B-cell lymphomas.131A compilation of recent publications reporting diagnostic,prognostic,or therapeutic associations in studies with over100patients is presented in Table3.80,104-119Such reports offer the scienti?c basis for the rational design of prospective large-scale studies needed to con?rm the multiple biological and clinical associations. Lung Cancer In both early-stage and late-stage lung cancer,there is an urgent need to?nd reliable discriminators for disease diag-nosis and prognosis.A large study of165lung adenocarcino-mas and125squamous cell carcinomas of the lung analyzed by miRNA array identi?ed a set of5miRNAs(let-7b,miR-103,miR-107,miR-181a,and miR-191)whose combina-tion was able to distinguish adenocarcinoma from squamous cell carcinoma(P global<.0001).132In addition,among male smokers with early-stage squamous cell carcinoma of the lung,the higher expression of5miRNAs(miR-25,miR-34a,miR-34c-5p,miR-191and let-7e)was predictive of a low mortality risk(hazard ratios[HRs]ranged between 0.38-0.51;P values ranged between.0001-.011).132These data suggest that miRNA signatures can discriminate between cancer histotypes and correlate with the most important clinical parameter(eg,the mortality risk). In3independent cohorts from different countries (United States[89patients],Norway[37patients],and Japan[191patients]),higher expression of miR-21was TABLE3.Continued CANCER TYPE NO.OF PATIENTS miRNA ROLE REFERENCE Lung cancer25paired NSCLC paracancerous tissues and serum,103control sera, and201patients with NSCLC miR-19a High serum miR-19a expression may be an independent poor prognostic factor for survival in patients with NSCLC 115 Lung cancer30NSCLC patients and75 individuals without tumor pathology (eg,inflammatory-interstitial diseases,infections,nontumor lung nodules,hemotypsis and other diseases)Exosomes isolated in plasma and BAL In plasma,a higher percentage of miRNAs with increased levels compared with tumor BAL or in nontumor plasma;the data reveal differences between BAL and plasma exosome amount and miRNA content 116 Glioblastoma Phase1:122patients with untreated WHO grade3to4disease and123 control serum samples Phase2:55WHO grade2,15WHO grade1,11astrogliosis,42other primary brain tumors serum samples, and8WHO grade2to4 astrocytoma tumor tissues miR-15b*,miR-23a, miR-133a,miR-150*, miR-197,miR-497,and miR-548b-5p,and the 7-miRNA panel The7-serum miRNA panel demon- strated a high sensitivity and specific- ity for the prediction of malignant astrocytomas;a marked difference in serum miRNA profile was observed between high-grade astrocytomas and normal controls 117 Prostate cancer Prebiopsy serum samples of133 enrolled patients from3study centers miR-26a-1and miR-141The analysis of circulating miRNAs does not appear to help identify patients with cancer undergoing prostate biopsy;however,their levels may be useful to identify patients with high-risk prostate cancer 118 Ovarian cancer360patients with epithelial ovarian cancer and200controls from2 institutions miR-205,let-7f Plasma miR-205and let-7f are bio- markers for ovarian cancer detection that complement CA125;let-7f may be predictive of ovarian cancer prognosis 119 BAL indicates bronchoalveolar lavage;HCC,hepatocellular carcinoma;miRNA,microRNA;NSCLC,non-small cell lung cancer;WHO,World Health Organization. VOLUME64_NUMBER5_SEPTEMBER/OCTOBER2014321 found to be signi?cantly associated with cancer-related mortality(HR,2.06;95%con?dence interval[95%CI], 1.13-3.75[P5.018];HR, 2.78;95%CI, 1.22-6.31 [P50.014];and HR,2.82;95%CI,1.57-5.07[P<.0005], respectively).133The higher levels were detected in more advanced-stage tumors,consistent with the?ndings that miR-21is involved in disease progression.Importantly, multivariable Cox regression analysis for just those cases with TMN stage I disease showed that higher miR-21 expression levels were associated with poor cancer-speci?c mortality in the cohorts from the US and Norway (HR,2.16;95%CI,1.11-4.21[P5.025])and worse relapse-free survival in the Japanese cohort(HR,3.40; 95%CI,1.57-7.36[P5.001])independently of all clini-cal covariates,suggesting the use of miR-21as an early-stage prognostic biomarker for patients with TMN stage I disease at high risk of metastases.133 An interesting study evaluated the potential of a previ-ously identi?ed24-plasma miRNA signature classi?er (MSC)134for use as a noninvasive screening tool for lung cancer in the Multicenter Italian Lung Detection cohort of current and former smokers.Plasma samples from69 patients with lung cancer and870disease-free individuals (652of whom had been screened with low-dose computed tomography[LDCT]and287from an unscreened observa-tion group)were analyzed by qRT-PCR.All the patients were categorized as belonging to one of the3cancer risk groups(low,intermediate,and high)on the basis of prede-?ned cutoff points of positivity for4different expression ratio signatures of24miRNAs,de?ned as risk of disease, risk of aggressive disease,presence of disease,and presence of aggressive disease.The study con?rmed the diagnostic, prognostic,and predictive value of this MSC.The diagnostic performance of MSC for lung cancer detection was87%sen-sitivity(SE),81%speci?city(SP),27%positive predictive value(PPV),and99%negative predictive value(NPV) within the group of69lung cancer patients when compared with disease-free patients overall(n5870),88%SE,80% SP,31%PPV,and99%NPV within the LDCT arm (patients who developed lung cancer versus healthy individu-als),and82%SE,83%SP,16%PPV,and99%NPV within the observational arm as speci?ed above.For all patients, MSC had an NPV of99%and99.86%,respectively,for dis-ease detection and disease-related death,whereas LDCT had a79%SE and81%SP with a false-positive rate of 19.4%.The combination of both MSC and LDCT provided a5-fold reduction(compared with LDCT alone)in the false-positive rate to3.7%,which means that MSC might complement LDCT screening,especially among individuals with a low MSC and good prognosis,thereby avoiding unnecessary invasive follow-up and optimizing health care costs.135Similarly,a combined17-miRNA risk score derived from serum samples of patients with advanced NSCLC (n5391)was found to accurately identify those at the high-est risk of death.In particular,individuals with a high-risk score had a2.5-fold increased risk of death,corresponding to a7.8-month decrease in median survival(P<.0000001) when compared with those with a low-risk score(95%CI, 1.8-3.4[P<.0000001]).136Thus,consistent with the data reported above,circulating miRNAs represent diagnostic and prognostic biomarkers that could be ef?ciently imple-mented in the clinical setting for lung cancer management. Breast Cancer Early-stage breast cancer is curable by surgery and by a com-bination of hormonal treatment,chemotherapy,and radia-tion.However,late relapses are frequent events after many years.MiRNA-based tests could potentially improve the prediction of OS as well as treatment response.An inte-grated miRNA/gene signature was recently reported in a group of466patients with breast cancer from The Cancer Genome Atlas(TCGA)(Table1).137The?nal signature, including30mRNAs and7miRNAs(miR-93,miR-103, miR-148b,miR-328,miR-484,miR-874,and miR-1307), proved to signi?cantly predict OS in a multivariable model independent of other clinical pathological characteristics of the tumors,and showed the highest prognostic value of distant relapse-free survival in patients with early stage I and II tumors(receiver operating characteristic[ROC]area under the curve[AUC],0.77;P<.001).Furthermore,the validation of this signature on8independent breast cancer cohorts,comprising a total of2399patients,demonstrated its superior performance for risk strati?cation with respect to other RNA predictors,including the mRNAs used in the MammaPrint(Agendia,Irvine,Calif)and Oncotype DX (Genomic Health,Redwood City,Calif)assays.137Sepa-rately,in a series of173triple-negative breast cancers,a 4-miRNA signature(miR-27a,miR-30e,miR-155,and miR-493)allowed the subdivision of triple-negative breast cancers not only into core basal(epidermal growth factor receptor and/or CK5or CK6positive)or5-negative(when all markers are negative)subgroups(75%SE and56%SP; AUC,0.74),but also into groups with high and low risks of death.138In particular,the median