ORIGINAL ARTICLE
Oncolytic adenovirus co-expressing miRNA-34a and IL-24induces superior antitumor activity in experimental tumor model
Wenjia Lou &Qing Chen &Leina Ma &Jia Liu &Zhi Yang &Junjie Shen &Youhong Cui &Xiu-wu Bian &Cheng Qian
Received:1June 2012/Revised:30October 2012/Accepted:26November 2012/Published online:6January 2013#Springer-Verlag Berlin Heidelberg 2013
Abstract It has been demonstrated that numerous micro-RNAs (miRNAs)have potent tumor-suppressing effects on a variety of cancers,implicating a possible application of miRNA in tumor therapy.Oncolytic adenovirus is a suitable vector to deliver tumor suppressor genes for treatment of cancers.How-ever,it remains unknown whether co-expression of tumor suppressor genes and miRNAs can contribute to a more potent antitumor capacity within an oncolytic adenovirus delivery system.In this study,we found that expression of miRNA-34a was reduced in hepatocellular carcinoma (HCC),and the reduced expression of miRNA-34a was associated with worse outcome of HCC patients.Thus,we developed an oncolytic adenoviral vector,AdCN205,to co-express miRNA-34a and IL-24driven by an adenovirus endogenous E3promoter in HCC cells.High levels of miRNA-34a and IL-24expression were detected in AdCN205-IL-24-miR-34a-infected HCC cells.AdCN205-IL-24-miR-34a significantly induced dramatic antitumor activity,as compared with that induced by AdCN205-IL-24or AdCN205-miR-34a alone.Transfer of miRNA-34a into HCC cells inhibited the expression of its target genes,Bcl-2and SIRT1.Treatment of established xeno-graft HCC tumors with AdCN205-IL-24-miR-34a in a mouse model resulted in complete tumor regression without
recurrence.Taken together,our data provide a promising and reasonable delivery strategy of double-aimed cancer therapy,in which miRNAs and tumor-suppressing genes are used simultaneously.
Keywords Biological therapy of cancer .Co-expression of microRNA and therapeutic gene .Oncolytic adenovirus .Liver cancer
Introduction
MicroRNAs (miRNAs)are a class of endogenous non-encoding RNAs that function as important regulatory mol-ecules by inhibiting the expression of target genes.Aberrant expression of miRNAs has been well documented to show relevance in the pathogenesis of human cancers [1,2].miRNA profiling data show that tumors have a different miRNA expression pattern from corresponding normal tis-sues.Hepatocellular carcinoma (HCC)is one of the most lethal cancers worldwide.Abnormal expression of various miRNAs in HCC has been reported [3–11].These miRNAs participate in tumor initiation,progression,metastasis,tumor-related angiogenesis,cancer stem cell maintenance,and chemoresistance [3–11].Previous studies have shown that transcription of miRNA-34a is regulated by p53[12,13].miRNA-34a inhibits the expression of SIRT1,which physically interacts with p53and attenuates p53-mediated functions through deacetylation of p53[12–14].miRNA-34a induces cell apoptosis,G 1arrest,and senescence by inhibiting expression of several oncogenes,cell-cycle-regulated genes,and antiapoptotic genes [15].In addition,miRNA-34a suppresses tumor metastasis by inhibiting ex-pression of c-MET in HCC and melanoma [16].Both SIRT1and c-MET were considered as oncogenes that promote tumor progression [16,17].Recently,it has been found that
Electronic supplementary material The online version of this article (doi:10.1007/s00109-012-0985-x)contains supplementary material,which is available to authorized users.
W.Lou :Q.Chen :L.Ma :J.Liu :Z.Yang :J.Shen :Y .Cui :X.-w.Bian :C.Qian (*)
Lab.of Biotherapy of Cancer,Institute of Pathology and Southwest Cancer Center,Southwest Hospital,Third Military Medical University,Chongqing,China e-mail:cqian3184@https://www.doczj.com/doc/e612818303.html,
W.Lou
Xinyuan Institute of Medicine and Biotechnology,School of Life Sciences,Zhejiang Sci-Tech University,Zhejiang,Hangzhou,China
J Mol Med (2013)91:715–725DOI 10.1007/s00109-012-0985-x
miRNA-34a could inhibit self-renewal of prostate cancer stem cells by repressing the CD44molecule[18].These studies indicate that miRNA-34a may be applicable as a potent tumor-suppressing gene in cancer therapy.
