MicroRNAome Genome_A Treasure for Cancer Diagnosis and Therapy

OPENREVIEWMicroRNA regulons in tumor microenvironmentHI Suzuki 1,A Katsura,H Matsuyama 2and K MiyazonoINTRODUCTIONRecent advances in cancer therapeutics have yielded several new-generation therapeutic modalities,and cancer prognoses have improved signi ficantly.However,further advances are required before major improvements are achievable,especially in patients with advanced and/or intractable cancers,which are refractory to conventional therapeutic options.Although cancer therapies ideally seek eradication of all cancer cells in tumor tissues,recent work has emphasized that targeting of cancer cells is not equivalent to targeting of tumor tissues.The work suggests that cancer initiation and progression are de fined by not only the behavior of cancer cells per se but also the development of tumor tissues,controlled in turn by crosstalk between cancer cells and the surrounding microenvironment.1–3Recent cancer research has emphasized the signi ficance of constant evolution of the tumor microenvironment,facilitating tumor formation,metastasis and development of refractoriness to cancer therapy.3Tumor microenvironments are highly heterogeneous,contain-ing various cell types,including fibroblasts,endothelial cells,pericytes,immune cells and local and bone marrow-derived stromal stem and progenitor cells,and a surrounding extracellular matrix (ECM)(Figure 1).1–3These cells originate from normal cells but become altered during tumor development.For example,many types of solid tumor are accompanied by variable extents of stromal cell in filtration and ECM deposition,termed desmoplasia.Such cancer tissue remodeling allows tumor cells to grow and disseminate and contributes to an increase in interstitial fluid pressure,which can impede delivery of cancer drugs.2–4Cancer-associated fibroblasts (CAFs)make versatile contribution to these responses.2,3MicroRNAs (miRNAs)are small non-coding RNAs that function as major players of posttranscriptional gene regulation within diverse cell types.5Re flecting the importance in cell biology,they also have critical roles in cancer biology.In addition to various protein-coding oncogenes and tumor-suppressor genes,previous studies have demonstrated multifaceted roles of miRNAs in cancer cell behaviors,which constitute major aspects of conventional hallmarks of cancer,including sustained proliferation,resistance to cell death,evasion of growth suppressors,establishment of replicative immortality and acquisition of invasive phenotypes.1,6For example,miR-34a is associated with growth control,miR-21with cell proliferation and the miR-200family with epithelial –mesenchymal transition (EMT).7In addition,recent work has shown that miRNAs of cancer cells modulate the microenvironment via non-cell-autonomous mechanisms,and alterations in the miRNA pro files of neighboring cells that lack genetic abnormalities favor the acquisition of cancer hallmark traits,thereby unexpectedly expanding the roles of miRNAs in tumor microenvironments.8–12Such actions include regulation of tumor angiogenesis,tumor immune invasion and tumor –stromal interactions.The present review focuses on the relationship between miRNA alterations in cancer cells and tumor microenvironments,and the mechanisms of miRNA-mediated regulation of tumor microenvironments,with the perspective of possible therapeutic interventions.Department of Molecular Pathology,Graduate School of Medicine,The University of Tokyo,Tokyo,Japan.Correspondence:Professor K Miyazono,Department of Molecular Pathology,Graduate School of Medicine,The University of Tokyo,7-3-1Hongo,Bunkyo-ku,Tokyo 113-0033,Japan.E-mail:miyazono@m.u-tokyo.ac.jp 1Current address:David H.Koch Institute for Integrative Cancer Research,Massachusetts Institute of Technology,Cambridge,MA 02139,USA.2Current address:Otsuka Maryland Medicinal Laboratories,Inc.,9900Medical Center Drive,Rockville,MD 20850,USA.Received 4May 2014;revised 4June 2014;accepted 6June 2014Oncogene (2014),1–10©2014Macmillan Publishers Limited All rights reserved 0950-9232//oncDYSFUNCTION OF miRNAS IN CANCERIn mammalian cells,the primary transcripts of miRNAs (primary miRNAs;pri-miRNAs)are transcribed by RNA polymerase II or III from miRNA-coding sequences,residing principally in intergenic regions or within introns of genes.Most pri-miRNAs are processed into short hairpin RNAs (precursor miRNAs;pre-miRNAs)by the Drosha/DGCR8complex with RNase III activity in the nucleus.Pre-miRNAs are transported to the cytoplasm and further cleaved by a second RNase III Dicer to yield 21–25-nucleotide-long miRNA duplexes.Single RNA strands derived from such duplexes are incorporated as mature miRNAs within Argonaute proteins (Ago1-4)to form RNA-induced silencing complexes.In general,RNA-induced silencing complexes suppress the expression of various target genes through sequence complementarity between miRNAs and target mRNAs.5,13Various mechanisms contribute to aberrant miRNA expression in cancer.13,14As with abnormalities in oncogenes and tumor-suppressor genes,alterations in miRNAs can be explained in part by several mechanisms,including chromosomal deletion,ampli-fication,mutation,epigenetic silencing and transcriptional dysre-gulation of pri-miRNA transcripts.On the other hand,concerning the characteristic process of miRNA biogenesis,impairment of miRNA processing might explain the downregulation of both broad groups of miRNAs and certain speci fic miRNAs.13,15In addition,various miRNA-processing regulators,such as LIN28,KSRP and p53,have been identi fied to date at each step of miRNA biogenesis,suggesting multidimensional regulation of miRNA biogenesis.13,16–18Furthermore,a recent report showed that Hippo signaling pathway regulated pri-miRNA processing in a cell-density-dependent manner,linking cell –cell contact to miRNA processing.19NON-CELL-AUTONOMOUS FUNCTION OF CANCER-RELATED miRNASMost,if not all,of cancers are de fined by the evolution of genomic abnormalities.Thus,in theory,alterations of ostensibly normal cells in tumor microenvironments will be largely attributable to dysfunction of the gene regulatory network of cancer cells per se .Recent advances have shown that miRNA dysfunction in tumor cells modulates the tumor microenvironment via non-cell-autonomous mechanisms (Figure 2),thus supporting this concept.10In this context,the core targets of miRNAs,termed miRNA regulons,are currently being expanded to include various modulators of tumor microenvironments.8–12This type of regula-tion has been also described in the context of protein-coding oncogenes and tumor-suppressor genes.For example,it has been shown that KRAS,mutation in which is an early event in the development of pancreatic ductal adenocarcinoma,enhances granulocyte macrophage colony-stimulating factor (GM-CSF)production from pancreatic ductal epithelial cells and thus increases recruitment of immunosuppressive Gr1+CD11b +myeloid cells to suppress anti-tumor immunity.20,21In this section,we focus on such distal impacts of cancer-associated miRNAs onthreeFigure ponents of tumor microenvironment.Tumor micro-environment is very heterogeneous and comprised of various cell types,such as CAFs,endothelial cells,pericytes,immune cells,including various types of lymphocytes,Treg,TAMs and MDSCs,and local and bone marrow-derived stromal stem and progenitor cells,and surrounding ECM.Cancer cellMicroenvironmentFigure 2.Non-cell-autonomous roles of cancer cell miRNAs in regulation of tumor microenvironment.Alteration of miR-9(a ),miR-126(b ),miR-135b (c ),miR-29b (d ),miR-30b/d (e ),miR-34a (f )and miR-199a-5p/3p and miR-1908(g )in cancer cells elicit distal impacts on tumor microenvironment to promote tumor progression through various non-cell-autonomous mechanisms.Oncogenic miRNAs and tumor-suppressive miRNAs are represented by black and red,respectively.microRNAs in tumor microenvironmentHI Suzuki et al2Oncogene (2014),1–10©2014Macmillan Publishers Limitedmajor components of the tumor milleu;these are the vasculature,ECM and immune cells.Distal regulation of tumor angiogenesis by miR-9and miR-126 Epithelial–mesenchymal transition(EMT)is an important step incancer metastasis.Among a set of EMT-regulatory miRNAs, including miR-200,miR-103/107,miR-205,let-7and miR-9,22miR-9has been shown to simultaneously modulate tumorangiogenesis through regulation of vascular endothelial growth factor(VEGF)-A(Figure2a).23In breast cancer cells,miR-9,inducedby MYC and MYCN,targeted E-cadherin and increased cell motilityand invasiveness.E-cadherin downregulation by miR-9activated β-catenin signaling,in turn upregulating VEGF-A expression and increasing tumor angiogenesis.23This report raised the possibilitythat miRNA targets diversify onto cell-autonomous regulators andnon-cell-autonomous regulators in tumor progression.This possibility was further solidified by studies on miR-126,the expression of which is frequently suppressed in various types of human cancer,including breast,gastric and colon cancers.24–26 Tavazoie et al.24showed that miR-126non-cell-autonomously restricted metastasis via regulation of multiple targets involved in endothelial recruitment(Figure2b).27They showed that endo-genous miR-126in breast cancer cells contributed to suppression of metastatic colonization and that the metastatic nodules developing upon miR-126silencing displayed a denser vascula-ture.Mechanistically,miR-126downregulates secretion of a soluble form of c-Mer tyrosine kinase receptor(MERTK),and insulin-like growth factor binding protein2(IGFBP2),from metastatic cells,by suppressing MERTK and inhibiting IGFBP2 and a regulatory gene thereof,PITPNC1(encoding