OS times for the high-risk versus low-risk miRNA signature groups were75.5 months versus82months(HR,2.46;95%CI,1.43-4.12 [P5.001]).This panel also showed the ability to predict outcomes of treatment with the2most common chemotherapy regimens used in patients with triple-negative breast cancer(ie,anthracyclines or anthracyclines plus taxanes). Recently,in an attempt to identify circulating miRNA signatures in patients with breast cancer,a microarray anal-ysis of fresh-frozen preoperative sera as well as normal and breast cancer paired tissue samples was performed in a large 322CA:A Cancer Journal for Clinicians cohort of Asian-Chinese patients with breast cancer.139 The miRNA expression pro?les between sera and matched tumor samples were largely dissimilar,thereby con?rming the high tissue speci?city of miRNAs and their selective release from the primary tumor.Notably,among the result-ing23breast cancer-associated serum miRNAs,the ROC curves derived from the combinations of the overexpressed miR-1,miR-92a,miR-133a,and miR-133b exhibited AUCs of0.90to0.91,con?rming that miRNA models are robust diagnostic tools to apply in the clinic. In an independent study of48white patients with estrogen receptor-positive early stage breast cancer(24of whom were lymph node positive and24of whom were lymph node nega-tive),a novel circulating9-miRNAs pro?le provided for the ?rst time the ability to distinguish the serum of patients with breast cancer from that of healthy controls(24age-matched women).140This signature included both overexpressed (miR-15a,miR-18a,miR-107,and miR-425)and underex-pressed(miR-133a,miR-139-5p,miR-143,miR-145,and miR-365)miRNAs.The miRNA pro?le validation on a cohort of111serum samples as well as independent data from3publicly available databases further con?rmed the dis-criminatory power with an AUC of0.665(95%CI,0.562-0.768,[ROC test])and a P value https://www.doczj.com/doc/355952503.html,ing a probability cutoff of0.48,the SE was83.3%,the SP was41.2%,the PPV was62.5%,and the NPV was67.4%. If compared,in the studies reported above,only one miRNA was held in common between the2patient popula-tions analyzed.139,140Thus,there is evidently a high variabil-ity of miRNA expression in bodily?uids,which underlines the need to conduct large consortium studies that include samples from as many parts of the world as possible. Colon Cancer Genetic pro?ling has been found to be somewhat useful when determining prognosis for patients with intermediate-stage disease,although to date it is not widely used.141With regard to miRNA pro?ling,a microarray study of2colon adenocarcinoma patient cohorts(84from a Maryland test cohort and113from a Hong Kong independent validation cohort)compared tumors with adjacent nontumorous tis-sue.142In both cohorts,high miR-21levels were found to be predictive of poor survival in both the training cohort(HR, 2.5;95%CI,1.2-5.2)and the validation cohort(HR,2.4; 95%CI,1.4-3.9),independently of clinical stage.Moreover, in patients with stage II and III disease treated with 5-?uorouracil-based adjuvant chemotherapy(either intrave-nous5-?uorouracil or oral drugs including tegafur with uracil,levamisole or leucovorin),increased expression of miR-21correlated with poor response and worse OS(HR, 4.3;95%CI,1.1-16.4[P5.03]and HR,3.5;95%CI, 1.1-11.6[P5.04]for the Maryland and Hong Kong populations,respectively).miR-21was also found to be overexpressed both in tissue samples of CRC(46patients; P<0001)and related liver metastases(30patients; P<.0001)compared with normal paired tissues and correla- tion with a worse disease-free interval was noted in the CRC sample set(P5.0026).143Finally,2other miRNAs have been proposed to be good predictive markers for thera-peutic response in patients with CRC:miR-126144and miR-150.145miR-126was detected by in situ hybridization (ISH)assay in formalin-?xed paraf?n embedded tissue from primary tumors,and the expression levels indicated as area per image(m m2)by using image analysis.Higher levels of miR-126were predictive of a good response to?rst-line treatment with capecitabine and oxaliplatin in patients with metastatic CRC;the median miR-126expression level was signi?cantly higher in the responding patients,with an(area of the ISH signal)of3629m m2(95%CI,2566m m224846 m m2)compared with the nonresponding patients(area per image of1670m m2;95%CI,1436m m222041m m2 [P5531026]).Accordingly,lower expression levels of miR-126were predictive of a worse progression-free survival and OS,along with the association with the number of met-astatic sites.Speci?cally,the median progression-free sur-vival for patients whose tumors demonstrate high miR-126 expression levels was11.5months(95%CI,9.0months-12.7months)compared with6.0months(95%CI,4.8 months-6.9months)for patients with low expression levels. The median OS in the group with high miR-126expression was26.2months(95%CI,21.8months-32.8months)com-pared with16.8months(95%CI,13.8months-19.1 months)in the group with low miR-126expression.Con-versely,low expression levels of miR-150were found to be associated with worse prognosis(HR0.57;95%CI,0.33- 0.97[P5.037])and poor therapeutic outcome in patients with stage II and III CRC after?uoracil-based adjuvant therapy with or without leucovorin,levamisole or cisplatin (289patients in cohort1:HR,0.44;95%CI,0.20-0.93[P 5.032]and185patients in cohort2:HR,0.38;95%CI, 0.19-0.79[P5.009]). To date,the early detection of adenomas is based on invasive screening approaches(such as colonoscopy and biopsies)and on noninvasive approaches(such as fecal occult blood tests or stool DNA tests based on mutations and methylation)with limited diagnostic accuracy.Thus, there is a great need for new noninvasive methods for rou- tine clinical investigation.Recently,researchers embarked on a pilot study with the challenge of identifying speci?c biomarkers to detect precancerous lesions,colorectal adeno-mas,and CRCs.146The results of the investigation lead to a panel of8plasma miRNAs that distinguished patients with colorectal adenoma(n516)from controls(n526) with high accuracy(AUC,0.868[95%CI,0.76-0.98];88% SE and64%SP),and a panel of3plasma miRNAs that VOLUME64_NUMBER5_SEPTEMBER/OCTOBER2014323 could distinguish patients with stage IV CRC(n515)from controls(AUC,0.868[95%CI,0.76-0.98];93%SE and 74%SP).147A further6miRNA pro?les of upregulated miRNAs(miR-15b,miR-18a,miR-19a,miR-19b,miR-29a,and miR-335)was achieved when analyzing a total of 196plasma samples from123patients newly diagnosed with sporadic colorectal neoplasia(63with CRC and60 with advanced adenoma)and73healthy individuals(con-trols).The panel showed a high accuracy in discriminating patients with CRC from healthy controls(AUC ranging from0.80[95%CI,0.71-0.89]to0.70[95%CI,0.59-0.80]),whereas only miR-18a was con?rmed to be signi?-cantly upregulated in patients with advanced adenomas (AUC,0.64;95%CI,0.52-0.75).147 As underlined above for breast cancer,the last2studies reported for colon cancer exclusively share the clinical rele-vance of miR-15b.This again highlights the importance of performing wider trials to con?rm these data in larger cohorts and patient populations of different ethnicities. Ovarian Cancer Because the majority of patients with ovarian cancer are diagnosed with advanced-stage disease,identifying miRNA biomarkers capable of detecting early-stage disease would have great clinical value.Currently,evidence from microar-ray-based studies is being limited because of the small num-ber of patients,thus the certainty of the results hinge on this fact.Nevertheless,some exhibit great potential to address some clinical issues including characterization of different histologic and genetic subtypes,the identi?cation of markers for diagnosis and screening,prediction of clinical outcome, and better individualization of therapies.148Recently, miRNA expression analysis performed in a large multicenter cohort of198patients(86patients as a training set and112 patients as validation set)demonstrated that the downregu-lation of3miRNAs(miR-217,miR-484and miR-642)was predictive of resistance to platinum-based chemotherapy when responding and nonresponding individuals were com-pared(P5.046,P5.0007and P5.04,respectively).149In this context,additional studies on miR-484demonstrated its capability to interfere