From a clinical point of view,for miRNA-based therapeutics to be effective,the efficient and functional delivery of miRNA mimics and/or antagonists into solid tumors remains a great challenge[19,20].Viral vector-directed methods have been shown to have high gene transfer efficiency into cancers[21]. Our previous study has shown that adenoviral vectors could efficiently deliver miRNA-122into various types of cancer cells,which resulted in strong antitumor efficacy[22,23].Kota et al.demonstrated that adenovirus-associated virus-mediated transfer of miRNA resulted in inhibition of cell proliferation, induction of cell apoptosis,and inhibition of disease progres-sion without toxicity in a mouse model of HCC[24].These studies provide evidence of a fundamental basis for the appli-cation of viral vectors in miRNA delivery to solid tumors.
Oncolytic adenoviruses have been considered as highly eligible vehicles for delivery of therapeutic genes to treat can-cers because of their tumor-restricted replication capabilities [25,26].High antitumor activity could be achieved by selec-tively eliminating tumors via viral replication and highly expressing therapeutic genes within cancer cells.Currently, protein-encoding cDNA sequences are still the most widely used therapeutic agents in oncolytic adenoviral vectors.How-ever,it is still not known whether oncolytic adenovirus is suitable for simultaneously expressing tumor-suppressing genes and miRNAs within infected cancer cells.IL-24is a cytokine belonging to the IL-10family.It exerts antitumor activity by inducing tumor cell apoptosis and inhibiting tumor angiogene-sis[27].Our previous studies have indicated that oncolytic adenovirus-mediated transfer of IL-24could exert a dramatic antitumor effect[25,28].However,many tumor cells were resistant to IL-24due to their overexpression of antiapoptotic genes including Bcl-2[29].In this study,we explored whether IL-24and miRNA-34a could be efficiently co-expressed by oncolytic adenovirus and whether the combination of IL-24 with miRNA-34a could exhibit an enhanced additive or syner-gistic antitumor activity.
Materials and methods
Cell culture
Human HCC cell lines(Huh7,PLC/PRF/5,and BEL7404) and human normal liver cell line(L-02)were purchased from Shanghai Cell Collection(Shanghai,China). HEK293(human embryonic kidney cells)was obtained from Microbix Biosystems,Inc.(Toronto,Ontario,Canada). Cells were banked after receipt and were cultured for less than6months before use in experiments.Cells were originally validated by analysis of their short tandem repeat profile.All cell lines were cultured in Dulbecco's modified Eagle's medium(GIBCO-BRL,Grand Island,NY)supple-mented with10%heat-inactivated fetal bovine serum (GIBCO-BRL)at37°C under a5%CO2condition.
Generation of plasmids and recombinant adenoviruses
The pri-miRNA-34a gene was obtained by PCR amplification using primers(forward:5′-ATACCGCTCGAGCCTCCTG CATCCTTTCTTT-3′;reverse:5′-GGCAATTGATACCG CTCGAGCCTGTGCCTTTTTCCTTCC-3′)with human ge-nomic DNA as template.The full length of IL-24cording sequence was obtained by PCR amplification using primers (forward:5′-CGTACGCCGCCACCATGAATTTTCAA CAGA-3′;reverse:5′-GCC ATCGAT TCAGAGCTT GTAGA-3′)with an in-house plasmid AdCN205-IL-24as template.The PCR products were cloned into pMD18-T vector(TaKaRa).The pri-miRNA-34a sequence was released from pMD-18T-vector by digestion with Kpn I and Mun I and was then cloned into plasmid pCN203[25]to generate pCN203-miR-34a.The full length of IL-24sequence was released from pMD-18T-vector after digestion with BsiW I and Cla I and was cloned into pCN203-miR-34a to obtain pCN203-IL-24-miR-34a.The2,690-bp adenoviral sequence was obtained from pTGC3602[25]after digestion with Mlu I and Spe I and was cloned into pCN203-miR-34a and pCN203-IL-24-miR-34a to obtain pCN204-miR-34a and pCN204-IL-24-miR-34a,respectively.Then homologous recombination between pCN204-miR-34a(or pCN204-IL-24-miR-34a)and pCN103plasmids carrying oncolytic adenoviral backbone was done in E.coli strain BJ5183to create pCN205-miR-34a(or pCN205-IL-24-miR-34a).