phosphatidy-linositol transfer protein,cytoplasmic1),respectively.Subse-quently,downregulation of endogenous miR-126promotes endothelial recruitment by increasing IGFBP2/IGF1/IGF1R signal-ing and attenuating GAS6/MERTK signaling,regulated by compe-tition between the MERTK ligand GAS6and MERTK,in endothelial cells.They further showed that overexpression of eight target genes of miR-126,including MERTK,IGFBP2and PITPNC1,in primary breast cancer,was associated with shorter metastasis-free survival in multiple cohorts.27This set of eight genes constitutes the core target genes of miR-126,namely the miR-126regulon,which links the anti-metastatic activity of miR-126to cancer–endothelial interactions.In addition,Wang and colleagues reported a unique function of miR-126in breast cancer metastasis.28Although the precise mechanism(s)determining asymmetric miRNA biogenesis from a single miRNA precursor have not yet been defined,the miR-126 precursor produces comparable amounts of5p and3p miRNA species.They showed that the two mature miRNAs derived from pre-miR-126,miR-126and miR-126*,cooperatively targeted expression of stromal cell-derived factor-1alpha(SDF-1α)/ chemokine(C-X-C motif)ligand12(CXCL12),and subsequently chemokine(C-C motif)ligand2(CCL2),in cancer cells.Through these inhibitory effects,they suppressed recruitment of mesench-ymal stem cells and inflammatory monocytes into tumor stroma, ultimately suppressing lung metastasis by breast cancer cells.In their report,miR-126/-126*appeared to exert metastasis-suppressive activities predominantly in primary tumor site,rather than metastatic nodules.28Regulation of tumor cell immunophenotype by miR-135b Although the roles of miRNAs in tumor microenvironments have emerged in recent reports,especially about solid tumors,8–11our group discovered a unique environmental involvement of miRNAs in hematological malignancies,where miRNA alteration accounts for regulation of immunophenotype of cancer cells per se and modulation of tumor microenvironments(Figure2c).29We showed that miR-135b,overexpressed in various types of cancer,including colon and lung cancers,30,31was highly expressed in nucleophosmin-anaplastic lymphoma kinase(NPM-ALK)-positive anaplastic large cell lymphomas(ALCLs)and mediated down-stream signaling of the NPM-ALK-STAT3(signal transducer and activator of transcription factor3)pathway.29In this tumor type, NPM-ALK oncogene strongly promotes the expression of LEMD1,ahost gene of miR-135b,and that of miR-135b,through activationof STAT3.FOXO1was identified as a target of miR-135b, suggesting contribution to the oncogenic activities of NPM-ALK.In parallel,we demonstrated immunomodulatory properties ofmiR-135b.Interestingly,miR-135b conferred interleukin(IL)-17-producing immunophenotype,which has recently been demon-strated by genome-wide expression profiling of various peripheralT-cell lymphomas,on ALCL cells.29This skewing of ALCL immunophenotype,overlapping with that of T-helper(Th)17 cells,was associated with targeting of Th2master regulators STAT6and GATA3by miR-135b,indicating that miR-135b contributes to generation of an IL-17-producing immunopheno-type by perturbing mutually antagonistic differentiation programs active during normal lymphocyte differentiation.32,33miR-135b suppression inhibited expression levels of IL-17A,IL-17F,IκBζ,IL-6and IL-8in ALCL cells.miR-135b silencing also attenuated the expression of granzyme B and perforin1,cytotoxic molecules highly expressed in ALCLs,implying that miR-135b exerts a broad range of effects on the ALCL immunophenotype.In line with theinflammatory role of IL-17,miR-135b blockade attenuated the paracrine inflammatory response in co-cultures of ALCL cells andfibroblasts and reduced tumor angiogenesis and in vivo growth. Although the mechanisms underlying lymphoma immunopheno-types related to the corresponding normal lymphocytes are largely unclear,the study illuminated the unique contribution made by an oncogenic kinase-linked miRNA to the modulation of tumor immune phenotype.Distal impacts of miR-29on angiogenesis and ECM remodelingAn association between miRNAs and GATA3transcriptional factorhas also been described in the setting of breast cancer development.34In the mammary gland,GATA3is required for luminal epithelial cell differentiation and maintenance,and its expression progressively decreases during progression of luminal breast cancer in association with a worse prognosis.A recent report demonstrated that GATA3promoted differentiation of breast cancer cells,inhibited metastasis and modified the tumor microenvironment,through induction of miR-29b(Figure2d).34 Thus the GATA3–miR29b axis functions as a tumor-suppressive arm,and loss of miR-29b in cells expressing GATA3promotes a mesenchymal phenotype and metastasis.As mechanistic insights, various targets of miR-29b,including VEGF-A,ANGPTL4,platelet-derived growth factor,LOX and matrix metalloproteinase9 (MMP9),which are involved in angiogenesis,collagen remodelingand proteolysis to promote metastasis,were identified.miR-29b introduction in a murine orthotopic breast cancer model reduced blood vessel development and the extent offibrillar collagen synthesis without a concomitant effect on primary tumor size and reduced the incidence and size of metastasis.This metastasis inhibitory effect of miR-29b was mitigated by re-introduction ofthe miR-29b regulons VEGF-A,ANGPTL4,LOX and MMP9, strengthening the importance of microenvironmental target regulation by miR-29b.34This study revealed a pleiotropic rolefor miR-29b in the modulation of the tumor microenvironment;both angiogenesis and ECM remodeling were affected.AsmiR-29b was also suppressed in NPM-ALK-positive ALCLs,in which GATA3is downregulated,35a GATA3–miR-29b axis may be operational in a range of cancer types.Additionally,in nasophar-yngeal carcinomas,miR-29c downregulation has been reported to induce the expression of ECM proteins,including COL1A2, COL3A1,COL4A1and lamininγ1,as well.36microRNAs in tumor microenvironmentHI Suzuki et al3©2014Macmillan Publishers Limited Oncogene(2014),1–10Distal modulation of tumor–immune crosstalk by miRNAsAn important aspect of the evolution of tumor microenvironments is evasion of anti-tumor immune responses.3Recent work has suggested that such immune escape of tumors can be broadly classified into two categories,depending on the characteristics of the tumor microenvironment.37One major type exhibits a T-cell-inflamed phenotype with infiltration of T cells and a broad chemokine profile.Such tumors appear to endure immune attack predominantly by engaging inhibitory effectors of the immune system,such as PD-L1,IDO and regulatory T(Treg)cells.The other major type lacks this T-cell-inflamed phenotype and appears to prevent immunological attack through immune system ignorance and exclusion.In tumors in which the latter mechanism is operative,dense stroma and accumulation of immunosuppressive myeloid or macrophage populations might be observed,instead of T-cell infiltration.miRNA dysregulation in cancer cells appears to contribute to both types of immune escape.Regulation of SDF-1αby miR-126/126*and modulation of tumor stroma reactions by miR-29b may be associated with the latter category of escape.28,34Recent analyses have shed light on the contributions made by miRNAs to activation of the inhibitory arm of the immune system in the former category of escape.In human melanoma,high expression levels of miR-30b and miR-30d were associated with frequent metastasis,early recurrence and lower overall survival (Figure2e).38Functional analyses revealed that miR-30b/-30d directly targeted the GalNAc transferase GALNT7and subse-quently promoted the secretion of the immunosuppressive cytokine IL-10.38This reduced immune cell activation and enhanced recruitment of Treg cells,promoting metastasis.In addition,a study in hepatocellular carcinoma also revealed enhancement of Treg cell function by a miRNA-mediated non-cell-autonomous mechanism(Figure2f).39In hepatocellular carci-noma,portal vein tumor thrombus(PVTT)is associated with a poor prognosis.Persistent presence of heptitis B virus(HBV)in liver tissue enhances transforming growth factor(TGF)-βactivity, and in turn,TGF-βsuppresses miR-34a.39Although miR-34a is well known to be a transcriptional target of p53,and a representative tumor-suppressive miRNA,15miR-34a had no significant effect on cell proliferation but suppressed the production of CCL22 important for Treg cell recruitment.An inverse correlation between the expression level of miR-34a and CCL22or FoxP3 was observed in HBV+primary tumor and PVTT samples. Restoration of miR-34a inhibited the growth of murine liver tumor cells,infiltration of Treg cells and metastasis,in immune-competent mice.39Taken together,the data showed active participation of miRNAs in immune escape and also revealed one of the mechanisms of immune suppression by TGF-β,which is abundantly expressed in tumor stroma and has a versatile role in tumor development.40Furthermore,other reports have also revealed the involvement of multiple miRNAs in immune responses.In glioma,miR-124 downregulation is associated with immunosuppressive activities of glioma stem cells and suppression of the effector response of T cells.41miR-17-5p and miR-20in miR-17-92polycistronic cluster were shown to suppress cell migration and invasion by altering the secretion levels of IL-8,CK8and CXCL1from breast cancer cells.42In head and neck cancer,the tumor-suppressive miR-145 has been reported to target SOX9and ADAM17and subsequently suppress IL-6production.43Convergent modulation of environmental regulators by multiple miRNAsIndependent miRNAs potentially