Research On The Role Of Hsa-miR-184 In Hepatocellular Carcinoma Cell Line

Background Hepatocellular carcinoma(HCC)is the most common malignant tumor of the digestive tract in clinical practice,ranking second in the annual mortality rate of malignant tumors in China,and seriously threatening the lives and health of our people.The current treatment strategy for hepatocellular carcinoma is a comprehensive treatment based on surgical resection.However,most patients with HCC have lost the opportunity for surgery when they are diagnosed,and hepatocellular carcinoma is prone to relapse and metastasis after surgery,which greatly limits the further improvement of the overall efficacy of HCC.Therefore,in-depth study of the pathogenesis of HCC and screening of suitable molecular targets are very important for the treatment of HCC.MicroRNA(miRNA)is a type of endogenous non-coding small RNA,which is generally about 18-25 nt in length.It mainly works at the post-transcriptional level by regulating the messenger RNA of the target gene and binding to its specific sequence in the 3'-Untranslated Region(3'-UTR).Many studies have shown that miRNAs widely affects the biological functions of tumor cell proliferation,apoptosis,invasion and metastasis.Therefore,we tried to use miRNA expression profile chip data and high-throughput sequencing data in public databases to screen miRNAs with differential expression in hepatocellular carcinoma,in order to find new therapeutic targets for liver cancer.Objective Using public databases to screen out differentially expressed miRNAs,analyze their expression levels in HCC cell lines with different invasion and metastatic potentials,and further study the role of miRNAs in the proliferation,invasion and metastasis capacity of HCC cells,providing potential for targeted treatment of HCC and theoretical basis.Research methods 1.Download the miRNA expression profile chip data(GSE22058)from the GEO database and miRNA-seq(TCGA-LIHC)data from the TCGA database,analyze those data and select the hsa-miR-184 that is up-regulated in both data sets for follow up research.2.Real-time quantitative PCR was used to determine the expression level of hsa-miR-184 in three hepatocellular carcinoma cell lines SMMC-7721,Bel-7402 and SK-HEP-1 with different invasive potential and HL-7702 normal liver cell lines,and lentivirus-mediated gene transduction technology was used to construct hsa-miR-184 overexpression and control group of SMMC-7721 cell line.3.MTT growth curve,scratch assay,Transwell invasion assay and Transwell metastasis assay were used to detect the changes in cell proliferation,migration,invasion and metastasis ability of the two groups.Result 1.Analysis of data from public databases shows that hsa-miR-184 is upregulated in both GSE22058 and TCGA-LIHC data sets,hsa-miR-30e-3p,hsa-miR-429,hsa-miR-133 b,hsa-miR-142-5p and hsa-miR-326 were down-regulated in the two data sets.We selected hsa-miR-184 for further research.2.Real-time quantitative PCR results showed that the expression level of hsa-miR-184 in various hepatocellular carcinoma cell lines was significantly higher than that in normal liver cell lines(P<0.05),and the hsa-miR-184 expression level is highest in SK-HEP-1 HCC cell line with high invasion and metastatic potential(9.397 ± 0.107),followed by Bel-7402 HCC cell line with moderate invasion and metastatic potential(8.513 ± 0.085),the lowest among SMMC-7721 HCC cell lines with low invasive metastatic potential(7.900 ± 0.072).The expression level of hsa-miR-184 gene in SMMC-7721 hepatocellular carcinoma cells overexpressed with hsa-miR-184 was significantly higher than that in the control group(3.901 ± 0.828 vs.1.014 ± 0.210,P = 0.004),and both groups of cells grew well.3.Compared with the control group,the proliferation ability of SMMC-7721 cells in the overexpression group was significantly increased(cell proliferation fold in day 5: 5.199±0.038 vs.3.464±0.012,p<0.0001),and the migration ability was enhanced(48-hour mobility: 51.830±1.260 vs.35.540±2.290,p<0.0001),the metastasis ability(metastasis rate: 1.820±0.020 vs.1.000±0.040,p<0.0001),and invasive ability(invasive rate: 1.980±0.060 vs.1.000±0.040),p<0.0001)significantly enhanced.Conclusion 1.The expression of hsa-miR-184,hsa-miR-30e-3p,hsa-miR-429,hsa-miR-133 b,hsa-miR-142-5p and hsa-miR-326 in human HCC tissues is up-regulated or down-regulated,suggesting that these micro RNAs may be involved in the occurrence and development of HCC.2.The expression of hsa-miR-184 is upregulated in hepatocellular carcinoma cell lines,and the degree of upregulation is related to the invasion and metastatic potential of hepatocellular carcinoma cell lines.Overexpression of hsa-miR-184 in SMMC-7721 hepatocellular carcinoma cell line with low invasion and metastatic potential can promote the proliferation,invasion and metastasis of this cell line,suggesting that hsa-miR-184 may be a potential target for HCC treatment.