with the VEGFB and VEGFR2path-ways,thus indicating new putative targets for therapeutic approaches on tumor vasculature.149 Separate studies150,151showed the clinical relevance of miR-200c in ovarian cancer.Leskela et al showed that women with International Federation of Gynecology and Obstetrics stages III and IV serous ovarian carcinoma (n557)without a complete response to a regimen of paclitaxel and carboplatin have tumors with signi?cantly lower miR-200c levels when compared with those who achieved a complete response(HR,1.43;95%CI,1.02-1.99[P5.037]).150Downregulation of miR-200c was also identi?ed as being associated with OS(HR,0.094;95% CI,0.012-0.766[P5.0272])and progression-free survival (HR,0.035;95%CI,0.004-0á311[P5.0026])in a multi-variable analysis of144samples of stage I epithelial ovarian cancer.151Finally,the integrated analysis of miRNA and transcriptome pro?les from a TCGA ovarian cancer data set revealed that the expression levels of the miR-200fam-ily members are able to clearly differentiate between2clini-cally relevant subtypes(ie,mesenchymal and epithelial phenotypes)of serous ovarian cancer.152 Gliomas MiRNAs seem to have promising potential in gliomas,in which they might play an informative role in discriminating malignant phenotypes as well as predicting the outcome of surgical resection.Expression pro?les of261gliomas from the TCGA data set identi?ed121miRNAs that were able to segregate tumor samples into5genetically and prognosti-cally different subclasses.153Another comprehensive analysis in160glioma samples from Chinese patients(63with World Health Organization(WHO)grade2disease,33 with WHO grade3disease,and64patients with glioblas-toma multiforme[GBM])revealed that the combination of 21miRNAs(so-called“hub miRNAs”because of their reg-ulating more than30target mRNAs)was able to predict the survival of patients with all types of gliomas(HR,1.097 [95%CI,1.068-1.127];correlation coef?cient[R2],0.093 [P55.64310212]and HR,1.076[95%CI,1.044-1.109]; R2,0.074[P51.1231026]from univariable and multi-variable analyses,respectively).Among the21relevant miR-NAs,miR-524-5p(HR,0.849[95%CI,0.721-0.999]R2, 20.164[P5.044])and miR-628-5p(HR,0.679[95%CI, 0.470-0.982];R2,20.387[P5.036])expression levels rep-resented protective factors,whereas miR-938(HR,1.113 [95%CI,1.029-1.204];R2,0.107[P5.007]),miR-595 (HR,1.200[95%CI,1.059-1.360];R2,0.183[P5.004]), and miR-346(HR,1.631[95%CI,1.043-2.552];R2,0.489 [P5.029])expression levels represented risk factors.154 The possibility of detecting miRNAs in bodily?uids such as serum and/or cerebrospinal?uid(CSF)represents a novel,minimally invasive,experimental procedure that could possibly identify and characterize gliomas without requiring surgical intervention.For example,pilot studies on a limited number of patients have shown that miR-21 and miR-10b155-157are signi?cantly increased in the CSF of patients with GBM or those with brain metastases from breast and lung cancer when compared with CSF from patients whose tumors are in remission and those with non-neoplastic conditions.It is interesting to note that because miR-10b is generally expressed in extracranial tissues but is undetectable in the brain and CSF of patients without can-cer,probably because of the blood-brain membrane,its 324CA:A Cancer Journal for Clinicians speci?c abundance in the CSF from patients with GBM and brain metastases could be referred to as a speci?c signa-ture of brain pathology.157Conversely,members of the miR-200family(miR-200a,miR-200b,miR-200c,and miR-141)are found to be highly elevated in the CSF of patients with brain metastases but not in those with GBM (P<.0001)or other primary brain tumors,suggesting they could serve as speci?c markers of metastatic brain tumors.157In addition,through a genome-wide serum miRNA analysis by next-generation sequencing,serum samples from122patients with untreated astrocytomas and 122normal controls were evaluated,and a panel of7miR-NAs(miR-15b*,miR-23a,miR-133a,miR-150*,miR-197,miR-497,and miR-548b-5p)were found to be signi?-cantly decreased(P<.001)between patients with tumor grades in the range of WHO grade2to4and the control group.117The AUC for the combination of the7miRNAs was0.972(95%CI,0.954-0.990)for patients with malig-nant astrocytomas(all grades)and controls and with an optimal cutoff of5.6085,the SE was88%and the SP was 97.87%.Moreover,a marked increase in the levels of expression of the same group of miRNAs was detected after patients underwent tumor resection(P<.001).117 A study of exosomes isolated from patient sera demon-strated that the expression levels of RNU6-1,miR-320,and miR-574-3p were able to effectively discriminate patients with GBM from healthy individuals,suggesting their signif-icance as a novel potential diagnostic tool.158In particular, ROC curve analyses revealed that the expression levels of either RNU6-1alone or in combination with miR-320and miR-574-3p were useful and robust biomarkers for differen-tiating patients with GBM(n550)from healthy controls (n530),with an AUC of0.722(95%CI,0.60-0.84 [P5.0007])for RNU6-1and0.775(95%CI,0.65-0.90 [P5.0001])for the3markers together.Importantly,at a cutoff value of0.372for RNU6-1,the SE was66%and the SP was68%,whereas at a cutoff value of0.374for the miRNA signatures,the SE was70%and the SP was71%. Taken together,these data suggest alternative noninva-sive approaches for the prediction of malignant progression and detection of residual disease in these types of tumors. Chronic Lymphocytic Leukemia CLL has become the paradigmatic disease of miRNA involvement in cancer as the downregulation of the miR-15a/miR-16cluster in the majority of patients with CLL was the?rst report linking miRNAs to cancer.27A unique miRNA signature composed of13genes including miR-15a,miR-16,and miR-155as well as members of the miR-181family possesses the ability to distinguish aggressive from indolent CLL.43More recently,a scoring method combining miR-21expression with the routine cytogenetic procedures of?uorescence in situ hybridization(FISH)and Karyotype(KARYO)(21FK score:miR-21qRT-PCR, FISH,Karyotype)was developed in order to stratify patients with CLL according to survival.159When com-pared with the classic prognostic factors,including B2M, ZAP-70,IgVH,and CD191CD381,the21FK score was found to be the only rating able to distinguish patients with a good prognosis(low miR-21expression,low score)from those with poor prognosis(high miR-21expression,high score)both in a homogenously selected cohort of104 patients with17DEL CLL and nonhomogeneous sets of 80cases with either one chromosomal abnormality (13qDEL,11qDEL,T12,or17pDEL)or a normal karyo-type.Indeed,from the multivariable analysis using the 21FK score,IgVH,and ZAP-70,the21FK score was dem-onstrated to be the only signi?cant variable in both sets of patients(HR,3.514;95%CI,1.409-8.764[P5.007]and HR,5.217;95%CI,1.408-19.327[P5.013],respectively). In the same study,when patients with17p deletions were compared with those with normal karyotypes,miR-21, miR-34a,miR-155,and miR-181b were found to be differ-entially expressed as well.In particular,miR-181b was spe- ci?cally downregulated in patients whose disease was refractory to their treatment regimen,with a mean treatment-free survival of5.35months versus13.80months for patients with high miR-181b expression(HR,2.77; 95%CI,1.295-5.91[P5.006]).159 An interesting study successfully demonstrated the potential of using circulating miRNAs for both the detec- tion and strati?cation of patients with CLL.160In a cohort of CLL plasma samples,a number of detectable circulating miRNAs including miR-17,miR-19b,miR-92a,miR-150, miR-223,miR-320,and miR-484were found to be highly expressed when compared with normal control plasma. Among them,several miRNAs showed distinct pro?les between CLL and other hematological malignancies such as hairy cell leukemia or multiple myeloma.Circulating miR-20a(AUC,0.920)and miR-195(AUC,0.951),as well as the miR-29a,miR-195,and miR-222combination pattern(AUC,0.982)were found to be the best classi?ers for clearly distinguishing patients with CLL from healthy individuals. Finally,with the aim of investigating the mechanisms underlying the transition from a clinical premalignant lesion named monoclonal B lymphocytosis(MBL)to CLL,Ferrajoli