target a range of genes exerting various functions,including both tumor suppressors and promo-ters.The sets of miRNA regulons mentioned above serve as examples that the targets divergently biased for tumor promotion or suppression.On the other hand,another scenario has also been described:one set of miRNAs convergently target metastasis-associated genes.44In melanoma,a set of pro-metastatic miRNAs, including miR-199a-5p,miR-199a-3p and miR-1908,has been shown to combinatorially target apolipoprotein E(ApoE) (Figure2g).44Secretion of ApoE from cancer cells suppresses cancer cell invasion and endothelial cell recruitment,thereby inhibiting metastasis and angiogenesis.Regulation of SDF-1αby miR-126/126*is also categorized as such a type of regulation.28 When independent miRNAs lack the power to drive significant biological effects,a combination of alterations in multiple miRNAs may modulate the threshold to achieve metastasis. PROXIMAL ROLES OF miRNAS IN TUMOR STROMAL CELLS Various cell types,includingfibroblasts,endothelial cells,pericytes and immune inflammatory cells,contribute to the formation of tumor microenvironments favorable for cancer growth.Of these, CAFs,in contrast to normalfibroblasts,enhance ECM production and secrete cancer-activating cytokines and chemokines,signifi-cantly promoting tumorigenesis.3,45–48It remains largely unclear how CAFs arise from normalfibroblasts.In certain cancer types, CAFs are thought to be generated from normalfibroblasts by tumor-derived paracrine signals.45,46Nonetheless,it has also been known that the tumor-supporting capacity of CAFs is sustainable over multiple passages in the absence of cancer cells.45Although early studies suggested the presence of genetic abnormalities in tumor-suppressors p53and phophatase and tensin homolog (PTEN)in CAFs,49detailed genetic analyses have indicated that genetic alterations are in fact rare,50,51suggesting that other epigenetic mechanisms stabilize the CAF phenotype.In this context,recent studies have revealed that the miRNA profiles of normalfibroblasts and CAFs differ and that several features of the CAF phenotype are attributable to miRNA dysregulation.9In addition,miRNA function has been linked to variations in other tumor-associated cell types,including osteoclasts,myeloid-derived suppressor cells(MDSCs),and tumor-associated macro-phages(TAMs).We next focus on the dysregulation of miRNAs in stromal cells and the proximal effects of such changes in tumor microenvironments(Figure3).Reprogramming of CAFs by miR-31,miR-214,and miR-155Mitra et al.52recently conducted miRNA expression profiling of primary CAFs vs adjacent normalfibroblasts in ovarian cancer patients,and induced CAFs(iCAFs),generated from normal fibroblasts upon co-culture with tumor cells,vs normalfibroblasts(Figure3a).This study showed downregulation of miR-214and miR-31and upregulation of miR-155in CAFs.Interestingly, introduction of miR-214and miR-31and silencing of miR-155 converted the CAF phenotype to normality,and vice versa, suggesting a direct involvement of miRNAs in reversible conver-sion of normalfibroblasts into CAFs.Patient CAFs,iCAFs and miRNA-reprogrammed CAFs(miR-CAFs)all exhibited high expres-sion levels of several pro-tumorigenic chemokines,including CCL5, CCL20and CXCL8/L5was shown to be a direct target of miR-214.Moreover,patient CAFs and miR-CAFs enhanced the growth of co-injected ovarian cancer cells and increased invasion by co-cultured cancer cells;these effects were abrogated upon neutralization of CCL5,indicating that CCL5is a key tumor modulator in miR-CAFs.52The results indicated that ovarian cancer cells reprogramfibroblasts to CAFs via the action of miRNAs. In addition,downregulation of miR-31has been reported in CAFs isolated from endometrial cancers,and miR-31downregulation upregulated the target SATB2(special AT-rich sequence-binding protein2),enhancing tumor cell migration.53Downregulation of miR-148a in CAFs of endometrial cancer has also been reported.54microRNAs in tumor microenvironmentHI Suzuki et al4Oncogene(2014),1–10©2014Macmillan Publishers LimitedProximal roles of PTEN-miR-320axis in CAFCAFs frequently show characteristic upregulation of CAF-related genes,such as α-smooth muscle actin,fibroblast-speci fic protein,platelet-derived growth factor-B and fibroblast activation protein.47,55In addition,alterations in the expression levels of several other markers,including periostin,p53,PTEN and podoplanin,have been reported to correlate with the prognostic outcomes of solid tumors.55–57Accordingly,abrogation of PTEN in stromal cells has been shown to promote the development of epithelial mammary tumors.57A recent study found that a speci fic miRNA was responsible for the tumor-suppressive activity of PTEN (Figure 3a).58Of several miRNAs exhibiting distinct expression patterns in PTEN-depleted fibroblasts,miR-320was identi fied as a tumor suppressor acting downstream of PTEN.miR-320targets ETS2(v-ets erythroblastosis virus E26oncogene homolog 2),MMP9and Emilin2,and downregulation thereof induces a tumor-speci fic secretome,composed of MMP9,MMP2,BMP1and LOXL2,leading to enhanced angiogenesis and tumor cell invasion.Expression of the miR-320-Ets2-related secretory regulons sepa-rated human normal breast stroma from tumor stroma and correlated with the outcomes of breast cancer patients.58Co-downregulation of miR-15a and miR-16-1in tumor cells and CAFsAnother report demonstrated that certain miRNAs exhibit a pan-tumor-suppressive function in both cancer cells and CAFs.59miR-15a and miR-16-1are well-characterized tumor-suppressive miRNAs encoded in the chromosomal region 13q14and are frequently lost in chronic lymphocytic leukemia and prostate cancer.60,61miR-15a and miR-16-1function as tumor suppressors in prostate cancer by targeting BCL2,cyclin D1and Wnt3a.61Musumeci et al .59reported that miR-15and miR-16were also downregulated in the CAFs of most of prostate tumor patients (Figure 3a).Restoration of miR-15a and miR-16-1suppressed proliferation and migration of CAFs as similarly observed in cancer cells.Furthermore,miR-15a and miR-16suppressed two novel targets,fibroblast growth factor 2(FGF2)and its receptor FGFR1,which enhance the proliferation and migration of both stromal and cancer cells.Reconstitution of miR-15and miR-16suppressed the tumor-promoting activity of stromal cells in vivo .59These findings suggest that some miRNAs can function as pan-tumor suppressors in cancer cells and tumor microenvironments,although the mechanisms of deregulation may be distinctbetween cancer cells and microenvironments.From the stand-point of therapeutic intervention,simple restoration of this type of miRNA might aid co-targeting cancer cells and tumor microenvironment.miRNA-mediated osteoclast differentiation in bone metastasis Osteolytic bone metastasis is frequently observed in many cancer types,including breast and lung cancers,and is associated with aberrant osteoclast activation.48,62,63In a recent study,treatment of preosteoclast cells with conditioned media from highly bone metastatic breast and bladder cancer cell lines was shown to induce characteristic changes in miRNA expression,similar to those noted upon stimulation with RANKL (receptor activator of nuclear factor κB ligand),a major stimulator of osteoclast differentiation (Figure 3b).64Bone-metastatic cancer cells trig-gered such miRNA expression changes partly through soluble intercellular adhesion molecule 1(sICAM1)-mediated NF-κB activation.Overexpression of multiple miRNAs that were down-regulated during osteoclastogenesis suppressed various osteo-clast genes,including Calcr (encoding the calcitonin receptor)and TRAF6,and inhibited osteoclast differentiation.Intravenous delivery of these miRNAs such as miR-141and miR-219reduced osteolytic bone metastasis.Interestingly,the serum levels of two miRNAs miR-16and miR-378,which increased during osteoclas-togenesis,were associated with bone metastasis,suggesting that the miRNAs might serve as useful biomarkers.64These findings emphasize the key roles played by miRNAs in aberrant osteoclastogenesis during bone metastasis.Proximal functions of miRNAs in MDSCsIn tumor-in filtrating immune cells,MDSCs have key roles in immune suppression,in turn allowing tumor progression.48,62,65–68MDSCs are myeloid-related cells characterized by the expression of Gr-1and CD11b markers and the ability to suppress T lymphocyte activation.MDSCs are thought to arise upon deviation of immature myeloid progenitor cells continually generated in bone marrow from the normal differentiation programs toward macrophages,dendritic cells and granulocytes.67,68MDSC num-bers increase in the bone marrow and blood of tumor-bearing mice and cancer patients and accumulate in tumor microenvironments.62,65MDSCs are heterogenous in nature and are mainly divided into Ly6G low /Ly6C high monocytic MDSCs and Ly6G high /Ly6C low granulocytic or polymorphonuclearmiR-214miR-31miR-155CCL5CCL20IL-8miR-320Ets2MMP9PTEN FGFR1FGF2miR-15miR-16CAFCCL5CCL20, IL-8MMP2, MMP9BMP1, LOXL2FGF2Calcr TRAF6miR-141miR-219sICAM1TGF-βmiR-494arginaseMMP2MMP13MMP14SHIP1, PTENmiR-21miR-155STAT3OsteoclastMDSCMicroenvironmentimmunosuppressionbone metastasisMDSC expansiontumor progressionCancer cellCancer cellFigure 3.Proximal functions of miRNAs in tumor stromal cells.Speci fic changes of multiple miRNAs in CAFs (a ),osteoclasts (b )and MDSCs (c )induce phenotypic changes of these cell types,leading to tumor progression.Oncogenic miRNAs and tumor-suppressive miRNAs are represented by black and red,respectively.microRNAs in tumor microenvironment HI Suzuki et al5©2014Macmillan Publishers LimitedOncogene (2014),1–10。

