The Role Of MiR-134in Hepatocellular Carcinoma And Its Molecular Mechanism

【Background and Objective】Hepatocellular carcinoma (HCC) is one of the most common malignancy worldwideand represents high morbidiy and mortality. Although substantial progress has beem madein the treatment of HCC in the past decades, the prognosis for advanced HCC remains poor.Therefore, it is still urgent to develop novel therapeutic strategy.Hepatocyte nuclear factor4α (HNF4α), a member of the nuclear hormonereceptor superfamily, which highly expresses in well differentiated hepatocytes and playsan important role in differentiation of hepatic lineage and maintaining the function ofmature hepatocytes. Previous studies from our laboratory indicated that forcedre-expression of HNF4α dramatically decreased markers of cancer stem cells(CSCs),including CD90and CD133in HCC. Additionally, overexpresssion of HNF4α couldupregulate the expression of liver function related genes, leading to a “hepatocyte-like”differentiation and a loss of tumorigenicity. We also proved the strong anti-tumor effect ofHNF4α in vivo.As a mutil-function transcription factor, HNF4α could be regulated by some miRNAs.Meanwhile, HNF4α is also able to regulate part of miRNAs. Recent study demonstratedthat miR-629and miR-24initiated hepatocarcinogenesis by negatively regulating HNF4α;whereas miR-124, which upregulated by HNF4α, could suppress the occurrence anddevelopment of HCC. Our previous study indicated that HNF4α regulated thetranscriptional expression of miR-379~656cluster via directly binding to its responseelement in Dlk1-Dio3region. Most miRNAs in this cluster exhibited inhibition onproliferation, invasion and metastasis of HCC cells in vitro. As a representative miRNA inthis cluster, miR-134exerted dramatically suppressive effect on different HCC cell line. Based on the research background and previous experimental results, we will furtherexplore the mechanism of the anti-tumor effect of miR-134. Moreover, analysis thecorrelation of miR-134, HCC clinical malignant phenotype and prognosis, trying toprovide a new target for HCC therapy.【Methods】1. The clinical significance of differentially expression of miR-134in HCC patients.(1) Real-time PCR was carried out to detect the relative RNA levels of HNF4α andprimary miR-134(pri-miR-134) in DEN-induced rat HCC model and the correlationbetween HNF4α and pri-miR-134was analyzed.(2) The levels of HNF4α and pri-miR-134in human HCC samples (n=71) weredetected by RT-PCR. Clinicopathologic correlation of miR-134downregulation in humanHCCs was analyzed.2. miR-134effect on the biological characteristics of HCC cell line(1) The hepatoma cells YY-8103or LM3were transfected with miR-134mimic oras-miR-134in HCC YY-8103or LM3to examine the ability of proliferation, soft agarcolonies formation, migration, invasion and cell cycle arrest.a. For proliferation assay, HCC cells were plated into a96-well plate. Cell CountingKit-8(cck-8) was used to examine cell proliferation.b. HCC transfected with miR-134mimic/as-miR-134were plated into soft agarmedium to evaluate soft agar colony formation ability.c. HCC transfected with miR-134mimic/as-miR-134were plated into the upperchamber of the transwell without or with matrigel to assess migration and invasion.d. Cell cycle and apoptosis measured by FACS48or72h after transfection.(2) Effect of miR-134on the tumorigenicity of hepatoma cells in vivo2×10~6LM3or YY-8103cells infected with AdmiR-134or AdGFP were inoculatedsubcutaneously into both flanks of each mouse (10mice per group). Tumor size wasmeasured with calipers on a regular basis, and volume of tumors was determined using the following formula: Volume=width~2×length×0.5.(3) Effect of miR-134on HCC xenografts in nude mice2×10~6of LM3cells were injected into the armpits of mice to establish a subcutaneoustumor model. Once tumor xenografts reached about50mm~3, injection of AdmiR-134andAdGFP was administered intratumorally twice a week. Kinetics of tumor formation wasestimated as described previously. At the time of euthanasia, tumors were removed forinvestigating tumor weight and tumor size. Tissue RNA was isolated and real-time PCRwas carried out to detect the expression of miR-134in different flanks.Immunohistochemistry was taken to detect expression of Ki67and target genes. Assesstherapeutic effect of miR-134on xenografts model in nude mice.3. Identify the target gene of miR-134The target genes of miR-134were predicted by targetscan6.2. YY-8103wastransfected with miR-134/as-miR-134. Western blot and real-time PCR were carried out toverify the target genes of miR-134. In addition, target genes were further demonstrated byluciferase assay. YY-8103was cotransfected with miR-134and a plasmid for expressingKRAS without it’s3’UTR. Cell Counting Kit-8was used to examine cell proliferation, thecolonies formation ability was evaluated by soft agar colonies formation, cell migrationand invasion assay were carried out by transwell.4. The role of miR-134in