To generate recombinant adenoviral vectors,pCN205-miR-34a and pCN205-IL-24-miR-34a were digested with Pac I and then transfected into HEK293cells with Effectene Transfection Reagent(Qiagen).The adenoviruses were harvested and puri-fied with the CsCl gradient centrifugation method as previously described[30].The titration of recombinant adenoviruses was determined with TCID50assay[30]on HEK293cells. Human HCC sample
Fresh tumor specimens were obtained with written informed consent from all patients according to protocols approved by the Institutional Review Board of the Southwest Hospital,Third Military Medical University,and they were performed in ac-cordance with the1964Declaration of Helsinki.All patients underwent surgical resection of primary HCC at the Institute of Hepatobiliary Surgery,Southwest Hospital,Third Military Medical University.We collected two cohorts of clinical sam-ples from surgical resection in this study.Cohort1contains14 paired fresh frozen HCC samples and their corresponding non-
cancerous liver tissue.Cohort2includes52formalin-fixed, paraffin-embedded HCC samples.Pathological analysis was performed by two independent pathologists according to the Guideline from WHO Classification of Tumors.
Quantitative RT-PCR on human HCC samples
Total RNA was extracted from fresh frozen tissues and paraffin-embedded tissue sections with Trizol solution (Sigma-Aldrich,MO)and miRNeasy FFPE kit(Qiagen), respectively.For miRNA-34detection,reverse transcription reaction was performed with TaqMan?MicroRNA Reverse Transcription Kit(Applied Biosystems)following the man-ufacturer's instructions.Quantitative RT-PCR(qRT-PCR) was performed using TaqMan?Universal PCR Master Mix(Applied Biosystems)on CFX96?Real-Time PCR Detection System(Bio-Rad Laboratories,CA)supplied with analytical software.U6RNA level was used for internal control of each sample for normalization.The fold change for gene expression was calculated using2?△△Ct.Expression level of miRNA-34a was defined as high or low groups, depending on their values,as compared with the mean value of total samples.
For Bcl-2and SIRT1detection,cDNA was obtained using Primescript?RT Reagent kit(Takara,Japan)accord-ing to the manufacturer's instructions.qRT-PCR was per-formed with SYBR Premix Ex Taq II(Takara,Japan)on CFX96?Real-Time PCR Detection System(Bio-Rad Laboratories,CA)supplied with analytical software. The following primers were used:Bcl-2(F)5′-G C C T T C T T T G A G T T C G G T G G-3′a n d(R)5′-AT C T C C C G G T T G A C G C T C T3′,S i r T1(F)5′-TAGCCTTGTCAGATAAGGAAGGA-3′and(R)5′-TGTTCTGGGTATAGTTGCGAAGT-3′,and GAPDH(F) 5′-TCAGTGGTGGACCTGACCTG-3′and(R)5′-TGCTGTAGCCAAATTCGTTG-3′.GAPDH mRNA levels were used for normalization.The fold change for gene expression was calculated using2?△△Ct.
Western blotting
The HCC cells were infected with oncolytic adenoviruses at a multiplicity of infection(MOI)of five.After48h,cells were harvested,and total proteins were separated on10–12%polyacrylamide gels and then transferred onto0.45-μm nitrocellulose and blocked with5%BSA.The mem-branes were incubated with primary antibodies anti-IL-24 and anti-SIRT1(R&D),anti-Bcl-2,and anti-beta-actin (Santa Cruz,CA,USA)followed by the addition of anti-rabbit infrared(IR)dye700and anti-mouse IR dye800 (Li-Cor,Lincoln,Nebraska,USA).Anti-SIRT1is rabbit polyclonal antibody,and anti-IL-24,anti-Bcl-2,and anti-beta-actin are mouse monoclonal antibodies.Fluorescent signal was revealed through the Odyssey infrared imaging system(Li-Cor,Lincoln,Nebraska,USA).
Cytotoxicity assay
Cells at104per well were plated in96-well plates and treated with various adenoviruses.At the indicated times, the medium was removed and replenished with fresh medi-um containing1mg/ml3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyltetrazolium bromide(MTT).Cells were incubated at 37°C for4h;then,the medium was removed before adding 150μl DMSO and mixed thoroughly for10min.Absor-bance was read on TECAN DNA expert(TECAN)at 595nm.Cell viability at treatment with oncolytic adenovi-ruses was represented as a percentage to cells without ade-novirus infection.
