Analysis Of MicroRNA Expression Profile In Hepatocellular Carcinoma Tissues Based On TCGA Database And Preliminary Study On The Role Of MiR-589-5p In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is the most common liver cancers and accounts for about 80-90% of cancers in liver.HCC is the fifth common malignant tumor and the third leading cause of all cancer-related deaths.According to the statistics,it is approximately 0.75 million of new cases of HCC occurs per year,and more than half of them are in China.HBV infection is the primary etiology that leads to liver cirrhosis related carcinoma in China.The development and progression of HCC is a multifactor and multistage process.Because of its quick metastasis ability and lack of sensitive diagnostic marker,most HCC patients were diagnosed with intrahepatic or extrahepatic metastasis at advanced stage and lost the opportunity for optimal surgery treatment.Although there are liver transplantation,tumor resection,embolization and chemotherapy for the treatment of HCC,the prognosis is still poor and the rate of 5-year survival for advanced stage HCC patient is less than 5%.Now it is imperative to seek high sensitive and high specific tumor markers to improve survival and life quality of HCC patients.Micro RNA(miRNA)is a class of endogenous,small single-stranded non-coding RNA molecules approximately 18-25 nucleotides in length,and can inhibit the translation or accelerate the degradation of their targeted m RNAs by binding to the sequences in the 3' untranslated regions(3'UTR)of their targeted m RNAs.They play vital roles in gene expression,cell cycle,development and progression of disease and organism development.Studies have shown that many miRNAs expression in HCC are aberrant and play an important role in the proliferation,metastasis,invasion and apoptosis of HCC cells.Therefore,analysis of the miRNA expression profile in hepatocellular carcinoma and to clarify functions of miRNA in liver cancer is of great significance in the diagnosis and treatment of HCC.Materials and methods:In this study,we downloaded genome wide miRNA sequencing data and the corresponding clinical data of HCC patients selected from The Cancer Genome Atlas(TCGA).We explored differentially expressed miRNAs between cancer tissues and adjacent non-cancer tissues and identified micro RNAs associated with the corresponding clinical characteristics of the patients.Besides,we performed GO and KEGG Pathway analysis with extremely dysregulated micro RNAs.In addition,we studied potential roles of miR-589-5p in HCC.We transfected miR-589-5p inhibitors into HepG2 and Hu H-7 cells respectively and examined its effects on tumor cell proliferation by MTT,colony formation assays and flow cytometry.We also used bioinformatics method to predict target genes of miR-589-5p and verified it by q PCR and Western Blot.Results:By analyzing the HCC related data in The Cancer Genome Atlas(TCGA),we identified 49 miRNAs that were expressed abnormally in HCC tissues.Among them,14 were up-regulated and 33 were down-regulated in HCC tissues comparing to the adjacent non-cancer tissues.In combination with the clinical characteristics,we found that 21 of them were associated with the clinical characteristics of HCC patients.We selected miR-589-5p from these abnormal micro RNAs to study its function.Micro RNA sequencing data showed that the mean expression level of miR-589-5p in HCC tissues was 1.93 times(P < 0.001)higher than it in adjacent non-cancer tissues and the high expression level was correlated with low survival time of HCC patients(P=0.0477).Then quantitative real-time PCR(q PCR)was used to detect expression of miR-589-5p and we found it was significantly up-regulated in HCC cell lines HepG2 and Hu H-7 comparing to normal liver cell line L-O2.In addition,inhibiting the expression of miR-589-5p in HepG2 and Huh7 reduced their cell proliferation ability.MIG-6 was predicted as a potential target gene of miR-589-5p by bioinformatics method and we found that MIG-6 expression was increased when miR-589-5p inhibitors were transfected into HepG2 cells.Conclusions:1.Various micro RNAs expressed aberrantly in HCC tissues comparing to the adjacent non-cancer tissues.2.The expression of miR-589-5p was up-regulated in HCC tissues and cells, and its expression level was correlated with survival time of HCC patients. Inhibiting the expression of miR-589-5p decreased the proliferation ability of HCC cells.3.MIG-6 is a potential target gene of miR-589-5p.