microRNA ：一种新型肿瘤分子标志物MicroRNA:A Novel Biomarker of CancerLIANG Dong -yu ，HOU Yan -qiang梁冬雨综述，侯彦强审校（上海交通大学附属第一人民医院松江分院中心实验室，上海201600）摘要：早期诊断、早期治疗是降低肿瘤死亡率的有效途径。

microRNA 是一类内源性的长约18~22个核苷酸的非编码小RNA ，其在肿瘤组织以及循环中的特异表达，为肿瘤的早期诊断带来新的希望。

肿瘤中microRNA 的表达模式不但与肿瘤诊断有关，与肿瘤的分期、进展以及预后也密切相关。

全文就microRNA 在肿瘤早期诊断以及预后判断中的作用作一综述。

主题词：肿瘤；microRNA ；分子标志物中图分类号：R730.4文献标识码：A 文章编号：1671-170X （2011）07-0485-04基金项目：上海市卫生局局级项目（2010104）通讯作者：侯彦强，副主任技师，博士；上海交通大学附属第一人民医院松江分院中心实验室，上海市松江区中山中路746号（201600）；E -mail:houyanqiang@ 。

收稿日期：2011-04-14肿瘤标志物是反映肿瘤存在和生长的化学物质，临床上通过对这些化学物质的检测，可以对相应肿瘤作早期诊断、疗效评估和预后判定。

目前，有多种肿瘤标志物应用于临床，但大多数标志物仍然缺乏足够的组织特异性和肿瘤特异性。

microRNA 是一组长约22个核苷酸的非编码小分子RNA ，其通过转录后水平调控参与细胞的增殖、分化、凋亡等生命活动。

近几年的研究发现mi -croRNA 表达具有明显的组织细胞特异性，且与肿瘤的发生和发展有着密切联系。

最近，Lawrie 等［1］在外周血中检测到microRNA 的存在，且发现循环mi -croRNA 的表达与肿瘤也有一定的相关性，这提示microRNA 可作为一种新型的肿瘤标志物。

⼀键完成microRNA定量PCR引物设计尽管microRNA芯⽚和microRNA测序检测⽅法已经普遍使⽤，但qRT-PCR依旧是检验microRNA表达定量的⾦标准。

由于microRNA的结构特殊，长度只有18-25个碱基，⽆法直接采⽤常规的PCR技术扩增，因此RT-qPCR的引物设计对很多刚刚接触microRNA的同学都是⼀个难题。

在针对microRNA的PCR技术中，设计其引物的理念是基于延长待测microRNA的长度，构建出⼀个⾜够长的PCR模板，才能进⼀步应⽤PCR技术来定量分析。

最常⽤的microRNA 反转录PCR⽅法就是茎环法（stem-loop）和加尾法（poly-A tail）。

由于茎环法反转录的引物设计原理限制，加尾法的检测通量⽐茎环法更⾼，因此加尾法也被实验室普遍使⽤。

今天⼩编就给⼤家介绍⼀个批量设计microRNA加尾法反转录PCR引物的软件miRprimer，重⼀键完成哦！点是⼀键完成miRprimer 官⽅推荐下载⽹站：https:///projects/miRprimer/⽹站后台⽂件直接下载miRprimer地址：https:///project/miRprimer/miRprimer2_installer.zipmiRprimer2_installer.zip，整个软件的压缩包只有2.68M (2848kb)⼤⼩。

提醒：提醒1. 软件⽀持在Windows XP或更⾼系统中运⾏，还没在苹果电脑Mac OS系统测试（原因是⼩编的钱包羞涩~~）2. 经⼩编测试，最新版本miRprimer顺利运⾏，不需在电脑中安装Ruby脚本环境。

第⼀步miRprimer2_installer.zip压缩包2.68M⼤⼩，下载后解压缩⽣成同名⽂件夹，内有三个⽂件：input_miRs.txt、miRprimer2.exe、README.txt。