Tumor xenograft in nude mice
All animals used in these experiments were maintained at the institutional facilities according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals.The experiments were approved by the Institutional Review Board of the Southwest Hospital,Third Military Medical University.Male BALB/c nude mice(4–5weeks of age) were obtained from the Animal Research Committee of the Institute of Biochemistry and Cell Biology(Shanghai, China).Mice were inoculated subcutaneously with Huh7 cells(2×106).When the tumor volume reached100to 150mm3,animals were randomly divided into five groups, and each group contained seven mice.Intratumoral injection of different adenoviruses(7×108IU/dose)in100μl of PBS was performed once every other day for a total of three times.The tumor size was measured using a caliper.The tumor volume(in cubic millimeter)was calculated as fol-lows:(length×width2)/2.
Immunohistochemistry of tumor xenograft sections Tumor-bearing animals were treated with AdCN205-miR-34a,AdCN205-IL-24-miR-34a,AdCN205-IL-24, AdCN205-EGFP,or PBS.Six days after treatment,the animals were sacrificed,and the tumors were harvested. The tumor tissues were quickly fixed in formalin and embedded in paraffin.The tumor tissues were sectioned at6μM,and deparaffinized tumor sections were stained with hematoxylin–eosin.For immunohistochemical staining,the antigen retrieval procedure was performed by heating the samples in Dako antigen retrieval solu-tion containing10mM EDTA(pH8.0)with a pressure cooker.Rabbit anti-IL-24and anti-adenovirus hexon (diluted at1:100,Santa Cruz,CA)for overnight was used to detect expression of IL-24and hexon.After
incubation with rabbit secondary antibody(diluted at 1:100,Immunology Consultants Laboratory,Inc,OR), the staining signals were amplified by a biotinylated peroxidase-conjugated streptavidin system(Bio-Genex Laboratories,San Reman,CA).Slides were counter-stained with hematoxylin(Sigma,St.Louis,MO).
Statistical analysis
The statistical significance was calculated by analysis of variance when more than two groups were compared or by Student's t test when only two groups were compared.To study the relationship between miRNA-34a expression and other variables,either independent-sample t test or the non-parametric Mann–Whitney test for continuous variables was used.The statistical significance of correlation between miRNA-34a expression and survival was estimated by the log-rank test.
Results
Reduced expression of miRNA-34a was associated
with worse outcome of HCC patients
The abundance of miRNA-34a in HCC was first investigat-ed by measuring miRNA levels in14paired HCC samples and their corresponding non-HCC liver tissues.As shown in Fig.1a,there was a tendency of reduced expression of miRNA-34a in HCC tissues,as compared with their corresponding non-HCC tissues,although difference was not statistically significant.Among these14paired samples, ten patients showed the reduced miRNA-34a expression in HCC tissues,and only four patients exhibited increased miRNA-34a expression(Fig.1a and supporting Table1). Furthermore,we measured miRNA-34a level in52addi-tional HCC tissues.The clinical and pathological character-istics of these patients were summarized in supporting Table2.Our data showed that the level of miRNA-34a in HCC tissues was statistically and inversely correlated with histological staging,necrosis,and vascular invasion of tumor and recurrence after hepatectomy resection of tumors(Fig.1b and c,supporting Table2).No signif-icant association was found between miRNA-34a ex-pression and other parameters such as age,gender, metastasis,liver injury,hepatic fibrosis,or hepatic https://www.doczj.com/doc/e612818303.html,ing the Kaplan–Meier analysis method, we found that low expression of miRNA-34a in HCC had tendency for short overall and disease-free survival, when compared to the high expression of miRNA-34a in HCC(Fig.1d and e).These results indicated that the reduced expression of miRNA-34a was associated with worse outcome of HCC patients.Characterization of oncolytic adenoviral vectors
co-expressing miRNA-34a and IL-24
Previously,we constructed oncolytic adenoviral vectors AdCN205-EGFP and AdCN205-IL-24.In both adenoviral vectors,the adenoviral E1A promoter was replaced by the hTERT promoter,and the CR2region of E1A was deleted. Therefore,this modification makes virus replication spe-cific to tumor cells that express a high level of hTERT in conjunction with the lack of Rb tumor suppressor gene expression.Additionally,the endogenous E3pro-moter was used to control expression of EGFP or IL-24 by replacing the6.7k/gp19k gene in the E3region with EGFP and IL-24encoding sequences[25].In this study, we used the same strategy to construct adenoviral vec-tors,AdCN205-miR-34a and AdCN205-IL-24-miR-34a, for expression of miRNA-34a alone and co-expression of miRNA-34a and IL-24,respectively(Fig.2a).The mechanism for co-expression of miRNA-34a and IL-24 (with stop codon)from AdCN205-IL-24-miR-34a was due to the cleavage of pri-mirR-34a from single RNA transcribed by endogenous E3promoter via miRNA process by the microprocessor complex[1].