the malignant phenotypes reversion by HNF4αThe cell proliferation, soft agar colony formation, migration, and invasion of YY-8103cells infected with AdHNF4α or AdGFP and transfected with as-miR-134or scramblecontrol (Scr) were detected.5. Statistical analysisStatistical analyses were performed with SPSS software (16.0version), with a P value<0.05considered significant and P <0.01considered very significant. For experimentsinvolving only two groups, data were analyzed with Student’s unpaired t-test; The unequalvariances pairing using the Wilcoxon signed rank test; Mann-Whitney U test was used forcomparison of tumor weight and volume; RΧC tables using χ~2test; The Kaplan-Meier method was used to calculate survival, and significance was determined by log-rank test.Statistical significance was set at*P <0.05,**P <0.01,***P <0.001.【Results】1. The clinical significance of miR-134in HCC.(1) We examined the expression profiling of HNF4α and pri-miR-134in DENinduced-HCC rat model (n=4at the indicated time point). The results show that HNF4αand pri-miR134were suppressed in the process of hepatocarcinogenesis. A strikingpositive correlation between HNF4α and pri-miR-134level was also observed inDEN-treated rat liver.(2) In human HCC samples (n=71),63%(45/71) and70%(50/71) of the HCCtissues showed lower level of HNF4α and pri-miR-134expression, respectively, ascompared with their surrounding noncancerous tissues. Reduced pri-miR-134expressionwas more frequent in the HCCs with lower HNF4α level relative to those with intermediateand high HNF4α level (89%versus38%). The high level expression of pri-miR-134inHCCs was associated with less aggressive pathologic features including small tumor size(P=0.0271), less liver cirrhosis (P=0.0127), early tumor stage (P=0.0051), presenceof tumor encapsulation (P=0.0013).2. The inhibition of miR-134on the biological characteristics of HCC cells and itsmechanism(1) miR-134suppresses malignant properties of HCC in vitromiR-134exerted marked inhibition on proliferation, soft agar colonies formation,migration and invasion of YY-8103and LM3cells, whereas as-miR-134treatmentupgraded malignant properties of HCC in vitro. Transfection of miR-134in YY-8103cellsdecreased G0/G1population by35%(P <0.01) and increased G2/M population by117%(P <0.01).(2) miR-134suppresses tumorigenicity of HCC cells in mice.In mices receiving YY-8103cells infected with AdGFP, xenograft were detected in 10/10mices by day18after inoculation while xenografts were detected in only3/10micesinjecting YY-8103cells infected with AdmiR-134at the same time.In mices receiving LM3cells infected with AdGFP, xenografts was detected in8/10mices by day10after inoculation and in all subjects by day19. In mices receiving LM3cells infected with AdmiR-134, xenografts was detected in only1/10mice by day10afterinoculation and6/10mices by day25.(3) Anti-tumor effect of miR-134Intratumoral injection of AdmiR-134significantly reduced the growth and the weightof tumor nodules in compared with AdGFP delivery. RT-PCR revealed that miR-134expression was significantly elevated in AdmiR-134treated tumors. Immunohistochemicalstaining showed that treatment with AdmiR-134in tumors resulted in remarkable reducedKi67staining.2. KRAS is a direct target of miR-134A bioinformatics search with Target Scan identified proto-oncogene KRAS as apotential target of miR-134. In the experiments with cultured HCC cells, overexpression ofmiR-134significantly decreased both mRNA and protein level of KRAS. In contrast,as-miR-134increased KRAS expression. Luciferase assays revealed that miR-134diminished the activity of KRAS3′UTR in HCC cells, whereas mutation of motif1in theputative MREs of KRAS3′UTR abrogated its response to miR-134. Immunohistochemicalstaining showed that treatment with AdmiR-134in xenograft tumors resulted in remarkabledownregulation of KRAS expression.3. miR-134suppresses the malignant properties of HCC partially throughdownregulation of KRASOverexpression of non-targetable KRAS reversed the suppressive effects of miR-134on the malignant property of HCC cells, including proliferation, migration and invasion.4. HNF4α suppresses tumorigenic capacity of HCC partially through upregulation ofmiR-134as-miR-134reversed HNF4α-imposed defect of proliferation, colony formation, migration and invasion. Additionally, the repression of KRAS by ectopic HNF4α was alsorestored by as-miR-134at the mRNA and protein levels.【Conclusions】1. The expression of miR-134is positively correlated with HNF4α in HCC tissues andlow miR-134expression is associated with aggressive behaviors of human HCC.2. miR-134suppresses malignant properties of HCC, including proliferation, soft agarcolonies formation, migration,invasion.3. KRAS is the direct target gene of miR-134.4. HNF4α suppresses tumorigenic capacity of HCC partially through upregulation ofmiR-134.5. Inhibition of the endogenous miR-134partially reversed the suppressive effects ofHNF4α on KRAS expression and HCC malignancy.