特别提⽰：不要更改这些⽂件的⽂件名。

特别提⽰：第⼆步input_miRs.txt，fasta格式储存的是需要设计引物的microRNA名字和序列。

Leading EdgeReviewModifications of Small RNAsand Their Associated ProteinsYoung-Kook Kim,1Inha Heo,1and V.Narry Kim1,*1School of Biological Sciences and Center for National Creative Research,Seoul National University,Seoul151-742,Korea*Correspondence:narrykim@snu.ac.krDOI10.1016/j.cell.2010.11.018Small regulatory RNAs and their associated proteins are subject to diverse modifications that can impinge on their abundance and function.Some of the modifications are under the influence of cellular signaling,thus contributing to the dynamic regulation of RNA silencing.IntroductionThe past decade has witnessed an explosion of research on small regulatory RNAs that has yielded a basic understanding of the many types of small RNAs in diverse eukaryotic species, the protein factors involved,and the functions of key factors along the RNA silencing pathways.Much more remains to be learned,however,with recent studies unveiling interesting new layers of regulation and complexity associated with small RNAs.We now know that both small RNAs and their associated protein factors can be modified at multiple steps in their biogen-esis and effector pathways.Insight into modifications of small RNAs came initially from sequencing efforts,which made it clear that most microRNA (miRNA)loci generate multiple isoforms(called isomiRs)apart from the reference sequence(Morin et al.,2008).Alternative/ inaccurate processing partly explains the heterogeneity,but a substantial portion of the variation is due to RNA modifications. Small RNAs are modified either internally or externally by untem-plated nucleotide addition,exonucleolytic trimming,20-O-methyl transfer,and RNA editing.Protein factors in RNA silencing path-ways are also subject to various posttranslational modifications, including phosphorylation,hydroxylation,ubiquitination,and methylation.In this Review,we focus on the recent develop-ments in the modifications of RNAs and proteins in RNA silencing pathways.Small RNA BiogenesisRNA silencing is a widespread mechanism of gene regulation in eukaryotes.At the core of all RNA silencing pathways lie small RNAs(20–30nt in length)associated with the Argonaute family proteins(Kim et al.,2009).Small RNAs provide the specificity of regulation by base-pairing to the target nucleic acids while the Argonaute proteins execute the silencing effects.The Argo-naute(Ago)proteins are grouped into Ago and Piwi subfamilies, and in animals,three types of small RNAs have been described: microRNAs(miRNAs),small interfering RNAs(siRNAs),and Piwi-interacting RNAs(piRNAs).miRNAs( 22nt)induce mRNA degradation and/or transla-tional repression.Nucleotides2–7,from the50end of the miRNA, are referred to as the‘‘seed’’and are critical for hybridization to the targets(Bartel,2009).As a class,miRNAs are found in all tissues,although each miRNA species displays a unique spatio-temporal pattern of expression.An miRNA originates from a long primary transcript(pri-miRNA)containing a local hairpin struc-ture(Kim et al.,2009).In animals,the nuclear RNase III Drosha liberates the hairpin-shaped precursor miRNA(pre-miRNA) (Figure1).The cytoplasmic RNase III Dicer removes the terminal loop to produce a small RNA duplex,consisting of the functional miRNA strand and the passenger(*)strand(miRNA/miRNA*). The duplex then binds to the Argonaute loading complex (comprised of Dicer,TRBP,and Ago),whose action leads to the incorporation of the functional miRNA strand(mature miRNA) into Ago.The plant miRNA system differs from its animal coun-terparts in several aspects(Figure2).The plant homolog of Dicer, Dicer-like1(DCL1),cleaves both pri-miRNA and pre-miRNA in the nucleus.Plant miRNAs generally show extensive comple-mentary to their target mRNAs and induce endonucleolytic cleavage of the targets.Endogenous siRNAs(endo-siRNAs, 21nt)are similar to miRNAs in their binding to the Ago subfamily proteins,in their dependence on Dicer for biogenesis,and in exerting their regu-latory effects posttranscriptionally(Kim et al.,2009).But unlike miRNAs,endo-siRNAs originate from long double-stranded RNA precursors(dsRNAs),and their biogenesis does not require processing by Drosha.Endo-siRNAs are abundant in lower eukaryotes and in plants,whereas in mammals,they are found in restricted tissues such as the ovary.Piwi-interacting RNAs(piRNAs,21–30nt)associate with the Piwi subfamily of Argonaute proteins.piRNAs mediate the silencing of repetitive elements in gonads via transcriptional and posttranscriptional silencing mechanisms.Production of piRNAs is not dependent on RNase III nucleases,and the steps and factors involved in their biogenesis remain largely unknown. Modifications of Small RNAs30End Modifications:Uridylation,Adenylation,and20-O-MethylationThe30ends of mature miRNAs are highly heterogeneous, whereas the50ends are relatively invariable.The patterns and sources of heterogeneity seem to vary depending on the miRNA species and the cell types.The30end often contains extra1–3nucleotides that do not match the genomic DNA sequences.These untemplated nucleotides are added by Cell143,November24,2010ª2010Elsevier Inc.703terminal nucleotidyl transferases that preferentially introduce uridyl or adenyl residues to the 30terminus of RNA.The first indication of 30end modification of small RNA came from a hen1mutant of Arabidopsis (Li et al.,2005).HEN1is a methyl transferase that adds a methyl group to the 20-OH at the 30end of RNA (Yu et al.,2005).In hen1mutants,miRNAs are reduced in abundance and become heterogeneous in size due to uridylation at the 30end.Because U tailing correlates with the exonucleolytic degradation of mRNAs (Shen and Goodman,2004),it was postulated that uridylation induces degradation of plant miRNAs and that the 20-O-methyl moiety is required to protect small RNAs from uridylation and decay (see below).Consistent with this notion,in green algae Chlamy-domonas ,a nucleotidyl transferase,MUT68,uridylates the 30end of small RNA,and the RRP6exosome subunit facilitates small RNA decay in a manner dependent on MUT68in vitro (Ibra-him et al.,2010).Deletion of MUT68results in elevated miRNA and siRNA levels,indicating that MUT68and RRP6collaborate in the turnover of mature small RNAs in plants.Similar links between 20-O-methylation,uridylation,and decay appear to exist in animals.A recent study on the zebrafish Hen1homolog shows that piRNAs are uridylated and adenylated andthat piRNA levels are reduced in hen1mutant germ cells (Kam-minga et al.,2010).In flies and mice,piRNAs are methylated by HEN1orthologs,but the connection to stability control remains unclear (Horwich et al.,2007;Kirino and Mourelatos,2007;Ohara et al.,2007;Saito et al.,2007).In flies,dAgo2-bound RNAs (mostly siRNAs)are protected by 20-O-methylation from being uridylated/adenylated,which in turn induces 30exonucleolytic trimming (Ameres et al.,2010).In nematode worms,the role of 20-O-methylation has yet to be determined.However,a subset of endo-siRNAs associated with an Ago homolog CSR-1is uridylated at the 30end,and the uridyl trans-ferase CDE-1(also known as CID-1or PUP-1)negatively regu-lates these siRNAs,indicating that uridylation serves as a trigger for decay (van Wolfswinkel et al.,2009).Although mature miRNAs lack methylation in animals,uridyla-tion plays a significant role in the control of miRNA biogenesis.In mammalian embryonic stem cells,let-7biogenesis is sup-pressed by the Lin28protein that binds to the terminal loop of the let-7precursors (Heo et al.,2008;Newman et al.,2008;Rybak et al.,2008;Viswanathan et al.,2008).Of interest,Lin28induces 30uridylation of pre-let-7by recruiting the terminal nucleotidyl transferase TUT4(also known as ZCCHC11)(HaganFigure 1.Modifications in the AnimalMicroRNA Pathway(Left)MicroRNAs (miRNAs)are subject to diverse modifications.Pri-miRNAs are edited by ADARs,which convert adenosine to inosine (I).RNA editing inhibits processing and/or alters target specificity.Pre-let-7is regulated through uridylation.Lin28recognizes pre-let-7and,in turn,recruits a nucleo-tidyl transferase TUT4(mammal)or PUP-2(worms),which adds an oligo-uridine tail at the 30end of RNA.The uridylated pre-miRNA is resistant to Dicer processing and subject to decay.TUT4also uridylates mature miRNA (miR-26),which reduces miRNA activity.Another nucleotidyl trans-ferase GLD-2adenylates mature miRNAs,which reduces the activity of miRNA and/or increases the stability of specific miRNAs (such as miR-122).(Bottom)Mature miRNAs are degraded through several mechanisms.In worms,a 50/30exonu-clease XRN-2degrades miRNAs that are released from Ago.In flies and humans,extensive pairing between miRNA/siRNA and target RNA triggers tailing as well as 30/50trimming of miRNA/siRNA.(Right)Protein factors,which are involved in the miRNA pathway,are also subject to various post-translational modifications.Human Drosha is phosphorylated at two serine residues,S300/S302,by an unknown kinase.Phosphorylation localizes Drosha to the nucleus,where the pri-miRNA processing occurs.MAP kinases Erk1/2phosphorylate human TRBP at S142,S152,S283,and S286,which increases the protein stability of TRBP and Dicer.Ago2is regulated by multiple modifications.A prolyl hydroxylase C-P4H(I)hydroxylates P700in human Ago2,which enhances stability of Ago2and increases P body localization.Phosphorylation of human Ago2at S387by MAPKAPK2,which is induced by p38pathway,also promotes P body localization of Ago2.However,the biological significance of P body localization of Ago2remains unclear.In mice,a stem cell-specific E3ligase,mLin41,ubiq-uitinates Ago2and targets it for proteosome-dependent degradation.704Cell 143,November 24,2010ª2010Elsevier Inc.et al.,2009;Heo et al.,2009).The oligo U-tail added by TUT4blocks Dicer processing and facilitates the decay of pre-let-7.The homologs of TUT4may have related functions in other organisms.In nematode worms,PUP-2uridylates pre-let-7in vitro and suppresses the let-7function in vivo (Lehrbach et al.,2009).Let-7is unlikely to be the only miRNA uridylated at the pre-miRNA level.In support of this notion,untemplated 30uridine is frequently found in other mature miRNAs originating from the 30arm of pre-miRNAs (but significantly less frequently in those from the 50arm)(Burroughs et al.,2010;Chiang et al.,2010).Because untemplated uridylation is observed in cells lacking Lin28,it will be interesting to determine which pre-miRNAs other than pre-let-7are controlled by uridylation and to identify addi-tional factors required for pre-miRNA uridylation.Although uridylation is generally thought to induce the decay of small RNAs,adenylation may have the opposite conse-quence.In cottonwood P.trichoacarpa ,many miRNA families are adenylated at their 30ends,and adenylation prevents miRNA degradation in in vitro decay assay (Lu et al.,2009).In the case of mammalian miR-122,which is adenylated by cytoplasmic poly (A)polymerase GLD-2(or TUTase2),30end adenylation is also implicated in its stabilization (Katoh et al.,2009).In the liver of Gld-2knockout mice,the steady-state level of mature miR-122is reduced,and the abundance of target mRNAs of miR-122increases.However,a recent study indicates that GLD-2adenylates most miRNAs,and the adenylation may affect their