To examine whether these recombinant adenoviruses can effectively express mature miRNA-34a and/or IL-24 in HCC cells,we determined transgene expression by qRT-PCR and Western blotting in the HCC cells after infection with recombinant adenoviruses.As shown in Fig.2b,both of AdCN205-miR-34a and AdCN205-IL-24-miR-34a expressed comparable levels of miRNA-34a in cancer cells.The high level of IL-24expression was only observed in cancer cells after infection with AdCN205-IL-24or AdCN205-IL-24-miR-34a(Fig.2c). Our data indicated that miRNA-34a can be efficiently expressed by oncolytic adenovirus,and miRNA-34a and IL-24can be efficiently co-expressed by a single onco-lytic adenovirus.
To examine whether miRNA-34a interferes with the rep-lication of oncolytic adenovirus,HCC cells and normal cells were infected with AdCN205-IL-24-miR-34a,AdCN205-miR-34a,AdCN205-IL-24,AdCN205-EGFP,or wild-type adenovirus(Ad-WT),and the production of viral progeny was evaluated.As shown in Fig.2d,high level of viral production was observed in all HCC cells infected with A d C N205-I L-24-m i R-34a,A d C N205-m i R-34a, AdCN205-IL-24,or AdCN205-EGFP at a comparable level, as compared with tumor cells infected with Ad-WT.In contrast,AdCN205-IL-24-miR-34a,AdCN205-miR-34a, AdCN205-IL-24,and AdCN205-EGFP only replicated at extremely low level in normal cells,whereas Ad-WT repli-cated at a high level.This result indicates that expression of miRNA-34a and IL-24has no effect on adenovirus replica-tion in HCC tumor cells.
Inhibition of Bcl-2and SIRT1expression by miRNA-34a but not IL-24in vitro
In order to determine biological functions of miRNA-34a,we examined the expression of miRNA-34a target genes,Bcl-2and SIRT1,in cancer cells after infection with AdCN205-miR-34a and AdCN205-IL-24-miR-34a.Figure 3shows that dramatic inhibition of Bcl-2and SIRT1expres-sion in cancer cells could be achieved after infection of HCC cells with AdCN205-miR-34a and AdCN205-IL-24-miR-34a,but not AdCN205-IL-24.This indicates that exogenous miRNA-34a can exert biological function in cancer cells.Inhibition of HCC cell growth but not normal cells
by AdCN205-miR-34a and AdCN205-IL-24-miR-34a in vitro To evaluate the growth-suppressing effect of AdCN205-miR-34a and AdCN205-IL-24-miR-34a on HCC cells,we performed a cell viability assay on the HCC cells and normal liver cells after infection with adenoviruses AdCN205-IL-24-miR-34a,AdCN205-miR-34a,AdCN205-IL-24,AdCN205-EGFP,or wild-type adenovirus.As shown in Fig.4,both of AdCN205-miR-34a and AdCN205-IL-24could induce higher cytotoxicity to HCC cells than that induced by AdCN205-EGFP and Ad-WT.Moreover,the AdCN205-IL-24-miR-34a exerted dramatic increased antitumor activity,as compared with AdCN205-miR-34a,AdCN205-IL-24,AdCN205-EGFP,or Ad-WT.In contrast,neither AdCN205-IL-24-miR-34a nor AdCN205-miR-34a exhibited cytotoxicity to normal liver cells.Ad-WT was able to kill both tumor and normal cells because Ad-WT lacks selectivity for infected cells.These data indicate that miRNA-34a delivered by adenovirus can effectively kill liver cancer cells in vitro.In addition,AdCN205-IL-24-miR-34a exhibited remarkable antitumor ability in HCC cells and showed only a very low toxicity to normal liver
cells.
