Roles Of MicroRAN-503in Hepatocellular Carcinoma And Its Underlying Mechanisms

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The development and progression of HCC is typical of a multistage process, which is believed to involve the deregulation of genes that are critical to cellular processes, such as cell cycle control, cell growth, apoptosis and cell migration and spreading. In the past decades, studies have focused on investigating the genes and proteins underlying the development and progression of HCC. Recently, an increasing number of reports have described a new class of small regulatory RNA molecules termed microRNAs (miRNAs) that are implicated in HCC progression.MicroRNAs (miRNAs) belong to a large family of non-coding, single-stranded RNAs,18-25nucleotides long. There is growing evidence that miRNAs regulate the expression of protein-coding genes, either at the level of messenger RNA degradation or translation, by pairing with their target mRNAs3’UTR. In1993, the first miRNA lin-4was discovered. They are involved in many biological processes including cell cycle regulation, development, differentiation, metabolism, and ageing. miRNA dysregulation has been observed in many human malignancies and there is some evidence for their involvement in the progression of tumors, either as oncogenes or tumor suppressors. However, their exact biological role in HCC remains unclear. Further research is still needed to explore new deregulated miRNA and its functions, which provide new ideas for the treatment and prognosis of HCC.AimsThis study aimed to find differentially expressed miRNA in HCC, and then quantitative PCR was used to validate the miRNA expression level in HCC tissues and cell lines. Based on this we further investigated the biological function of miR-503in HCC cells by using gain-of and loss of function experiments, analyzed and verified its target genes and explored the possible molecular mechanisms of HCC in development and progress.Methods1. MicroRNA real-time quantitative PCR chips was used to explore differential expression miRNA in HCC cell lines and normal immortalized liver cells.2. We verified the expression of miR-503in125paired samples of clinical HCC tumor, adjacent normal liver tissues and7HCC cell lines using quantitative R-T PCR analysis and then investigated the correlation between clinicopathologic factors and miR-503expression.3. We conducted gain-of and loss of function analysis by transfecting HCC cell lines with chemically synthesized miR-503mimics or inhibitor. Methods including CCK-8, colony formation assays, Edu assays, tumor formation in nude mice and flow cytometry were used to measure cell proliferation and cell cycle.4. We investigated the direct target genes of miR-503involved in cell cycle arrest by a dual-luciferase reporter system, Western blot and qPCR.Results1MicroRNA array showed that miR-503was significantly downregulated in HCC cell line. 2. Further study verified that significant downregulation of miR-503was observed in HCC tissues compared with their adjacent non-tumor tissues. Furthermore, the expression of miR-503was noticeably reduced in five of seven (71.4%) HCC cell lines examined compared with normal liver cell line L023. Lower miR-503expression in HCC tissues significantly correlated with shorter survival of HCC patients, and miR-503was identified to be an independent predictor for prognosis of HCC patients.4. Restoration of miR-503dramatically suppressed HCC cell growth in vitro and in vivo by inducing G1phase cell cycle arrest.5. We confirm cyclin D3and E2F3are direct functional targets of miR-503by using a dual-luciferase reporter system.ConclusionsIn conclusion, we report the altered miR-503expression pattern in HCC, investigate the potential role of miR-503in tumorigenesis, and demonstrate that cyclin D3and E2F3are direct functional targets of miR-503. Our data demonstrate an important role for miR-503in the molecular etiology of cancer and suggest its potential application in cancer therapy.