et al evaluated the differential expression of previously mentioned miR-155in224samples from2 independent validated cohorts of patients with CLL and MBL.161MiR-155expression was higher in puri?ed B cells from patients with MBL when compared with normal B cells(P<.0001)and patients with CLL(P<.0001). Increased miR-155plasma levels,measured before treat-ment,were found to be associated with poor OS(P5.01) VOLUME64_NUMBER5_SEPTEMBER/OCTOBER2014325 and subsequent resistance to therapy with either?udara-bine,cyclophosphamide,or rituximab(P5.02)or lenalido-mide(P5.03)in nonresponding patients compared with individuals with complete responses.These data support the ability of miR-155to predict prognosis and response to treat-ment independent of the type of therapy administered.161 Acute Myelogenous Leukemia The pro?ling?ndings with acute myelogenous leukemia (AML)have highlighted the existence of a complex net-work comprising gene mutations,along with aberrantly expressed genes and miRNAs.All of them collectively de?ne the AML phenotype associated with clinically aggressive disease.High levels of miR-10a-5p in patients with NPM1-mutant AML were found to be associated with complete remission after chemotherapy with idarubi-cin and cytarabine(odds ratio[OR],1.33;P5.019).162 When NPM1mutation status and miR-10a-5p were included in a multivariable model,they demonstrated a sig-ni?cant interaction correlating with complete remission status(P5.01),suggesting a synergistic effect enabling the tumor to acquire sensitivity to treatment.162Furthermore, another study including a large cohort of patients with complex karyotype AML with known TP53status demon-strated that low levels of miR-34a expression such as TP53 alterations were predictive of response to chemotherapy (OR,0.78;95%CI,0.42-1.44[P5.42])and poor clinical outcome(OS:HR,1.47;95%CI,1.06-2.03[P5.02]and relapse-free survival:HR, 1.90;95%CI, 1.14-3.18 [P5.01]).163As in patients with CLL,high levels of miR-155have also been shown to be signi?cantly correlated with a shorter OS(HR, 1.62;95%CI, 1.25-2.09 [P<.001])and a complete remission rate of less than50% (OR,0.46;95%CI,0.27-0.81[P5.007]),independent of other strong clinical and molecular predictors.164 Recently,a global screening of670miRNAs in238 patients with intermediate-risk cytogenetic AML demon-strated that high levels of miR-644and miR-196b were otherwise independent miRNAs associated with shorter OS(OR,0.51;95%CI,0.51-0.85[P5.004];and OR, 0.42;95%CI,0.19-0.94[P5.036],respectively)and,in addition,increased levels of miR-135a and miR-409-3p were correlated with a higher risk of relapse at5years(OR, 0.23;95%CI,0.0863-0.624[P5.003];and OR,0.62;95% CI,0.3762-1.033[P5.067],respectively).165This4-miRNA scoring scale generated an independent prognostic score allowing a?nite strati?cation of patients with AML and intermediate-risk cytogenetic?ndings into different prognostic categories and facilitating the proper assignment of postremission therapy.165 Regarding circulating miRNAs in patients with AML, less is known about their potential clinical application. Advancements have been made in the past year by analyzing140patients newly diagnosed with AML and 135healthy adult donors.New data set analyses have led to the identi?cation of a6-miRNA panel consisting of miR-10a-5p,miR-93-5p,miR-129-5p,miR-155-5p,miR-181b-5p,and miR-320d,which are able to differentiate between patients with AML and healthy controls(AUC range,0.8129-0.9531).166Even more,high levels of miR-181b-5p in patient sera were found to be signi?cantly asso-ciated with a worse OS(P5.002).166Overall,these data demonstrated that the expression patterns of circulating miRNAs might be systematically altered in patients with AML and detecting their levels could provide clinically useful diagnostic and prognostic classi?ers. Acute Lymphoblastic Leukemia One of the?rst large-scale miRNA pro?ling analyses in a total of72cases of acute lymphoblastic leukemia(ALL),98 cases of AML,and13normal bone marrow controls identi-?ed a signature of27differentially expressed miRNAs that were able to separate patients with ALL from those with AML.Six miRNAs(miR-128a,miR-128b,miR-130b, miR-151*,j-miR-5,a newly identi?ed miR,and miR-210) were expressed at a signi?cantly higher level in patients with ALL compared with those with AML.167More importantly,4miRNAs(miR-128a,miR-128b,let-7b,and miR-223)revealed the strongest diagnostic performance: the combination of any2or3of these miRNAs were found to separate ALL from AML cases with an overall diagnos-tic accuracy of97%to99%.167 When referring to ALL,the pediatric population becomes one of clinical signi?cance because ALL is the most common malignancy diagnosed in children,represent-ing over25%of all pediatric cancers.In pediatric ALL bone marrow samples,it was shown that miR-100,miR-196b, and let-7e are downregulated,whereas miR-128a and miR-181b are upregulated when compared with normal pediatric control samples.168The subsequent correlation analysis with the clinical and biological features showed that miR-196b expression was signi?cantly elevated(P5.01)in the T-cell ALL phenotype and miR-100expression was elevated in patients with a white blood cell count less than50,000/mm3 at diagnosis,as well as in the presence of t(12;21)(P5.04) and the absence of a hyperdiploid karyotype(P5.04).168 Furthermore,patients with ALL with reduced miR-335 expression are likely to have a poorer5-year OS rate (74.2%)compared with patients with high miR-335expres-sion(OS rate of96.9%;P5.009).ROC curve analysis using the expression level of miR-335resulted in a highly signi?cant AUC of0.73(P5.003).In vitro studies sug-gested that the poor prognosis would be linked to the devel-opment of resistance to glucocorticoid therapy mediated by the derepression of MAP1K.169Indeed,after restoring 326CA:A Cancer Journal for Clinicians miR-335levels,cancer cells proved to acquire sensitivity to a prednisolone-based therapy because of the miR-335-mediated direct inhibition of MAPK1.These?ndings pro-vide suggestions for the development of new strategies that might work,for example,against the glucocorticoid resist-ance arising in treated patients with ALL.169 Multiple Myeloma Because of the inherent heterogeneity of multiple myeloma (MM),several groups have attempted to classify the disease based on gene expression pro?ling.One of the largest pro?ling studies was performed on163bone marrow sam-ples from patients with MM and revealed distinct miRNA signatures associated with the translocation/cyclin D-classi?ed MM subgroups.This study demonstrated the miRNA pattern correlation with clinical outcome in the different translocation/cyclin D prognostic groups of patients,including both the unfavorable4p16and MAF (v-maf musculoaponeurotic?brosarcoma oncogene homo-log[avian])and the favorable11q13groups.In particular, the speci?c upregulation of the miR-99b/let-7e/miR-125a cluster on19q was detected in the4p16group,and miR-150,miR-155,and miR-34a were upregulated in the MAF group.Upregulation of miR-1275and downregulation of miR-138were observed in the11q13cases.Furthermore, the upregulation of3clusters of miRNAs(miR-503-424, miR-17HG,and miR-106A-363)were found to be signi?