Fig.1Downregulation of miRNA-34a is correlated with poor prog-nosis of HCC patients.a miRNA-34a expression level in paired HCC patient sample and their corresponding non-HCC tissues (n 014).U6RNA was used as an endogenous control.No significance was ob-served between cancerous and non-HCC tissues in the expression level of miRNA-34a.b Median miRNA-34a expression profiles of samples with different grade of vascular invasion were shown in the scatter plot with added indicator bars showing the means .Outliers were also represented.Minus sign no vascular invasion (n 06),plus sign low
grade of vascular invasion (n 040),double plus sign high grade of vascular invasion (n 06).*p <0.05.c Median miRNA-34a expression profile of samples with or without recurrence was shown in scatter plot with added indicator bars showing the means .Outliers were also represented.Minus sign no recurrence (n 011);plus sign recurrence (n 021).**p <0.01.Overall survival (d )and disease-free (e )curves were shown between high-miR-34a (n 019)and low-miRNA-34a (n 030)patient groups
Antitumor effect of AdCN205-miR-34a and AdCN205-IL-24-miR-34a on established HCC tumors in an animal model In order to determine the antitumor potency of AdCN205-miR-34a and AdCN205-IL-24-miR-34a in vivo,we treated the established HCC tumors in an experimental animal model by intratumoral injection of AdCN205-IL-24-miR-34a,AdCN205-miR-34a,AdCN205-IL-24,AdCN205-EGFP,and PBS.As shown in Fig.5a ,animals receiving PBS experienced progressive tumor growth,and minor anti-tumor activity was observed in the animals treated with control vector AdCN205-EGFP.Antitumor activity induced by AdCN205-miR-34a was slightly higher than that induced by AdCN205-EGFP.Treatment with AdCN205-IL-24resulted in significant tumor inhibition.These data indicated that single gene therapy was not enough to abolish tumor growth.Impressively,the treatment with AdCN205-IL-24-miR-34a resulted in significant inhibition of tumor growth,compared with that induced by AdCN205-miR-34a,AdCN205-IL-24,and AdCN205-EGFP (p <0.01).All of animals treated with AdCN205-IL-24-miR-34a had com-plete tumor regression without tumor recurrence after 7weeks (Fig.5a ).
Immunohistochemistry analysis showed that expression of hexon was observed in the tumor sections from animals infected with AdCN205-IL-24-miR-34a,AdCN205-miR-34a,AdCN205-IL-24,and AdCN205-EGFP,indicating strong adenoviral replication in those tumors.Pathological analysis revealed that the necrotic area could be found in the tumor treated with AdCN205-IL-24-miR-34a,AdCN205-miR-34a,AdCN205-IL-24,and AdCN205-EGFP,whereas the necrotic area was hardly observed in the tumors treated with PBS (Fig.5b ).The upregulation of IL-24was observed only in the tumor sections from animals treated
with
Fig.2Construction and characterization of oncolytic adenoviruses.a Schematic structure of oncolytic adenoviruses involved in this study.AdCN205-miR-34a,AdCN205-IL-24,AdCN205-EGFP,and AdCN205-IL-24-miR-34a carried miRNA-34a,IL-24,EGFP,and IL-24plus miRNA-34a under control of adenovirus E3endogenous promoter,respectively.b Expression of mature miRNA-34a in HCC cells at 48h after infection with oncolytic adenoviruses at MOI of five by real-time PCR.c Expression of IL-24in Huh7cells at 48h after infection with oncolytic adenoviruses at MOI of five by Western blotting.d Selective replication of oncolytic adenoviruses in vitro.HCC cells (Huh7and PLC/PRF/5)and normal cells (L-02)were infected with oncolytic adenoviruses at MOI of five,and the viral titers were measured in cell lysates at 48h after infection.The data were presented as mean±SD of three independent
experiments
Fig.3Expression of Bcl-2and SIRT-1in HCC cells after in-fection with oncolytic adenovi-ruses.Huh7cells were infected with oncolytic adenoviruses at MOI of five,and expression of Bcl-2and SIRT-1was measured at 48h after infection by West-ern blotting
AdCN205-IL-24and AdCN205-IL-24-miR-34a,as com-pared with those treated with PBS,AdCN205-miR-34a,and AdCN205-EGFP (Fig.5b ).In addition,our data showed that miRNA-34a was significantly higher in tumors treated by AdCN205-miR-34a and AdCN205-IL-24-miR-34a than those treated by AdCN205-IL-24and AdCN205-EGFP (Fig.6a ).Furthermore,we found that expression of miR-34a targeted gene Bcl-2and SIRT1was significantly re-duced in tumors treated by AdCN205-miR-34a and AdCN205-IL-24-miR-34a (Fig.6b and c ).These results indicate that treatment with oncolytic adenoviral vectors can induce adenoviral replication and therapeutic gene ex-pression in cancer cells under in vivo conditions.