activity rather than stability (Burroughs et al.,2010).Deep sequencing of Ago-associated small RNAs shows that adenylated miRNAs are relatively depleted in the Ago2and Ago3complexes,suggesting that adenylation may interfere with Ago loading.Similarly,it has been reported that uridylation of mature miR-26by TUT4results in the reduction of miR-26’s activity without altering the miRNA levels (Jones et al.,2009).Therefore,it remains an inter-esting but yet unresolved issue whether or not uridylation/adenylation affects the stability of miRNAs in animals.One may speculate that 30modified miRNAs enter the silencing complex with altered frequencies,which in turn affects the small RNA’s sensitivity to nucleases.Further examination is needed to iden-tify the players involved in these processes,particularly the nucleases that recognize a U/A tail,and to dissect their action mechanisms.miRNA DecaySeveral nucleases degrade small RNAs (Figures 1and 2).An Arabidopsis enzyme SDN1(small RNA degrading nuclease,a 30-to-50exonuclease)degrades single-stranded miRNAs in vitro (Ramachandran and Chen,2008).miRNAs accumulate in a mutant lacking SDN1and its related nucleases SDN2and SDN3,indicating that the SDN proteins may act redundantly to degrade plant miRNAs.The 20-O-methyl group at the 30end of miRNAs,which is a general feature of plant miRNAs,has a protective effect against SDN1in in vitro assays.Of note,uridy-lation causes a small but detectable protective effect in the same in vitro assay,indicating that SDN1is unlikely to be the nuclease responsible for U-tail-promoted degradation.Given that RRP6(a 30-to-50exonuclease)facilitates decay of small RNAs in a MUT68-dependent manner in Chlamydomonas extracts,multi-ple enzymes may be involved in small RNA decay in plants,playing partially overlapping but differential roles (Ibrahim et al.,2010).In C.elegans ,XRN-2(a 50-to-30exonuclease)is involved in the degradation of mature miRNAs (Chatterjee and Grosshans,2009).Because miRNAs are tightly bound to and protected by Ago,it is unclear how XRN-2accesses the 50end of an miRNA for decay.Of interest,larval lysate promotes efficient release of miRNA in vitro,implicating an as yet unknown factor that assists the release of miRNA from the otherwise tightly associated Argo-naute protein (Chatterjee and Grosshans,2009).In Arabidopsis ,two XRN-2homologs,XRN2and XRN3,degrade the loop of miRNA precursor following processing,but they do not affect mature miRNA levels (Gy et al.,2007).In mammals,a general nuclease for miRNAs has yet to be identified.Knockdown of XRN-1or an exosome subunit in human cells results in only partial upregulation of miR-382,and XRN-2depletion does not have a significant effect (Bail et al.,2010).Thus,it awaits further investigation whether or not there is one major conserved pathway for miRNA decay in mammals.There have been intriguing reports of regulated decay of miRNAs.For instance,miR-29b is degraded in dividing cells more rapidly than in mitotically arrested cells (Hwang et al.,2007).In the central nervous system of Aplysia ,the levels of miR-124and miR-184decrease in 1hr after treatment with the neurotransmitter serotonin (Rajasethupathy et al.,2009).Figure 2.RNA Modifications in the Plant miRNA PathwayIn plants,both pri-miRNA and pre-miRNA are cleaved by DCL1/HYL1complex.After cleavage,30ends of miRNA duplex are 20-O-methylated by a methyl transferase HEN1.The methylation protects miRNAs from uridylation and exonucleolytic degradation.In the green algae Chlamydomonas ,the nu-cleotidyl transferase MUT68attaches uridine residues at the 30end of mature miRNA lacking a methyl group.Then,the RRP6exosome subunit,a 30-to-50exonuclease,degrades the uridylated miRNAs.In Arabidopsis ,a 30/50exonuclease SDN1is reported to degrade mature miRNAs.Cell 143,November 24,2010ª2010Elsevier Inc.705Because U0126,an inhibitor of mitogen-activated protein kinase (MAPK),blocks the reduction of miR-124,the decay process may be dependent on the MAPK pathway.Of interest,a study on mammalian neuronal cells shows that most miRNAs turn over more rapidly in neurons than in other cell types (Krol et al.,2010).Neuronal activation accelerates decay of the miRNAs,whereas blocking neuronal activity stabilizes the miRNAs.It will be exciting to discover the nuclease(s)and the upstream signals for miRNA degradation in these systems.Recently it has been shown that a polynucleotide phosphory-lase (PNPase,a type I interferon-inducible 30-to-50exonuclease)binds specifically to several miRNAs (miR-221,miR-222,and miR-106b)and induces rapid turnover in a human melanoma cell line (Das et al.,2010).Because there is no apparent commonality in terms of the sequences,it is unclear how PNPase recognizes the miRNAs specifically.As mentioned above,there is substantial evidence linking uri-dylation/adenylation and exonucleolytic attack on small RNAs.A recent study provides evidence that extensive complemen-tarity between a small RNA and its target RNA triggers uridyl/adenyl tailing as well as 30/50trimming in flies and humans (Figure 1)(Ameres et al.,2010).Animal small RNAs with high complementarity to the targets,such as piRNAs and fly endo-siRNAs,appear to be generally protected by 20-O-methylation at the 30end like plant small RNAs.It has been postulated that animal miRNAs,which do not carry methylation,maintain only partial complementarity with their targets so as to avoid tailing and trimming of miRNAs.Of note,viruses seem to exploit a related miRNA decay pathway to invade host cells more effec-tively.Herpesvirus saimiri ,a family of primate-infecting herpesvi-ruses,expresses viral noncoding RNAs called HSURs (H.saimiri U-rich RNAs).A recent report reveals that HSURs rapidly down-regulate host miR-27and that base-pairing between HSUR and miR-27is required for the degradation (Cazalla et al.,2010).These discoveries imply an additional layer of stability controlof small RNAs,which is influenced by the interaction with the target RNA.miRNA EditingAdenosine deaminases acting on RNAs (ADARs)convert adenosine to inosine on the dsRNA region of small RNA precur-sors (Figure 1and Figure 3A).Because inosine (I)pairs with cytosine instead of uridine,such edits could alter the structure of small RNA precursor,thereby interfering with processing.For instance,editing of pri-miR-142by ADAR1and ADAR2suppresses Drosha processing (Yang et al.,2006),whereas that of pre-miR-151by ADAR1interferes with Dicer processing (Kawahara et al.,2007a ).Because hyperedited dsRNAs can be targeted by the nuclease Tudor-SN,RNA editing may also desta-bilize small RNA precursors (Scadden,2005).In rare cases,RNA editing occurs in the seed sequence of miRNA,changing the targeting specificity.In the brain,where ADAR is abundant,miR-376cluster miRNAs are frequently edited in the seed region and are redirected to repress a different set of mRNAs (Kawahara et al.,2007b ).High-throughput sequencing of the fly endo-siRNA pool also reveals evidence for RNA editing (Kawamura et al.,2008).The precursors of endo-siRNAs (long hairpins and sense-antisense pairs)may be targeted by ADARs,although the functional significance of this siRNA modification is unknown.Posttranslational Protein Modifications Phosphorylation of RNase III EnzymesHuman Dicer interacts with two related dsRNA-binding proteins,TRBP and PACT.Although they do not influence Dicer process-ing itself,TRBP and PACT stabilize Dicer and may also function in RISC assembly (Chendrimada et al.,2005;Haase et al.,2005;Lee et al.,2006).A recent study indicates that four serine residues of human TRBP (S142,S152,S283,and S286)are phosphorylated by the MAP kinase Erk,which controls cell proliferation,survival,and differentiation (Figure 1)(Paroo etal.,Figure 3.Modifications in the Endo-siRNA and piRNA Pathways(A)Endogenous small interfering RNAs (endo-siRNAs)are processed from long dsRNAs in a Dicer-dependent manner and are loaded onto Ago proteins.High-throughput sequencing data show that the adenosine-to-inosine (I)editing occurs in fly endo-siRNAs,likely by ADAR,although the role of RNA editing is unknown.Fly endo-siRNAs bound to dAgo2are 20-O-methyl-ated by HEN1homolog,which protects RNAs from uridyl/adenyl tailing and degradation.In worms,a subset of endo-siRNAs,which are asso-ciated with an Ago homolog CSR-1,is uridylated at the 30end by the nucleotidyl transferase CDE-1.(B)piRNAs are generated from single-stranded RNA precursors that are processed by primary processing and/or secondary processing (ping-pong amplification cycle).piRNAs are associated with Piwi subfamily proteins (PIWI).Animal piRNAs are 20-O-methylated by HEN1orthologs.In zebra-fish,depletion of hen1induces uridylation of piRNAs and facilitates decay,suggesting that methylation stabilizes piRNAs.However,the phys-iological significance of piRNA methylation in fliesand mammals remains unclear.PIWI proteins are methylated at arginine residues (sDMA,symmetrical dimethyl arginine)at their N termini by orthologs of the methyl transferase PRMT5.In flies and mice,TDRD proteins interact with PIWI proteins through sDMA and may play important roles in piRNA metabolism.706Cell 143,November 24,2010ª2010Elsevier Inc.2009).Phosphorylation enhances protein stability of TRBP, consequently elevating Dicer protein levels.Intriguingly,TRBP phosphorylation preferentially increases growth-promoting miRNAs such as miR-17,whereas tumor-suppressive let-7is reduced.The mechanism of selective downregulation of let-7 is unclear,but it may be an indirect effect.An interesting implica-tion of thesefindings is that the MAPK/Erk pathway exerts its effects,in part,by regulating miRNA biogenesis.Drosha,a nuclear enzyme for pri-miRNA processing(Lee et al.,2003),has recently been shown to be a direct target of posttranslational modification(Tang et al.,2010).Mass spec-trometry and mutagenesis studies reveal that human Drosha is phosphorylated at serine300(S300)and serine302(S302) (Figure1).Phosphorylation of these residues is essential for the nuclear localization of Drosha and is required for pri-miRNA processing.Because both endogenous and overexpressed Drosha localize to the nucleus constitutively,it is unclear whether or not the phosphorylation at S300/S302is a regulated process. Understanding the physiological significance of this regulation will require the identification of the kinase that phosphorylates Drosha.Argonaute2Is a Target of Multiple ModificationsAgo2is subject to multiple posttranslational modifications (Figure1).Human Ago2binds to the type I collagen prolyl-4-hydroxylase(C-P4H(I))that hydroxylates Ago2at proline700 (Qi et al.,2008).Depletion of C-P4H(I)reduces the stability of the Ago2protein and,accordingly,downregulates siRNA-medi-ated silencing.Furthermore,hydroxylation is required for Ago2 localization to the processing body(P body),a cytoplasmic granule that is thought to be a site for RNA storage and degrada-tion.P body localization of Ago2is also enhanced by phosphor-ylation at serine387,which is mediated by the p38MAPK pathway(Zeng et al.,2008).However,given the controversy over the direct role of P body in small RNA-mediated silencing, the biological significance of P body localization of Ago2remains unclear.Ubiquitination also plays a part in the control of Ago2.Mouse Lin41(mLin41or Trim71),a