-cantly associated with decreased OS.More importantly, after a stepwise selection in a multivariable analysis,miR-886-5p and miR-17expression levels were used to con-struct a miRNA-based risk predictor that was able to strat-ify patients into3risk groups(median OS times of19.4 months,40.6months,and65.3months,respectively; P5.001).This scale was demonstrated to ameliorate the classi?cation of patients according to OS by improving the predictive power of the conventional International Staging System/FISH approach(P5.0004).Indeed,the expression value of this miRNA risk estimator identi?ed a separate class of patients with a signi?cantly worse prognosis within a group de?ned as“low risk”by the International Staging System/FISH classi?cation system.170 Dysregulated patterns of circulating serum miRNAs in comparison with healthy controls have also been identi?ed in patients with MM.When using a multivariate logistical regression model on a set of103newly diagnosed patients, 18patients in relapse,57patients with monoclonal gamm-opathy of undetermined signi?cance,and30healthy donors,a combination of2miRNAs(miR-34a and let-7e) proved to be able to distinguish patients with MM and monoclonal gammopathy of undetermined signi?cance from healthy donors(AUC,0.898[80.6%SE and86.7% SP];and AUC,0.976[91.1%SE and97.6%SP],respec-tively).171In same study,lower levels of miR-744and let-7e were found to be associated with shorter OS and remission:the1-year mortality rate for miR-744and let-7e was41%(95%CI,28.8%-57.9%)and34.6%(95%CI, 23.4%-49.2%),respectively,in the low miR-expressing patients and3.3%(95%CI,0.8%-12.7%)and3.9%(95% CI,1.0%-14.8%),respectively,in the patients with high expression.The Cox model also showed the prognostic impact on time to disease progression for both miR-744 (HR,0.690;95%CI,0.584-0.817[P<.0001])and let-7e (HR,0.552;95%CI,0.424-0.718[P<0001]),overall sug-gesting they might be useful as a determinant of patient sur-vival rates.171It was also proven that miR-29a is upregulated in the serum of patients with MM,and that this dysregula- tion could potently discriminate patients with MM from healthy donors with a SE of88%and an SP of70%.172 Carcinoma of an Unknown Primary Metastatic CUP represents one of the10most frequent cancer diagnoses worldwide(3%-5%of newly diagnosed cancer).173Patients with CUP present with metastases (late-stage disease)without an identi?ed primary tumor. MiRNAs are of particular importance in the identi?cation of the site of origin for CUP because their expression levels and pro?les re?ect with great?delity the tissue origin,and also because they are highly stable in formalin-?xed paraf- ?n-embedded(FFPE)tissue blocks(the most available specimen type in pathology).MiRNA pro?ling may be helpful in further discriminating the histological origin of the tumor,as well as its optimal treatment.In an attempt to classify tumors based on their tissue-speci?c miRNA sig-natures,miRNA expression pro?ling was performed in400 paraf?n-embedded and fresh-frozen samples from22dif-ferent tumor tissues and metastases by a custom-made microarray.174The authors built an miRNA-based classi?er including a total of48differentially expressed miRNAs, which was demonstrated to successfully classify most of the samples with a high con?dence(accuracy of89%).174This 48-miRNA panel was further validated on a cohort of204 FFPE tumor samples comprising25different classes as accurately strati?ed by the array.Indeed,for124of204the samples(60%),the sensitivity of the array was90%(111of 124of the classi?cations agreed with the reference diagno-sis),and it exceeded90%for most tissue types.Speci?city (negative agreement)in this group ranged from95%to 100%and averaged above99%.175A new enriched miRNA-based assay(second-generation array)comprising a higher number of miRNAs(64)and able to discriminate a greater number(48)of tumor types has been devel-oped.176As reported in a cohort of84FFPE samples from patients with CUP,data generated by this assay have been shown to agree with the clinical diagnosis at the time of presentation and after patient management in70%and 89%of patients,respectively;moreover,it agreed with the VOLUME64_NUMBER5_SEPTEMBER/OCTOBER2014327 ?nal clinical diagnosis reached with supplemental immuno-histochemical stains in92%of patients,indicating a22% improvement in agreement from diagnosis at presentation to the?nal clinical diagnosis.177In conclusion,these novel miRNA-based approaches demonstrate high accuracy in identifying the?nal clinical diagnosis in patients with CUP,thereby providing a useful tool for the better man-agement and rational therapeutic intervention of this par-ticularly challenging tumor category. MiRNAs as Targets for Anticancer Drug Therapy RNA Inhibition With the Use of MiRNAs By blocking the function of a mRNA,miRNAs could rep-resent a valid option for treating patients with speci?c can-cers in the near future.178-180MiRNAs can be separated into2groups:tumor suppressive or oncogenic,depending on their mRNA targets.Both groups are very well suited for therapeutic purposes,and they are represented by patients with a negatively correlated expression between a speci?c miRNA and its experimentally proven target(s)in the tumors.With the objectives of recovering tumor sup-pressor proteins and inhibiting oncogenic ones,the strategy would involve restoring the miRNAs that are downregu-lated and silencing the miRNAs that are upregulated(Fig. 4).There are2major advantages to using miRNAs(com-pared with other gene-silencing therapies explained in detail in Table4)181-195:?rst,miRNAs are“natural”mole-cules produced in human cells and second,they can target multiple genes from the same pathway and therefore their action can occur at multiple levels in the same pathway, thereby signi?cantly reducing the development of resistance because multiple mutations in multiple genes are needed. For example,miR-15a and miR-16-1,both of which have reduced expression in patients with CLL,have2antiapop-totic targets:the mRNAs encoded by the oncogenes for BCL2and MCL1.196Therefore,a personalized therapy based on the identi?cation of patients with reduced miR-15a/miR-16-1cluster expression and BCL2and MCL1 protein overexpression in CD5/CD19-positive B lympho-cytes can be envisioned.Note how these types of therapies are very speci?c for patients having these molecular charac-teristics in the same cells.Patients with low levels of miR-15a/miR-16but normal levels of BCL2or MCL1or conversely with normal levels of miRNAs but abnormal levels of these antiapoptotic oncogenes should be excluded from clinical trials targeting miR15a/miR-16. Strategies for MiRNA Targeting Strategies for miRNA targeting are based on either restor-ing miRNA levels or blocking miRNA function with oligonucleotide-based strategies(Table4).181-195A targeted approach to replenish the expression levels of par-ticular miRNAs is restoring the level and function of one or a limited number of miRNAs,usually located within a cluster (such as miR-15a and miR-16-1at13q14.3),either with miRNA mimics or with miRNAs encoded in expression vec-tors.MiRNA mimic molecules are double-stranded sequen-ces with100%similarity to the endogenous miRNA,which can be delivered by nanoparticles and thereby are expressed in the cells.This approach is particularly attractive because nanoparticles can be coated with antibodies that recognize tumor-speci?c antigens,therefore allowing a tumor-speci?c delivery of the miRNA of interest.A recent study reported the delivery of miR-34a using nanoparticles coated with a neuroblastoma-speci?c antidisialoganglioside GD2antibody. With this design,the therapy resulted in the inhibition of neuroblastoma tumor growth in a murine orthotopic xeno-graft model.197Moreover,MRX34,a liposome-formulated mimic of the tumor suppressor miR-34,has been developed in a clinical phase1trial for patients with advanced or meta-static liver cancer.Preclinical studies have already shown that tail vein injection of MRX34reduced tumor growth and sig-ni?cantly enhanced survival with a favorable safety pro?le in orthotopic mouse models of hepatocellular carcinoma.198 Another mechanism has been targeting new blood vessel formation in tumors.Positively charged liposomes have been investigated for the ef?cient delivery of drugs to tumor blood vessels.The negative charge of the proteogly-cans on the endothelial cell membranes can bind and help cells internalize cationic liposome due to their electrostatic interaction.199This approach has been proven to be effec-tive in the delivery of miRNA mimics,such as was observed in a NSCLC xenograft murine model.MiR-29b has been shown to be signi?cantly reduced in patients with NSCLC, consequently increasing the expression of its oncogenic tar-get cyclin-dependent kinase6.Through cationic lipoplexes (LPs)-based carriers,miR-29b was successfully delivered, resulting in inhibited tumor growth by60%compared with a LP-miR-negative control.200These results demonstrated that cationic LPs target tumor vasculature in a speci?c manner,and thereby achieve a greater therapeutic potential for lung cancer treatment. Current strategies for inhibitory targeting of microRNAs are mainly based on antisense oligonucleotides(so-called anti-miRNAs),comprised of locked nucleic acids(LNA) along with tiny LNA anti-miRNA constructs,antagomirs, and miRNA