Discussion
Different therapeutic strategies have been developed to modulate miRNA functions in cancer therapies.For onco-genic miRNAs,which promote cancer initiation and pro-gression,an antagomiR should be used to inhibit the effects of the oncomiR.The antagomiR downregulates the onco-genic properties of the miRNA,resulting in suppression of cancer progression [19].For tumor-suppressing miRNAs,miRNA mimics should be used to restore their expression to achieve decreased cancer development [20].Our data showed that miRNA-34a level was reduced in tumor tissues from HCC patients and that the reduced level of miRNA-34a was associated with histological staging,necrosis,and vascular invasion of tumor and recurrence after hepatectomy resection of tumors.These results coincided with previous studies showing that miRNA-34a was downregulated in
colon cancer,prostate cancer,and HCC [15,16,18].These data suggest that miRNA-34a can be considered a tumor-suppressive miRNA and that providing recovered expres-sion of miRNA-34a could be a therapy of HCC.
Previous studies have shown that adenovirus endogenous promoter could express exogenous therapeutic genes in a predicted manner [25].In this study,we constructed several oncolytic adenovirus vectors to deliver miRNA-34a for the treatment of HCC.Our data showed that miRNA-34a could be efficiently expressed in tumor cells by oncolytic adeno-viruses.The expressed miRNA-34a could exert biological functions by downregulating expression of miRNA-34a-targeted genes.In addition,the expressed miRNA-34a did not alter adenovirus replication in cancer cells.These data indicated that functional miRNA-34a could be delivered and expressed from oncolytic adenovirus-based systems.Fur-thermore,our data showed that AdCN205-miR-34a caused cytotoxic effects on tumor cells,but it did not elicit cytotox-icity on normal liver cells,indicating that AdCN205-miR-34a is a safe vector for HCC treatment.
In the present study,miRNA-34a appears to function as an effective gene for cancer therapy.The antitumor activity induced by miRNA-34a may be explained by multiple mechanisms.It has been reported that transactivation of miRNA-34a by p53influences gene expression and pro-motes apoptosis of tumor cell [13].Bcl-2,an inhibitor of cell apoptosis,is regulated directly by miRNA-34a [31].Repression of SIRT1by miRNA-34a regulates apoptosis [14].Additionally,recent studies also show that miRNA-34a can inhibit migration and invasion by downregulating c-MET expression in tumors [16].Previous studies also indi-cate that tumor-suppressing gene p53,antiapoptotic
gene
Fig.4Induction of cytotoxicity by oncolytic adenoviruses in vitro.HCC cell lines and normal cells were infected with oncolytic adenoviruses at MOI of five,and cell viability was determined by MTT assay at different time points after infection.Data are shown as the means ±SD of three independent experiments.*p <0.05and **p <0.01
Bcl-2,and oncogene SIRT1and c-Met play important roles in the pathogenesis of HCC [16,17].In this study,we found that overexpression of miRNA-34a induced downregulation of Bcl-2and SIRT1expression.Our data suggest that miRNA-34a may exert antitumor activity through down-regulation of Bcl-2and SIRT1.Further studies are war-ranted to demonstrate whether AdCN205-miR-34a can inhibit tumor invasion and metastasis.