stem cell-specific Trim-NHL protein, inhibits the miRNA pathway(Rybak et al.,2009).As an E3ubiq-uitin ligase,mLin41ubiquitinates Ago2and targets it for protea-some-dependent degradation.Of interest,mLin41is a target of let-7miRNA,suggesting that mLin41and let-7may be engaged in a reciprocal negative feedback loop.Recently,other Trim-NHL proteins have been reported to associate with the Argonaute proteins and affect miRNA pathway.Mei-P26(fly)inhibits miRNA biogenesis,whereas TRIM32(mouse)and NHL-2(worm)acti-vate the miRNA pathway(Hammell et al.,2009;Neumu¨ller et al.,2008;Schwamborn et al.,2009).Their mechanism of action appears to be different than that of mLin41because the E3ligase activity of Mei-P26and TRIM32is dispensable for their effects and because NHL-2enhances miRNA activity without a change in miRNA levels.Tudor Regulates PIWI ProteinsThe PIWI(P element-induced wimpy testis)clade proteins bind to Piwi-interacting RNAs(piRNAs)and silence transposable elements in gonads.Mouse has three PIWI homologs(MILI, MIWI,and MIWI2),and there are three PIWI proteins inflies (Aubergine[Aub],AGO3,and Piwi)(Kim et al.,2009).Recent studies have revealed that PIWI proteins carry symmetrical dimethyl arginine(sDMA)at their N termini.Arginine methylation of PIWI is mediated by a methyl transferase PRMT5(dPRMT5/ capsuleen[csul]/dart5in Drosophila)(Figure3B)(Heo and Kim, 2009;Siomi et al.,2010).sDMA is recognized by Tudor domain-containing proteins(TDRDs),which are critical for germ-line development.In bothflies and mice,deletion of TDRDs alters piRNA abundance and/or composition,indicating that TDRDs play important roles in the piRNA metabolism through specific binding to the sDMAs of PIWI proteins.How TDRDs act in the piRNA pathway at a molecular level awaits further investigation. PerspectivesAs we delve deeper and wider into the small RNA world,the emerging landscape becomes ever more complex on both the RNA and protein sides.High-throughput analyses have uncov-ered a considerable heterogeneity in small RNA populations. Some isomiRs are expressed differentially in certain tissues, suggesting that these variations may be associated with specific regulatory functions(Chiang et al.,2010).Biochemical and genetic studies also provide substantial evidence for the regula-tory roles of the modifications discussed in this Review.Thus,it is likely that at least some of the observed heterogeneity reflects multiple layers of regulation.We should be cautious,however,in extrapolating the current evidence because it is unclear how much fraction of the small RNA and protein modifications trans-late into functional consequences and whether certain modifica-tions simply reflect the noise of RNA metabolism.In addition to the functionality issue,a number of key questions remain to be answered.Are there conserved pathways and enzymes for RNA and protein modifications?If so,what are the similarities and differences?20-O-methylation is applied to many small RNA pathways,but the details differ significantly in different systems.For instance,plant HEN1acts on dsRNA duplexes,whereas animal HEN1homologs methylate ssRNA loaded on Argonaute proteins.Uridylation/adenylation is carried out by a family of ribonucleotidyl transferases.How each member selectively recognizes its substrates is largely unknown. RNA stability is likely to play important roles in RNA silencing pathways.Decay pathways of small RNA are beginning to be unraveled,but there is no consensus between different species as yet.One possibility is that multiple enzymes act in parallel as in the mRNA decay pathway,which involves several30exonucle-ases,50exonucleases,and endonucleases.Some of the decay enzymes may function redundantly,and it remains one of the major challenges in thefield to identify them.Protein modifica-tion is also emerging as one of the key regulatory layers. Outstanding questions include which enzymes are involved, what the in vivo significance of such modifications is,and whether the protein modifications are developmentally regu-lated.Future studies will reveal new types of modifications,addi-tional regulatory factors,and their biological relevance.The RNA silencing machinery should respond accurately to developmental and environmental cues.Most signaling path-ways are thought to be connected to RNA silencing,but we are just beginning to understand the molecular links between RNA silencing and cell signaling.What the upstream signals are,how certain RNAs and proteins get specifically recognized, Cell143,November24,2010ª2010Elsevier Inc.707and what the downstream effects of the modifications are await elucidation.We also need to understand the interplay between different modifications.There appears to be a crosstalk between certain modifications of RNA(such as methylation,uridylation, and decay),which may influence their fate and function.It is likely that there is a crosstalk between the different posttranslational modifications in the proteins involved in the biogenesis and effector functions of small RNA silencing pathways.Under-standing these networks will undoubtedly provide ample oppor-tunities to manipulate RNA silencing and will reveal new lessons about gene regulation.ACKNOWLEDGMENTSWe thank members of V.N.K.’s laboratory for helpful discussions and comments.This work was supported by the Creative Research Initiatives Program(20100000021)and the National Honor Scientist Program (20100020415)through the National Research Foundation of Korea(NRF) and the BK21Research Fellowships(I.H.)from the Ministry of Education, Science and Technology of Korea.We apologize to authors whose work has not been covered because of space limitations.REFERENCESAmeres,S.L.,Horwich,M.D.,Hung,J.H.,Xu,J.,Ghildiyal,M.,Weng,Z.,and Zamore,P.D.(2010).Target RNA-directed trimming and tailing of small silencing RNAs.Science328,1534–1539.Bail,S.,Swerdel,M.,Liu,H.,Jiao,X.,Goff,L.A.,Hart,R.P.,and Kiledjian,M. (2010).Differential regulation of microRNA stability.RNA16,1032–1039.Bartel,D.P.(2009).MicroRNAs:target recognition and regulatory functions. Cell136,215–233.Burroughs,A.M.,Ando,Y.,de Hoon,M.J.,Tomaru,Y.,Nishibu,T.,Ukekawa, R.,Funakoshi,T.,Kurokawa,T.,Suzuki,H.,Hayashizaki,Y.,and Daub,C.O. (2010).A comprehensive survey of30animal miRNA modification events and a possible role for30adenylation in modulating miRNA targeting effectiveness. Genome Res.20,1398–1410.Cazalla,D.,Yario,T.,Steitz,J.A.,and Steitz,J.(2010).Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA.Science328, 1563–1566.Chatterjee,S.,and Grosshans,H.(2009).Active turnover modulates mature microRNA activity in Caenorhabditis elegans.Nature461,546–549.Chendrimada,T.P.,Gregory,R.I.,Kumaraswamy,E.,Norman,J.,Cooch,N., Nishikura,K.,and Shiekhattar,R.(2005).TRBP recruits the Dicer complex to Ago2for microRNA processing and gene silencing.Nature436,740–744.Chiang,H.R.,Schoenfeld,L.W.,Ruby,J.G.,Auyeung,V.C.,Spies,N.,Baek, D.,Johnston,W.K.,Russ,C.,Luo,S.,Babiarz,J.E.,et al.(2010).Mammalian microRNAs:experimental evaluation of novel and previously annotated genes. Genes Dev.24,992–1009.Das,S.K.,Sokhi,U.K.,Bhutia,S.K.,Azab,B.,Su,Z.Z.,Sarkar,D.,and Fisher, P.B.(2010).Human polynucleotide phosphorylase selectively and preferen-tially degrades microRNA-221in human melanoma cells.Proc.Natl.Acad. A107,11948–11953.Gy,I.,Gasciolli,V.,Lauressergues,D.,Morel,J.B.,Gombert,J.,Proux,F., Proux, C.,Vaucheret,H.,and Mallory, A.C.(2007).Arabidopsis FIERY1, XRN2,and XRN3are endogenous RNA silencing suppressors.Plant Cell19, 3451–3461.Haase,A.D.,Jaskiewicz,L.,Zhang,H.,Laine´,S.,Sack,R.,Gatignol,A.,and Filipowicz,W.(2005).TRBP,a regulator of cellular PKR and HIV-1virus expression,interacts with Dicer and functions in RNA silencing.EMBO Rep. 6,961–967.Hagan,J.P.,Piskounova,E.,and Gregory,R.I.(2009).Lin28recruits the TUTase Zcchc11to inhibit let-7maturation in mouse embryonic stem cells. Nat.Struct.Mol.Biol.16,1021–1025.Hammell,C.M.,Lubin,I.,Boag,P.R.,Blackwell,T.K.,and Ambros,V.(2009). nhl-2Modulates microRNA activity in Caenorhabditis elegans.Cell136, 926–938.Heo,I.,and Kim,V.N.(2009).Regulating the regulators:posttranslational modifications of RNA silencing factors.Cell139,28–31.Heo,I.,Joo,C.,Cho,J.,Ha,M.,Han,J.,and Kim,V.N.(2008).Lin28mediates the terminal uridylation of let-7precursor MicroRNA.Mol.Cell32,276–284.Heo,I.,Joo,C.,Kim,Y.K.,Ha,M.,Yoon,M.J.,Cho,J.,Yeom,K.H.,Han,J., and Kim,V.N.(2009).TUT4in concert with Lin28suppresses microRNA biogenesis through pre-microRNA uridylation.Cell138,696–708.Horwich,M.D.,Li,C.,Matranga,C.,Vagin,V.,Farley,G.,Wang,P.,and Zamore,P.D.(2007).The Drosophila RNA methyltransferase,DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC.Curr.Biol. 17,1265–1272.Hwang,H.W.,Wentzel,E.A.,and Mendell,J.T.(2007).A hexanucleotide element directs microRNA nuclear import.Science315,97–100.Ibrahim,F.,Rymarquis,L.A.,Kim,E.J.,Becker,J.,Balassa,E.,Green,P.J., and Cerutti,H.(2010).Uridylation of mature miRNAs and siRNAs by the MUT68nucleotidyltransferase promotes their degradation in A107,3906–3911.Jones,M.R.,Quinton,L.J.,Blahna,M.T.,Neilson,J.R.,Fu,S.,Ivanov,A.R., Wolf,D.A.,and Mizgerd,J.P.(2009).Zcchc11-dependent uridylation of micro-RNA directs cytokine expression.Nat.Cell Biol.11,1157–1163.Kamminga,L.M.,Luteijn,M.J.,den Broeder,M.J.,Redl,S.,Kaaij,L.J., Roovers,E.F.,Ladurner,P.,Berezikov,E.,and Ketting,R.F.(2010).Hen1is required for oocyte development and piRNA stability in zebrafish.EMBO J. 29,3688–3700.Katoh,T.,Sakaguchi,Y.,Miyauchi,K.,Suzuki,T.,Kashiwabara,S.,Baba,T., and Suzuki,T.(2009).Selective stabilization of mammalian microRNAs by30 adenylation mediated by the cytoplasmic poly(A)polymerase GLD-2.Genes Dev.23,433–438.Kawahara,Y.,Zinshteyn,B.,Chendrimada,T.P.,Shiekhattar,R.,and Nishi-kura,K.(2007a).RNA editing of the microRNA-151precursor blocks cleavage by the Dicer-TRBP complex.EMBO Rep.8,763–769.Kawahara,Y.,Zinshteyn,B.,Sethupathy,P.,Iizasa,H.,Hatzigeorgiou,A.G., and Nishikura,K.(2007b).Redirection of silencing targets by adenosine-to-inosine editing of miRNAs.Science315,1137–1140.Kawamura,Y.,Saito,K.,Kin,T.,Ono,Y.,Asai,K.,Sunohara,T.,Okada,T.N., Siomi,M.C.,and Siomi,H.(2008).Drosophila endogenous small RNAs bind to Argonaute2in somatic cells.Nature453,793–797.Kim,V.N.,Han,J.,and Siomi,M.C.(2009).Biogenesis of small RNAs in animals.Nat.Rev.Mol.Cell Biol.10,126–139.Kirino,Y.,and Mourelatos,Z.(2007).The mouse homolog of HEN1is a poten-tial methylase for Piwi-interacting RNAs.RNA13,1397–1401.Krol,J.,Busskamp,V.,Markiewicz,I.,Stadler,M.B.,Ribi,S.,Richter,J., Duebel,J.,Bicker,S.,Fehling,H.J.,Schu¨beler,D.,et al.(2010).Characterizing light-regulated retinal microRNAs reveals rapid turnover as a common property of neuronal microRNAs.Cell141,618–631.Lee,Y.,Ahn,C.,Han,J.,Choi,H.,Kim,J.,Yim,J.,Lee,J.,Provost,P., Ra˚dmark,O.,Kim,S.,and Kim,V.N.(2003).The nuclear RNase III Drosha initi-ates microRNA processing.Nature425,415–419.Lee,Y.,Hur,I.,Park,S.Y.,Kim,Y.K.,Suh,M.R.,and Kim,V.N.(2006).The role of PACT in the RNA silencing pathway.EMBO J.25,522–532.Lehrbach,N.J.,Armisen,J.,Lightfoot,H.L.,Murfitt,K.J.,Bugaut,A.,Balasu-bramanian,S.,and Miska,E.A.(2009).LIN-28and the poly(U)polymerase PUP-2regulate let-7microRNA processing in Caenorhabditis elegans.Nat. Struct.Mol.Biol.16,1016–1020.708Cell143,November24,2010ª2010Elsevier Inc.。