sponges(Table4).181-195Miravirsen (SPC3649),an LNA against miR-122,is the?rst miRNA target therapy tested in a clinical trial for the treatment of hepatitis C virus infection.The recently completed phase 2a trial showed that SPC3649exhibited robust antiviral activity in a dose-dependent manner.201More impressively, 4of9patients treated at the highest dose(7mg/kg)with SPC3649reached undetectable levels of hepatitis C virus 328CA:A Cancer Journal for Clinicians RNA (consequently decreasing the possibility of developing hepatocellular carcinoma).202The effectiveness of antimiR-122treatment proved the plausibility of the LNA-based therapeutics and encourages the development of other spe-ci?c miRNA-targeting therapeutic strategies. Combination Approaches With MiRNA Therapeutics The introduction of combination chemotherapy by Freir-eich and Frei in the early 1960s contrary to the initial opinion in the medical community,not only did not harm children with acute lymphocytic leukemia but moreover represented a huge step forward,and was responsible for the excellent outcome of this otherwise 100%lethal disease.203Therefore,we emphasize the need to use combination therapies of various miRNA agents in “cocktails”to be delivered to patients along with chemotherapeutics (therapies with miRNA mimetic and anti-miRNA agents).With regard to CLL for exam-ple,there are 2hypotheses that drive our multimodal approach,in which we could achieve the inhibition of mRNAs and therefore protein expression.130First,a “sandwich RNA inhibition strategy”comprising the use of multiple agents could focus on a major molecular alteration clearly linked to CLL pathogenesis.Given the fact that for this disease the overexpression of antiapop-totic factor BCL2has been correlated with poor patient outcome,we could target this protein by designing regi-mens using a cocktail of anti-BCL2antisense oligonu-cleotides and miR-15a and miR-16(which are already known to target BCL2mRNA).Published studies have already proven that oblimersen sodium (an antisense oli-gonucleotide compound designed to speci?cally bind to human BCL2mRNA)has relative ef?cacy in treating patients with relapsed or refractory CLL 204;therefore,our expectations for this multimodal approach appear to be promising for this indolent disease.Second,“the mul-tiplex RNA-inhibition strategy”targets various molecu-lar defects that are part of the same pathway (such as apoptosis).In this case,multiple synthetic miRNAs tar-geting overexpressed regulators of apoptosis such as BCL2(miR-15a and miR-16)and MCL1(the miR-29family)may be more ef?cient at consistently and robustly reducing the expression levels of these proteins versus a single therapeutic agent. FIGURE 4.MicroRNAs as Targets for Anticancer Drug Therapy.In order to restore tumor suppressor protein levels,oncomirs can be targeted with therapies such as antagomirs or locked nucleic acids anti-microRNAs.When aiming to target oncogenic proteins,the levels of tumor suppressor miR-NAs can be restored by using miRNA mimics.TRBP indicates human immunodeficiency virus-1transactivating response RNA-binding protein;RISC,RNA-induced silencing complex. VOLUME 64_NUMBER 5_SEPTEMBER/OCTOBER 2014 329 TABLE4.The Principal Types of RNA Therapeutic Drugs Antisense oligonucleotides(ASOs) Definition:A single-stranded chemically modified DNA-like molecule17to22nucleotides in length designed to be complementary to a selected messenger RNA (mRNA). Mechanism of action:Specifically inhibits expression of that gene mainly through formation of an mRNA-ASO duplex by sequence complementarity,leading to cleavage of the mRNA of target gene. Example:NCT00030641181 Oligonucleotide:Oblimersen,phase2/3 Condition:Lung cancer:stage IIIB or IV relapsed or refractory disease Molecular target:BCL-2 Rationale:Oblimersen may increase the effectiveness of docetaxel by making the tumor cells more sensitive to the drug. Purpose:Randomized phase2/3trial to compare the effectiveness of docetaxel with or without oblimersen in treating patients who have relapsed or refractory non-small cell lung cancer that has been previously treated. Other examples: NCT01563302:A Phase1/2Study of ISIS481464,an Antisense Oligonucleotide Inhibitor of STAT3,Administered to Patients With Advanced Cancers.182 NCT01780545:The Borealis-2Clinical Trial:A Randomized Phase2Study Comparing Docetaxel Alone to Docetaxel in Combination With OGX-427in Patients With Relapsed or Refractory Metastatic Urothelial Carcinoma After Receiving a Platinum-containing Regimen:Hoosier Oncology Group GU12-160.183 NCT01839604:A Phase I/Ib,Open-Label,Multicentre Study to Assess the Safety,Tolerability,Pharmacokinetics and Preliminary Anti-tumour Activity of AZD9150 in Patients With Advanced/Metastatic Hepatocellular Carcinoma.184 Ribozymes or DNAzymes Definition:A ribozyme,or RNA enzyme,is an RNA molecule that can catalyze a chemical reaction.A DNAzyme,or deoxyribozyme,is a catalytic DNA that is site specifically cleaving the target RNA. Mechanism of action:Consists of3steps,cyclically repeated:base pairing to a complementary target sequence followed by site-specific cleavage of the substrate and finally release of the cleavage products. Example:NCT00021021185 Oligonucleotide:Angiozyme phase2 Condition:Kidney cancer Molecular target:Vascular endothelial growth factor Rationale:RPI.4610may stop the growth of metastatic kidney cancer by stopping blood flow to the tumor. Purpose:Phase2trial to study the effectiveness of RPI.4610in treating patients who have metastatic kidney cancer. Other examples: NCT01449942:Phase I/II Study of EBV-LMP1Targeted DNAzyme in Nasopharyngeal Carcinoma.186 Small interfering RNAs(siRNAs) Definition:A double-strand RNA homologous to an mRNA of a target gene. Mechanism of action:The siRNAs are incorporated into a multiprotein RNA-induced silencing complex,leaving the antisense strand to guide this complex to its homologous mRNA target for endonucleolytic cleavage of mRNA. Example:NCT01591356187 Oligonucleotide:siRNA-EphA2-DOPC phase1 Condition:Advanced cancers Molecular target:EphA2 Rationale:siRNA targeting EphA2may decrease tumor growth in cancer relapse. Purpose:The goal of this clinical research study is to learn about the safety of siRNA-EphA2-DOPC when given to patients with advanced,relapsed cancer. Researchers also want to learn the highest tolerable dose of this drug that can be given.siRNA-EphA2-DOPC is designed to shut down the activity of a gene that causes tumor growth. Other examples: NCT00672542:Phase I Study of Active Immunotherapy of Metastatic Melanoma With Mature Autologous Dendritic Cells Transfected With Tumor Antigen RNA and Small Inhibitory RNAs to Alter Proteasomal Antigen Processing.188 NCT02110563:Phase I,Multicenter,Cohort Dose Escalation Trial to Determine the Safety,Tolerance,and Maximum Tolerated Dose of DCR-MYC,a Lipid Nano particle(LNP)-Formulated Small Inhibitory RNA(siRNA)Oligonucleotide Targeting MYC,in Patients With Refractory Locally Advanced or Metastatic Solid Tumor Malignancies,Multiple Myeloma,or Lymphoma.189 NCT00257647:Use of SV40siRNA Vectors to Treat CML.190 MicroRNA mimics Definition:A microRNA(miRNA)mimic is a small single-strand19to24nucleotide RNA with an identical sequence to the miRNA of interest(to be reexpressed). Mechanism of action:Mimic the effects of an endogenous miRNA with consequent inhibition of protein production by either transcriptional inhibition or translational block or both. Example:NCT01829971191 Oligonucleotide:MRX34(miR-34mimic)phase1 Condition:Primary liver cancer,solid tumors,lymphoma,AML,ALL,CLL,CML(in accelerated or blast phase) Molecular target:MYC,MET,BCL2,b-catenin. Rationale:MRX34will block tumor development and progression by targeting a variety of cancer related pathways. Purpose:This is a study to evaluate the safety of MRX34in patients with primary liver cancer or those with liver metastasis from other cancers.The drug is given intravenously,twice per week for3weeks in a row and then1week off.In addition,patients with hematologic malignancies will be evaluated on a treatment schedule of5days in a row with2weeks off. ASOs/AMOs,LNAs anti-miRNAs,and antagomirs Definition:Small single-stranded oligonucleotides specifically designed to block the function of miRNA. The ASOs/AMOs are single-stranded,chemically modified DNA-like molecules that are17to22nucleotides in length and designed to be complementary to a selected miRNA and specifically inhibit its expression. The LNAs anti-miRNAs represent LNA-modified ASOs. The antagomirs are single-stranded23-nucleotide RNA molecules complementary to the targeted miRNA that have been modified to increase the stability of the RNA and protect it from degradation. 