Because of the complexity of cancer pathogenesis,our previous studies are consistent with others that have sug-gested a single therapeutic agent is not enough to eradicate tumors [25,32].Nevertheless,our previous study and others have generated some single vectors co-expressing shRNA and therapeutic genes [32,33].In these studies,shRNA sequence carried by miRNA backbone was embedded into therapeutic genes.Thus,in this study,we constructed an oncolytic adenoviral vector,AdCN205-IL-24-miR-34a,in which miRNA-34a and IL-24were simultaneously expressed.Our data showed that infection of tumor cells with AdCN205-miR-34a could express both miRNA-34a and IL-24effectively.Our data further showed that AdCN205-IL-24-miR-34a dramatically inhibited the tumor cell growth in vitro.Moreover,the treatment of established HCC with AdCN205-IL-24-miR-34a resulted in complete tumor regression without tumor recurrence.It has been demonstrated that IL-24can secrete a soluble form of
IL-
Fig.5Treatment of established tumors by oncolytic
adenoviruses in vivo.a Tumors were established in Balb/c nude mice by implantation of Huh7cells.The tumor-bearing animals were treated with oncolytic adenoviruses.The tumor size was measured and the tumor volume was calculated.The data are expressed as the means of the tumor volume ±SD (n 07).*p <0.05,compared with
AdCN205-miR-34a,AdCN205-EGFP or PBS;**p <0.01,com-pared with AdCN205-IL-24,AdCN205-miR-34a,AdCN205-EGFP ,or PBS.b Immunohisto-chemical staining for hexon and IL-24and pathologic examina-tion of tumor tissues after treat-ment with oncolytic
adenoviruses.Arrows indicated hexon and IL-24-positive cells and white line labeled necrotic area in tumors
24protein that can selectively kill cancer cells.In addition to its direct antitumor activity,IL-24also exerts the antiangio-genic activity in vivo [27].Our previous study is consistent with others who have indicated that oncolytic adenovirus-mediated transfer of IL-24could exert dramatic antitumor activity [25,28].However,many tumor cells were resistant to IL-24due to their overexpression of antiapoptotic genes
[29].Thus,combined therapy with IL-24and other thera-peutic genes or chemotherapy would enhance IL-24-mediated antitumor activity [34,35].It has been demon-strated that inhibition of Bcl-2expression in tumor cells could render them sensitive to IL-24[29],which would explain why a better antitumor activity could be achieved by a combination of miRNA-34a and IL-24.We
therefore
Fig.6Expression of miRNA-34a and Bcl-2and SIRT1in the established tumors after treat-ment with oncolytic adenovi-ruses.Expression level of
miRNA-34a,Bcl-2,and SIRT1in vector-treated tumors was represented as a percentage of tumors treated with PBS.Data are shown as the means ±SD of four animals per groups.
*p <0.01,compared with either AdCN205-IL-24or AdCN205-
EGFP
Fig.7The illustrative
description of antitumor activity induced by AdCN205-IL-24-miR-34a.In this model,the oncolytic adenovirus can spe-cifically replicate in the tumor cells and lead to cell lyses.Subsequently,the viral endoge-nous promoter can promote the transcription of IL-24mRNA and pri-miR-34a.IL-24can,in turn,induce apoptosis and in-hibit metastasis as well as an-giogenesis.Meanwhile,mature miR-34a induces apoptosis by downregulation of Bcl-2and suppresses metastasis and an-giogenesis by silencing c-MET and SIRT1genes
propose an illustrative description showing antitumor activ-ity induced by combination of miRNA-34a and IL-24 (Fig.7).In this model,the oncolytic adenovirus can specif-ically replicate in the tumor cells and induce cell lysis. Secondly,the viral endogenous promoter can induce the transcription of IL-24mRNA and pri-miR-34a.IL-24can elicit apoptosis and inhibit the metastasis as well as angio-genesis.Meanwhile,the mature miRNA-34a induces apo-ptosis by downregulating Bcl-2and suppresses metastasis and angiogenesis by targeting c-MET and SIRT1genes. Downregulation of Bcl-2induced by miRNA-34a can over-come tumor cell resistance to IL-24.However,a future study is required to determine the detailed mechanisms.
In conclusion,this study provides a potent vector for simultaneous delivery of miRNAs and therapeutic genes in the field of cancer therapy.And more importantly,the com-bined treatment with miRNA and therapeutic gene develops a novel strategy for cancer therapy.
Acknowledgments This work was supported by funds from National Natural Sciences Foundation of China(30872984,81020108026,and 81090423)and National Basic Research Program of China(973Pro-gram,no.2010CB529406).
Disclosure statement All authors have no potential conflicts of interest.
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