Cell：哺乳动物microRNA表达图谱-生物研究-生物谷生物谷报道：在6月29日著名国际杂志Cell上发表的一篇文章中，发布了一张microRNA表达图谱，该结果显示，哺乳动物为数众多的microRNA只是起源于少数几个microRNA基因，而且组织或细胞特异的microRNA很少。

该图谱对来自人类和啮齿类的26种正常、肿瘤组织的细胞的microRNA表达进行了定量测定。

“他们掌握了一种极为有用的资源，”Dartmouth医学院Victor Ambros说（未参与实验），他们在研究中收集的组织种类非常多，并对这些组织中的microRNA的特征进行了系统性分析。

新绘制的microRNA表达图谱含有许多关于microRNA结构、序列、表达方式和进化的有用信息。

Ambros认为这是一个基本数据库，为研究发育和疾病有关的microRNA世界提供了重要线索。

microRNA是动物、植物和病毒中普遍存在的非编码小RNA，通过干扰mRNA的功能调节基因表达。

科研人员已经鉴定出多种哺乳动物microRNA，但对这些microRNA中大多数的表达水平和特征都是未知的。

洛克菲勒大学Pablo Landgraf率领的研究小组克隆并测序了256个哺乳动物小RNA数据库中的30多万个序列，找到了大约400个在任何组织都表达的microRNA——比之前预期的要少。

约翰霍普金斯大学Joshua Mendell说，这个结果提示在其它研究中找到的几个百microRNA是未知的。

Landgraf等还发现他们测序的microRNA克隆中，97％的来自不到300个microRNA。

文章共同作者、瑞士Basel大学Mihaela Zavolan认为许多microRNA在各种组织间是低背景水平表达的（low background level），现在不能确定这些“痕量”microRNA的功能，但很有可能大多数microRNA的功能是不相关的。

m ic ro RNA的生物形成参与细胞生长/分化和肿瘤形成徐广峰,付海龙△(综述),赵亚萍※(审校)(解放军第82医院检验科,江苏淮安223001)中图分类号:R318 文献标识码:A 文章编号:1006-2084(2012)03-0368-03摘要:micr oRNA(miRNA)作为一类非编码小分子调控RNAs,参与调节多种生理和病理过程。

在动物细胞中,miRNA基因的转录初产物pri-miRNA被RNaseⅢ家族酶成员Drosa和DGCR8催化加工成为m iRNA前体(pr e-miRNA),然后由细胞核转运至细胞质中,经另一种核糖核酸酶ⅢDicer识别剪切为成熟m iRNA。

在pri-miRNA向成熟miRNA加工转化过程中发生的改变参与细胞生长、分化以及肿瘤形成。

关键词:microRNA生物形成;细胞生长;细胞分化;肿瘤形成;调节MicroRNA Bio genes is Participating in Cell G ro wth,Different iat ion and Tumorigenesis XU Guang-feng1,FU Hai-long2,ZH AO Ya-ping1.(Department Laboratory,the82nd Hos pital of the People′s L iberation Ar my,H uaian223001,China)Abst rac t:Micr oRNA(miRNA),as a kind of noncoding,small,regulatory RNAs,par ticipates in r egulatinga variety of physiolog ical and pathological process.In anim al cells,prim ary tra nscript of miRNA gene(pr i-miR-NA)is processed by RNaseⅢfamily members Drosa and DGCR8to produce the miRNA pr ecursor(pre-miR-NA).S ubsequently,the pre-miRNA is tr ansported from the nucleus to the cytoplasm,w her e it is shar ed by a second RNaseⅢ-like enzyme,ter med Dicer,into mature miRNA.The changes that take place in miRNA pro-cessing ar e involved in cell g rowth,differ entiation and tumor igenesis.Key words:MicroRNA biog enesis;Cell g rowth;Cell differentia tion;Tumorigenesis;Regulationmicr oRN A(miRN A)是真核生物中一类参与基因转录后水平调控的非编码小分子RNAs。