330CA:A Cancer Journal for Clinicians 成都工业学院·计算机工程学院 《程序设计基础》实验报告 1.实验目的 （1）熟练掌握while语句、do…while语句和for语句格式及使用方法，掌握三种循环控制语句的循环过程以及循环结构的嵌套，利用三 种循环语句实现循环结构； （2）掌握简单、常用的算法，并在编程过程中体验各种算法的编程技巧； （3）进一步学习调试程序，掌握语法错误和逻辑错误的检查方法。2.实验内容 （1）输出两个整数m和n，求它们的最大公约数和最小公倍数。 要求： ①画出算法流程图，从键盘输入m和n； ②对负数和零可不做考虑； ③运行程序，对m>n、m ③按照数字、大写字母、小写字母及其他字符数的顺序输出结果 3.流程图 4.源程序 5. 运行结果 （1 ） 求最大公约数和最小公倍数 （2）求1000内最大的10个素数之和（3）计算π值 for循环结构程序设计教案 课程名称：Ｃ语言程序设计 本课内容：循环结构程序设计——for 语句 一、教学目标 1、领会程序设计中构成循环的方法 2 、掌握for 语句的用法 二、教学重点 1、for 语句的一般格式 2、for语句的执行过程 三、教学难点 for 语句的综合利用 四、教学方法 1、课堂讲授，给出主要内容。 2、讲解其基本格式。 3、应用示例，结合相应的知识讲解。 4、执行过程用流程图和例题用(演示法和讲解法)进行详细说明。 五、教学过程 （一）导入 1、复习前两节课我们学习的两种循环语句——while语句和do~while 语句的基本格式和执行过程和特征。 2、给出例子如下： main( ) {int x=1; (表达式１) s=0; while(x<=100) (表达式2) {s=s+x; x=x+1; (表达式3) } printf(“%d”,s); } 分析用while语句来完成的程序，主要完成1到100的累加和，对一个循环程序来说最关键的三个部分：循环变量的赋初值、控制循环条件、循环控制变量的更新。 （二）教学过程 1、写出本节课要介绍的for语句的一般格式 for(表达式1；表达式2；表达式3) 循环体； 2、掌握：基本格式和各个表达式的功能和特征 强调：for语句中的所有表达式和while语句中的表达式所完成的功能是相同的。 表达式1：循环变量的初始化（初始值） 表达式2：循环条件（终止值） 表达式3：循环变量自增 3、for语句的执行过程 for ( int i = 0 ; i < 10 ; i++) { printf("我最棒"); } 一、循环控制 (一)、break语句 break语句通常用在循环语句和开关语句中。当break用于开关语句switch中时，可使程序跳出switch而执行switch以后的语句；如果没有break语句，则将成为一个死循环而无法退出。break在switch中的用法已在前面介绍开关语句时的例子中碰到，这里不再举例。 当break语句用于do-while、for、while循环语句中时，可使程序终止循环而执行循环后面的语句，通常break语句总是与if语句联在一起。即满足条件时便跳出循环。 例如： int main(int argc, char *argv[]) { int sn=0,i; for(i=1;i<=100;i++) { if(i==51) break; /*如果i等于51，则跳出循环*/ sn+=i; /*1+2+……+50*/ } printf(%d\n,sn); } 可以看出，最终的结果是1+2+……+50。因为在i等于51的时候，就跳出循环了。自己写写怎样在while和do--while循环中增加break语句。 注意: 1. break语句对if-else的条件语句不起作用。 2. 在多层循环中，一个break语句只向外跳一层。 例如： int main(int argc, char *argv[]) { int i,j; printf(i j\n); for(i=0;i<2;i++) for(j=0;j<3;j++) { if(j==2) break; printf(%d %d\n,i,j); } } 输出结果为: i j 0 0 0 1 1 0 1 1 当i==0,j==2时，执行break语句，跳出到外层的循环，i变为1。 (二)、continue语句 总结归纳了C语言的32个关键字 第一个关键字：auto 用来声明自动变量。可以显式的声明变量为自动变量。只要不是声明在所有函数之前的变量，即使没加auto关键字，也默认为自动变量。并且只在声明它的函数内有效。而且当使用完毕后，它的值会自动还原为最初所赋的值。自动变量使用时要先赋值，因为其中包含的是未知的值。 例：auto int name=1; 第二个关键字：static 用来声明静态变量。可以显式的声明变量为静态变量。也为局部变量。只在声明它的函数内有效。它的生命周期从程序开始起一直到程序结束。而且即使使用完毕后，它的值仍旧不还原。即使没有给静态变量赋值，它也会自动初始化为0. 例：static int name=1. 第三个关键字：extern 用来声明全局变量。同时声明在main函数之前的变量也叫全局变量。它可以在程序的任何地方使用。程序运行期间它是一直存在的。全局变量也会初始化为0. 例：extern int name； 第四个关键字：register 用来声明为寄存器变量。也为局部变量，只在声明它的函数内有效。它是保存在寄存器之中的。速度要快很多。对于需要频繁使用的变量使用它来声明会提高程序运行速度。 例：register int name=1; 第五个关键字：int 用来声明变量的类型。int为整型。注意在16位和32位系统中它的范围是不同的。16位中占用2个字节。32位中占用4个字节。还可以显式的声明为无符号或有符号： unsigned int或signed int .有符号和无符号的区别就是把符号位也当作数字位来存储。也可用short和long来声明为短整型，或长整行。 例：int num; 第六个关键字：float 用来声明变量的类型。float为浮点型，也叫实型。它的范围固定为4个字节。其中6位为小数位。其他为整数位。 例：float name; 《C语言循环程序设计—for语句》教学案例漠河县职业技术学校尘威威 《C语言循环程序设计—for语句》教学案例 漠河县职业技术学校尘威威 C语言基础是中职计算机专业的一门必修课，也是要求计算机专业学生学习、掌握的一门重点课程，这门课程核心内容就是要让学生掌握一门编程的语言，学会编写简单的程序，能读懂C语言源程序。 案例背景： 在计算机应用专业教学中，C语言是一门理论与实践结合得比较紧的课程。要掌握和使用好这门语言，既要求学生有比较扎实的理论基础，又要具备较强的应用实践能力。如果只是按照传统的知识体系照本宣科，让学生理解这些枯燥的概念都难，更不要说达到良好的教学效果，而且易挫伤学生学习编程的积极性。因此，在教学中可以改为从案例入手，通过给学生演示、让学生模仿，在实际应用中去探究和领悟这些概念，并适时地加以归纳总结和进行概念的延伸，让学生在轻松愉快的气氛中学习新知识。所以从课程内容而言，案例教学是适用的，是切合学生的。 循环结构是程序设计三种基本结构的重中之重，而循环中的for 循环是程序中运用最多的，也是较灵活的语句之一，它既是前面知识的延续，又是后面知识的基础，在知识构架中起着重要的衔接作用，如果不采用一些恰当有效的方法，学生在学习过程中会难以掌握。在教学过程中教师应结合一些有趣的程序，提高学生的学习兴趣，引导 学生全身心地投入课堂。本文针对学生的实际情况，具体阐述for循环语句在具体编程时的灵活应用。 教学目标确定 （一）知识与技能 1、领会程序设计中构成循环的方法 2、能使用for循环语句编写C语言语句，并能运用for循环语句编写出正确的程序。 （二）过程与方法 C语言程序设计中for循环语句教学以行动导向教学为主线，通过“提出问题―分析问题―解决问题―问题扩展―讨论―总结归纳―实践”的程序，过渡到知识应用和练习。 本课采用多媒体课件进行教学，通过课件把文字和图片有机的结合，使学生在学习过程中更加容易理解，学习效率高。在课堂讨论和实践过程中，教师适当引导，学生主动探究、归纳总结学习内容，既有利于领会掌握新知识点，又能充分发挥学生的主体作用。在重点的突破上，采用范例比较教学法，给出具体的案例，让学生通过典型的例子掌握知识，同时通过用while、do while语句的所编写的程序进行比较，加深学生印象，让学生快速的掌握for循环语句的基本结构及使用方法。 （三）情感与价值观 1．让学生在自主解决问题的过程中培养成就感，为今后自主学习打下良好的基础。 第5章循环结构程序设计 练习题 1. 单项选择题 （1）语句while (!e);中的条件!e等价于 A 。 A. e==0 B. e!=1 C. e!=0 D. ~e （2）下面有关for循环的正确描述是 D 。 A. for循环只能用于循环次数已经确定的情况 B. for循环是先执行循环体语句，后判定表达式 C. 在for循环中，不能用break语句跳出循环体 D. for循环体语句中，可以包含多条语句，但要用花括号括起来 （3）C语言中 D 。 A. 不能使用do-while语句构成的循环 B. do-while语句构成的循环必须用break语句才能退出 C. do-while语句构成的循环，当while语句中的表达式值为非零时结束循环 D. do-while语句构成的循环，当while语句中的表达式值为零时结束循环 （4）C语言中while和do-while循环的主要区别是 A 。 A. do-while的循环体至少无条件执行一次 B. while的循环控制条件比do-while的循环控制条件严格 C. do-while允许从外部转到循环体内 D. do-while的循环体不能是复合语句 （5）以下程序段 C 。 int x=-1; do { x=x*x; } while (!x); A. 是死循环 B. 循环执行二次 C. 循环执行一次 D. 有语法错误 （6）下列语句段中不是死循环的是__C__。 第5章循环结构程序设计35 A. i=100; while (1) { i=i%100+1; if (i==20) break; } B.for (i=1;;i++) sum=sum+1; C.k=0; do { ++k; } while (k<=0); D.s=3379; while (s++%2+3%2) s++; （7）与以下程序段等价的是__A__。 while (a) { if (b) continue; c; } A. while (a) B. while (c) { if (!b) c; } { if (!b) break; c; } C. while (c) D. while (a) { if (b) c; } { if (b) break; c; } （8）以下程序的输出结果是_B___。 #include 精心整理 第5章循环结构程序设计 5.1基本知识点 ?while语句的使用格式和注意事项 ?do-while语句的使用格式和注意事项 ?for语句的使用格式和注意事项 ?break和continue语句在循环语句中的应用 ? ? ? ? 1. C. A.do-while的循环体至少无条件执行一次 B.while的循环控制条件比do-while的循环控制条件严格 C.do-while允许从外部转到循环体内 D.do-while的循环体不能是复合语句 （5）以下程序段C。 intx=-1; do { x=x*x; } while(!x); A.是死循环 B.循环执行二次 C.循环执行一次 D.有语法错误 （6）下列语句段中不是死循环的是__C__。 A.i=100; while(1) { i=i%100+1; if(i==20)break; } B.for(i=1;;i++) sum=sum+1; C. C. COUT< num++; cout< for循环使用详解(c语言版) 说明:c语言的很多语法部分都和JAVA,AS等相同特别是for的用法. c语言中的for循环语句使用最为灵活,不仅可以用于循环次数已经确定的情况,而且可以用于循环次数不确定而只给出循环结束条件的情况,它完全可以代替while语句. for(表达式 1;表达式 2;表达式 3)语句 它的执行过程如下: (1)先求表达式 1. (2)求表达式2,若其值为真(值为非0),则执行for语句中指定的内嵌语句,然后执行下面第三步做若为 假(值为0),则结束循环,转到第5步. (3)求解表达式3 (4)转回上面第(2)步骤继续执行; (5)结束循环,执行for语句下面的一个语句; for(循环变量赋初值;循环条件;循环变量增值)语句 如 for(i=1;i =100;i++)sum=sum+i; 它的执行相当于 i=1; while(i =100){ sum=sum+i; i++; } 显然,用for语句更简单、方便。 说明： (1)for语句的一般形式中的”表达式1”可以省略,此时应在for语句之前给循环变量赋初值.注意省略表达式1时,其后的分号不能省略.如for(;i =100;i++){....}; (2)如果表达式2省略即不判断循环条件,循环无终止地循环下去,也就是认为表达式2始终为真. 例如:for(i=1;;i++){.....}; 相当于 i=1; while(1) {sum=sum+1; i++; } (3)表达式3也可以省略,但此时程序设计者应另外设法保证循环能正常结束.如: for(i=1;i =100;) {sum=sum+1; i++; C语言的32个关键字和9种控制语句 C语言的关键字共有32个，根据关键字的作用，可分其为数据类型关键字、控制语句关键字、存储类型关键字和其它关键字四类。 1 数据类型关键字（12个）： (1) char ：声明字符型变量或函数 (2) double ：声明双精度变量或函数 (3) enum ：声明枚举类型 (4) float：声明浮点型变量或函数 (5) int：声明整型变量或函数 (6) long ：声明长整型变量或函数 (7) short ：声明短整型变量或函数 (8) signed：声明有符号类型变量或函数 (9) struct：声明结构体变量或函数 (10) union：声明共用体（联合）数据类型 (11) unsigned：声明无符号类型变量或函数 (12) void ：声明函数无返回值或无参数，声明无类型指针（基本上就这三个作用） 2控制语句关键字（12个）： A循环语句 (1) for：一种循环语句(可意会不可言传） (2) do ：循环语句的循环体 (3) while ：循环语句的循环条件 (4) break：跳出当前循环 (5) continue：结束当前循环，开始下一轮循环 B条件语句 (1)if: 条件语句 (2)else ：条件语句否定分支（与if 连用） (3)goto：无条件跳转语句 C开关语句 (1)switch :用于开关语句 (2)case：开关语句分支 (3)default：开关语句中的“其他”分支 D返回语句 return ：子程序返回语句（可以带参数，也看不带参数） 3 存储类型关键字（4个） (1)auto ：声明自动变量一般不使用 (2)extern：声明变量是在其他文件正声明（也可以看做是引用变量） 循环控制结构（又称重复结构）是程序中的另一个基本结构.在实际问题中，常常需要 进行大量的重复处理，循环结构可以使我们只写很少的语句，而让计算机反复执行，从而完成大量类同的计算. C语言提供了while语句、do...while语句和for语句实现循环结构. 3.4.1while语句 while语句是当型循环控制语句，一般形式为: while<表达式>语句; 语句部分称为循环体，当需要执行多条语句时，应使用复合语句. while语句的流程图见图3-8，其特点是先判断，后执行，若条件不成立，有可能一次也不执行. [例3-11]求n！ 分析：n！=n*（n-1）*（n-2）*..2*1，0!=1.即S0=1，Sn=Sn-1*n.可以从S0开始，依次求出S1、S2、...Sn. 统一令S等于阶乘值，S的初值为0！=1；变量i为计数器，i从1变到n，每一步令S=S*i，则最终S中的值就是n！. 流程图见图3-9，程序如下： 考察图3-9中循环部分的流程图可以看出，在循环前各变量应有合适的值(s=1)，另外，控制循环结束的变量(此处为i)必须在循环体中被改变，否则，循环将无限进行下去，成为死循环. 本题中，将多项式的每一项用t表示，s代表符号，在每一次循环中，只要改变s、n的值， 就可求出每一项t. 一般情况下，while型循环最适合于这种情况：知道控制循环的条件为某个逻辑表达式的值，而且该表达式的值会在循环中被改变，如同例3-12的情况一样. 3.4.2do...while语句 在C语句中，直到型循环的语句是do...while，它的一般形式为： do语句while<表达式> 其中语句通常为复合语句，称为循环体. do...while语句的流程图见图3-10，其基本特点是：先执行后判断，因此，循环体至少被执行一次. 但需要注意的是，do...while与标准的直到型循环有一个极为重要的区别，直到型循环是当条件为真时结束循环，而do...while语句恰恰相反，当条件为真时循环，一旦条件为假，立即结束循环，请注意do...while语句的这一特点. 例[3-13]计算sin(x)=x-x3/3!+x5/5!-x7/7!+... 直到最后一项的绝对值小于1e-7时为止. 分析：这道题使用递推方法来做. 让多项式的每一项与一个变量n对应，n的值依次为1，3，5，7，...，从多项式的前一项算后一项，只需将前一项乘一个因子： (-x2)/((n-1)*n) 用s表示多项式的值，用t表示每一项的值，程序如下: #include 2015-2016(2)循环结构练习题 一、单选题（共40题,共100分） 1. 以下正确的描述是（）（2分） 语句的作用是结束整个循环的执行 B.只能在循环体内和switch语句体内使用break语句 C.在循环体内使用break语句或continue语句的作用相同 D.从多层循环嵌套中退出时，只能使用goto语句 标准答案：B 考生答案： 2. 下面的关键字中，不能够从循环体中跳出循环体外的是（）（2分） 标准答案：D 考生答案： 3. 以下叙述中错误的是（）（2分） A.语句标号是由合法的标识符后面加一个冒号构成 语句的作用是把程序的执行转向语句标号所在的位置 语句必须与语句标号在同一个函数内 D.