MiR-203Inhibits Proliferation Of HCC Cells By Targeting Survivin

To validate whether down-regulation of microRNA-203(miR-203) inhepatocellular carcinoma (HCC) is involved in HCC progression by targetingsurvivin.INTRODUCTIONHCCHepatocellular carcinoma (HCC) is the fifth most common malignancyworldwide, with a5-year survival rate of9%. The disease is more common inmen than in women, with the highest incidence rates reported in East Asia. Recentstudies indicate that the incidence of HCC in the US and UK has increasedsubstantially over the last two decades. Although a small subset of HCC patientsqualify for surgical intervention, many patients with advanced HCC have littlechance of survival. The extremely poor prognosis of HCC is largely the result of ahigh rate of recurrence and metastasis. The generally poor clinical outcome ofindividuals diagnosed with HCC underscores the importance in For Peer Reviewobtaining a better understanding of the transcriptional activation of oncogenicsignaling pathways, and the control of cancer-associated genes.miRNA-203MicroRNAs,a class of small non-coding RNAs,have been identified as geneexpression regulators by targeting to the3’-untranslated region (UTR) of mRNAs fortranslational repression, or mRNA degradation. In the recent years, a lot of data havesuggested that microRNAs are involved in essential tumor cell biological processes,including proliferation, invasion, and apoptosis. It was reported that microRNA-203 (miR-203) exhibited significantly down-regulated expression in some tumorssuch as prostate cancer, hematopoietic malignancy and colon cancer.Other studiesshowed that the low level expression of miR-203is invoved into cell proliferation inhuman head and neck squamous cell carcinoma, chronic myelogenous leukemiaand B cell leukemia.SurvivinSurvivin is a multitasking protein that has dual roles in promoting cellproliferation and preventing apoptosis, may plays a role in neoplasia.Survivin maycounteract a default induction of apoptosis in G2/M phase.It is essential forchromosome alignment and segregation during mitosis and cytokinesis and acts as animportant regulator of the localization of the chromosomal passenger complex.Thisprotein may be a inhibitor of CASP3and CASP7. Target prediction using thecomputational algorithm Targetscan (http://www.targetscan.org/) suggests thatsurvivin may be one of the target genes of miR-203. The over-expression of survivinwas also reported previously.This study will investigate whether miR-203may inhibit cell proliferation ofHCC cells by targeting survivin.METHODSThe internet resource TargetScan (http://www.targetscan.org/) was used topredict potential mir-203targets, using “hsa-mir-203” as a search term.MiR-203mimics was transfected into HepG2cells to enhance miR-203expression, andmiR-203inhibitor was transfected into HepG2cells to inhibit miR-203expression.Quantitative real-time PCR (Q-PCR) analyses miR-203and mRNA expression.Theeffect of up-and down-regulation of miR-203on survivin expression of HepG2cellswas evaluated using western blot assay. The effect of miR-203or survivin expressionon the proliferation of HepG2cells was detected by CKK-8assay. All data were presented as means±SD. The differences between groups wereanalysed using one-way analysis of variance and LSD method, with p<0.05considered statistically significant (two-tailed).RESULTSTo determine the mRNA target of miR-203, we performed target predictionusing TargetScan5.2. The survivin was predicted to be one of the targets of miR-203.One conserved miR-203-binding site located within the3’UTR (nt1034-1041) ofsurvivin was found. To demonstrate the functional relationship between miR-203andsurvivin in HepG2cells, Q-PCR quantitation of miR-203demonstrated thattransfection of miR-203mimics increased the expression of miR-203by400%, whiletransfection of miR-203inhibitor inhibited the intracellular level of miR-203by80%(p＜0.05). Western-blot result showed that survivin expression decreasedsignificantly when miR-203was over expressed (p＜0.05), while no significantlychange by miR-203inhibition. We made the conclusion that over-expression ofmiR-203significantly inhibited the expression of survivin in HepG2cells (P <0.05),and down-expression of miR-203did not significantly promote the expression ofsurvivin in HepG2cells (P>0.05).CCK-8result showed miR-203inhibition had noeffect on cell proliferation (p＞0.05), while miR-203over-expression decreased cellproliferation significantly (p＜0.05). We concluded from these that Bothover-expression of miR-203and down-regulation of survivin suppressedproliferation of HepG2cells significantly compared with negative control. Q-PCRresult showd that the expression of survivin decreased by80%in siRNAtransfection24h (p＜0.05). The proliferation of HepG2cells with survivin inhibitionwas detected by CCK-8. The result showed survivin inhibition decreased HepG2cellsproliferation significantly (p＜0.05). DISCUSSIONIdentification of molecular mechanism of HCC is the basement for the treatmentof HCC. Increasing evidence supports that dysregulation of microRNA mayfunctionally contribute to HCC progression. We and other group found miR-203expression is very low in HCC tissue, suggesting that the low-expression of miR-203in HCC may contribute to its progression. However, the detail function of miR-203isnot clear. In this study, a quantitative real-time PCR (Q-PCR) microRNA array wasused to detected the differential expressed microRNAs in tumor tissue compared withthe matched normal tissue. It showed that the expression level of miR-203wassignificantly down-regulated in tumor tissue. It has also been reported the samefinding. In contrast, the expression level of survivin (BIRC5) was significantly higherin tumor tissue than in the matched normal tissue.In light of the previous reports,however, the effect of miR-203and survivin on the proliferation of HCC cells, aswell as the functional relationship between miR-203and survivin in HCC cells hasnot been documented. To verify that survivin is regulated by miR-203in HCC cells,miR-203expression was increased in HepG2cells by transfection of miR-203mimic.Our result showed survivin was detected markedly downregulated by miR-203over-expression.Further, we detected the effect of miR-203and survivin on theproliferation of HCC cells. Our result showed both miR-203over-expression andsurvivin inhibition inhibited the proliferation of HepG2cells significantly comparedwith negative control.This demonstrated the functional relationship between miR-203and survivin. In summary, our results indicate that low expression of miR-203doesnot cause the increaseing expression of survivin and may not, thus,promote HCCproliferation. The reason of the low expression of miR-203in HCC tissue, however, isstill unknown and needs further investigation.CONCLUSIONIn HCC,survivin expression decreased significantly when miR-203was over expressed, while no significantly change by miR-203inhibition.MiR-203inhibitionhad no effect on HepG2cells proliferation (p＞0.05),while miR-203over-expressiondecreased HepG2cells proliferation significantly. Survivin inhibition decreasedHepG2cells proliferation significantly.