结构化程序设计主张使用goto语句，使用goto语句的程序可读性强标准答案：D 考生答案： 4. 有以下程序段，while 循环执行的次数是（） int k=0; while(k=1)k++; （2分） A.无限次 B.有语法错，不能执行 C.一次也不执行 D.执行1次 标准答案：A 考生答案： 5. 运行以下程序的结果为（） main() { int num=0; while (num<=2) {num++; printf("%d",num); } } （2分） 标准答案：B 考生答案： 6. 下述程序的输出结果是（）。 #include<> main() { int Y=100; while(Y--); printf("y=%d",Y); （2分） =0 =1 =随机值 =-1 标准答案：D 考生答案： 7. 执行下面的程序后的输出结果为（） int x,y,z; x=20,y=40,z=15; while(y>x) x+=4,y-=4; z/=2; printf("%d,%d,%d",x,y,z); （2分） ,36,30 ,8,7 ,28,7 ,28, 标准答案：C 考生答案： 8. 以下程序段的输出结果是（） int x=0,y=5,z=3; while(z-->0&&++x<5) y=y-1; printf("%d,%d,%d\n",x,y,z); （2分） ,2,0 ,2,-1 ,3,-1 ,-2,-5 标准答案：B 考生答案： 9. 下述语句执行后，变量k的值是（） int k=1; while(k++<10); （2分） D.此为无限循环，值不定 标准答案：B 考生答案： 10. 下面程序的运行结果是（）。 main() { int c1=2, c2=0; while(!c2--) printf("%d,%d", c1--, c2); } （2分） ,-1 B.不输出任何内容 ,-1 ,0 标准答案：C 考生答案： 11. 运行以下程序后,如果从键盘上输入china#main( ) { int v1=0,v2=0; char ch ; while((ch=getchar())!='#') switch(ch) { case 'a': case 'h': default: v1++; case '0': v2++; 本来说好讲讲除了scanf和printf以外例如gets、puts，petchar、putchar等输入输出函数。但准备了半天东西发现牵扯的知识太多，并且很多东西我自己也没有弄清楚。所以啦，我就打算先讲讲程序中最常见的两种循环语句，分别是while 循环语句和for循环语句。 这个while啊，我们都学过英语都知道有“当....的时候”的意思。对，学c语言时就当这个意思就行。 这个例题也没找到什么好的，就搬来了《c程序设计语言》上的例子，如果看过来就当是复习吧。 请看题： 使用公式℃=(5/9)(℉-32),打印下列华氏温度与摄氏温度的对照表。 0 -17 20 -6 40 4 60 15 80 26 100 37 120 48 140 60 160 71 180 82 200 93 220 104 240 115 260 126 280 137 300 148 我们的答案如下 #include } 值得高兴的是，我们又遇到了很多没有见过的东西，总是能见到新东西总是让人感到高兴的。 先是fahr、celsius等几个没见过的单词。这个其实不用说也都知道是啥东西，也就是几个可能原来不认识的变量名，并不是函数。 接下来是/**/格式的几个句子 /*当fahr=0，20，40，...，300时，打印华氏温度与摄氏温度对照表*/ /*华氏温度下限*/ /*华氏温度上限*/ /*步长*/ 这种在/*和*/之间加东西的东西叫做注释。和它的名字一样，仅作为注释，在程序运行过程就会被编译器忽略，因为编译器只对文章正文感兴趣。 这东西存在的主要价值基本上就是帮助看你程序的人或在你检查自己程序时可以快速理解你写的这一部分是干啥用的。因此注释在每个语句的句尾都可以加，在任何可以跳格也就是可以打空格或制表符的地方也都可以加。 剩下我们可能看不懂的大概也就剩while的循环语句了：while(fahr<=upper) { celsius=5*(fahr-32)/9; 三种基本的循环语句: for语句、while语句和do-while语句。 一、循环语句 (一)、for循环它的一般形式为: for(<初始化>;<条件表过式>;<增量>) 语句; 初始化总是一个赋值语句，它用来给循环控制变量赋初值；条件表达式是一个关系表达式，它决定什么时候退出循环；增量定义循环控制变量每循环一次后按什么方式变化。这三个部分之间用;分开。 例如: for(i=1;i<=10;i++) 语句; 上例中先给i赋初值1，判断i是否小于等于10，若是则执行语句，之后值增加1。再重新判断，直到条件为假，即i>10时，结束循环。 注意: (1).for循环中语句可以为语句体,但要用{和}将参加循环的语句括起来。 (2).for循环中的初始化、条件表达式和增量都是选择项，即可以缺省，但;不能缺省。省略了初始化，表示不对循环 控制变量赋初值。省略了条件表达式，则不做其它处理时便成为死循环。省略了增量，则不对循环控制变量进行操作，这时可在语句体中加入修改循环控制变量的语句。 (3).for循环可以有多层嵌套。 例如： for(;;) 语句; for(i=1;;i+=2) 语句; for(j=5;;) 语句; 这些for循环语句都是正确的。 main() { int i,j; printf(i j\n); for(i=0;i<2;i++) for(j=0;j<3;j++) printf(%d %d\n,i,j); } 输出结果为: i j 0 0 0 1 0 2 1 0 1 1 1 2 用for循环求1+2+……+100的和： main() { int sn=0,i; for(i=1;i<=100;i++) sn+=i; /*1+2+……+100*/ printf(%d\n,sn); } 从程序可以看出，使用循环语句可以大大简化代码。(二)、while循环它的一般形式为: while(条件) 语句; while循环表示当条件为真时，便执行语句。直到条件为假才结束循环。并继续执行循环程序外的后续语句。 例如： #include stdio.h main() { 1以下不是死循环的语句是 C 。 A、for(i=10; ; i++ ) sum+=i; B、while (1){x++;} C、for(; (c=getchar())!=?\n?;) printf(“%c”, c); D、for(;; x+= i); 2以下程序的输出结果是 A main() { int x, i; for(i = 1; i <= 100; i++) { x = i; if( ++x % 2 == 0) if( ++x % 3 == 0 ) if( ++x % 7 == 0) printf(“%d “, x); } printf(“\n”); } A、28 70 B、39 81 C、42 84 D、26 68 3. 以下程序段的输出结果是 B int k, j, s; for(k = 2; k < 6; k++, k++) { s = 1; for(j = k; j < 6; j++) s += j; } printf(“%d\n”, s); A、15 B、10 C、24 D、9 4以下程序的输出结果是 B main() { int i, sum; for(i = 1; i < 6; i++) sum += i; printf(“%d\n” sum); } A、15 B、不确定 C、16 D、0 有以下程序 main() { int i; for(i=0; i<3; i++) switch(i) { case 1: printf("%d", i); case 2: printf("%d", i); default : printf("%d", i); } } 执行后输出结果是 A A、011122 B、120 C、012020 D、012 以下程序段的输出结果是 C 。 int n=10; while(n>7) { n--; Pr intf(“%d”, n ); } A、1098 B、10987 C、987 D、9876 下面程序的运行结果是 A #include 第5章循环结构程序设计 5.1基本知识点 ?while语句的使用格式和注意事项 ?do-while语句的使用格式和注意事项 ?for语句的使用格式和注意事项 ?break和continue语句在循环语句中的应用 ?循环结构的嵌套 ?使用goto语句实现循环结构 ?穷举法程序设计方法 ?迭代程序设计方法 5.2练习题5 1.单项选择题 （1）语句while(!e);中的条件!e等价于A。 A.e==0 B.e!=1 C.e!=0 D.~e （2）下面有关for循环的正确描述是D。 A.for循环只能用于循环次数已经确定的情况 B.for循环是先执行循环体语句，后判定表达式 C.在for循环中，不能用break语句跳出循环体 D.for循环体语句中，可以包含多条语句，但要用花括号括起来（3）C语言中D。 A.不能使用do-while语句构成的循环 B.do-while语句构成的循环必须用break语句才能退出 C.do-while语句构成的循环，当while语句中的表达式值为非零时结束循环 D.do-while语句构成的循环，当while语句中的表达式值为零时结束循环 （4）C语言中while和do-while循环的主要区别是A。 A.do-while的循环体至少无条件执行一次 B.while的循环控制条件比do-while的循环控制条件严格 C.do-while允许从外部转到循环体内 D.do-while的循环体不能是复合语句 （5）以下程序段C。 intx=-1; do { x=x*x; } while(!x); A.是死循环 B.循环执行二次 C.循环执行一次 D.有语法错误（6）下列语句段中不是死循环的是__C__。 A.i=100; while(1) { i=i%100+1; if(i==20)break; } B.for(i=1;;i++) sum=sum+1; C.k=0; do { ++k; }while(k<=0); D.s=3379; while(s++%2+3%2) s++; （7）与以下程序段等价的是__A__。 while(a) { if(b)continue; c; } A.while(a) B.while(c) {if(!b)c;} {if(!b)break;c;} C.while(c) D.while(a) 第5章循环结构程序设计 基本知识点 while语句的使用格式和注意事项 do-while语句的使用格式和注意事项 for语句的使用格式和注意事项 break和continue语句在循环语句中的应用 循环结构的嵌套 使用goto语句实现循环结构 穷举法程序设计方法 迭代程序设计方法 练习题5 1. 单项选择题 （1）语句while (!e);中的条件!e等价于 A 。 A. e==0 B. e!=1 C. e!=0 D. ~e （2）下面有关for循环的正确描述是 D 。 A. for循环只能用于循环次数已经确定的情况 B. for循环是先执行循环体语句，后判定表达式 C. 在for循环中，不能用break语句跳出循环体 D. for循环体语句中，可以包含多条语句，但要用花括号括起来 （3）C语言中 D 。 A. 不能使用do-while语句构成的循环 B. do-while语句构成的循环必须用break语句才能退出 C. do-while语句构成的循环，当while语句中的表达式值为非零时结束循环 D. do-while语句构成的循环，当while语句中的表达式值为零时结束循环 （4）C语言中while和do-while循环的主要区别是 A 。 A. do-while的循环体至少无条件执行一次 B. while的循环控制条件比do-while的循环控制条件严格 C. do-while允许从外部转到循环体内 D. do-while的循环体不能是复合语句 （5）以下程序段 C 。 int x=-1; do { x=x*x; } while (!x); A. 是死循环 B. 循环执行二次 C. 循环执行一次 D. 有语法错误（6）下列语句段中不是死循环的是__C__。 A. i=100; while (1) { i=i%100+1; if (i==20) break; } B. for (i=1;;i++) sum=sum+1; C. k=0; do { ++k; } while (k<=0); D. s=3379; while (s++%2+3%2) s++; （7）与以下程序段等价的是__A__。 while (a) { if (b) continue; c; } A. while (a) B. while (c) { if (!b) c; } { if (!b) break; c; } C. while (c) D. while (a) { if (b) c; } { if (b) break; c; }（8）以下程序的输出结果是_B___。 Turbo C 2.0提供三种基本的循环语句: for语句、while语句和do-while语句。 一、循环语句 (一)、for循环它的一般形式为: for(<初始化>;<条件表过式>;<增量>) 语句; 初始化总是一个赋值语句，它用来给循环控制变量赋初值；条件表达式是一个关系表达式，它决定什么时候退出循环；增量定义循环控制变量每循环一次后按什么方式变化。这三个部分之间用;分开。 例如: for(i=1;i<=10;i++) 语句; 上例中先给i赋初值1，判断i是否小于等于10，若是则执行语句，之后值增加1。再重新判断，直到条件为假，即i>10时，结束循环。注意: (1).for循环中语句可以为语句体,但要用{和}将参加循环的语句括起来。 (2).for循环中的初始化、条件表达式和增量都是选择项，即可以缺省，但;不能缺省。省略了初始化，表示不对循环控制变量赋初值。省略了条件表达式，则不做其它处理时便成为死循环。省略了增量，则不对循环控制变量进行操作，这时可在语句体中加入修改循环控制变量的语句。 (3).for循环可以有多层嵌套。 例如： for(;;) 语句; for(i=1;;i+=2) 语句; for(j=5;;) 语句; 这些for循环语句都是正确的。 main() { int i,j; printf(i j\n); for(i=0;i<2;i++) for(j=0;j<3;j++) printf(%d %d\n,i,j); } 输出结果为: i j 0 0 0 1 0 2 1 0 1 1 1 2 用for循环求1+2+……+100的和： main() { int sn=0,i; for(i=1;i<=100;i++) sn+=i; /*1+2+……+100*/ printf(%d\n,sn); } 从程序可以看出，使用循环语句可以大大简化代码。 (二)、while循环它的一般形式为: while(条件) 语句; while循环表示当条件为真时，便执行语句。直到条件为假才结束循环。并继续执行循环程序外的后续语句。 例如： #include stdio.h main() { char c; c='\0'; /*初始化c*/ while(c!='\n') /*回车结束循环*/ c=getche(); /*带回显的从键盘接收字符*/ } 上例中，while循环是以检查c是否为回车符开始，因其事先被初始化为空，所以条件为真，进入循环等待键盘输入字符；一旦输入回车，则c='\n'，条件为假，循环便告结束。与for循环一样，while循环总是在循环的头部检验条件，这就意味着循环可能什么也不执行就退出。 循环习题集锦 一．选择题 1．for(i=0;i<10;i++);结束后，i 的值是；（10） A 9 B 10 C 11 D 12 2.下面程序的循环次数是：（D） int k=0; while(k<10) {if(k<1) continue; if(k==5) break; k++; } A. 5 B 6 C 4 D 死循环，不能确定循环次数 3.下面程序的输出结果是（10） main() { int s,k; for(s=1,k=2;k<5;k++) s+=k; printf(“%d\n”,s); } A 1 B 9 C 10 D 15 4.要使下面程序输出10 个整数，则在下画线处填入正确的数是：（c） for(i=0;i<= ;) printf(“%d\n”,i+=2); A 9 B 10 C 18 D 20 5.运行下面程序：（B） main() { int i=10,j=0; do { j=j+i; i--; }while(i>5); printf(“%d\n”,j); } 输出结果是： A 45 B 40 C 34 D 55 6.运行下面程序 #include “stdio.h” main() { int k=0,a=1; while(k<10) { for( ; ;) { if((k%10)==0) break; else k--; } k+=11; a+=k; } printf(“%d %d\n”,k,a); } 则输出的结果是（C） A 21 32 B 21 33 C 11 12 D 10 11 7.以下叙述正确的是：（D） A.do---while 语句构成的循环不能用其他语句构成的循环来代替。 B.do---while 语句构成的循环只能用break 语句退出。 C.用do---while 语句构成的循环，在while 后的表达式为非零时结束循环。 D.用do---while 语句构成的循环，在while 后的表达式为零时结束循环。 8.有如下程序： main() 循环控制练习题 1.设有程序段int k=10; while (k=0) k=k-1; 则下面描述中正确的是（） A while循环执行10次 B 循环是无限循环 C 循环体语句一次也不执行 D 循环体语句执行一次 2.设有以下程序段int x=0,s=0; while(!x!=0)s+=++x;printf("%d",s) ;则（） A 运行程序段后输出0 B 运行程序段后输出1 C 程序段中的控制表达式是非法的 D 程序段执行无限次 3、语句while(!E);中的表达式!E特等价于（） Ａ、Ｅ＝＝０Ｂ、Ｅ！＝１Ｃ、Ｅ！＝０Ｄ、Ｅ==１ ４、下面程序段的执行结果是（） a=1;b=2;c=2; while(a while(x<15) y++,x+=++y; printf("%d,%d",y,x); A、20,7 B、6，12 C、20，8 D 8，20 6、下面程序段的运行结果是（） int n=0; while (n++<=2); printf("%d",n); A,2 B、3 C、4 D 有语法错误 7、设有程序段 t=0; while(printf("*")) {t++; if(t<3) break;}下面描述正确的是（） A、其中循环控制表达式与0等价 B、其中循环控制表达式与’0‘等价 C 、其中循环表达式是不合法的 D、以上说法都不对 8、下面程序的功能是将从键盘输入的一对数，由小到大排序输出。当输入一对相等的数时结束循环，请选择填空。 main()大一上期C语言实验报告5 循环控制语句
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