Effect Of MiR-30A-5P On The Proliferation, Apoptosis, Invasion And Migration Of SMCC-7721 Human Hepatocellular Carcinoma Cells

Background and Objectives:Hepatocellular carcinoma is the sixth most common neoplasm and the third most frequent cause of cancer death. More than half of those people are in China. The prognosis of HCC is poor and most of patients die within months after diagnosis. Although medical science has made great progress, the metastatic mechanism remains unclear, the recurrence and metastasis are common and the 5-year survival rate is still low. Thus, Innovative therapeutic strategies are urgently needed for liver cancer treatment and will necessarily take advantage of progresses in the comprehension of the molecular pathogenesis of HCC.MicroRNAs (miRNAs), an abundant family of short, endogenous and non-coding RNAs, modulate gene expression are post-transcriptionally by binding to complementary sequences in the 3’untranslated region(UTR) of the target mRNA to induce target degradation or translational repression. It has been proved that miRNAs play an important role in HCC, such as development, proliferation, apoptosis, invasion, and metastasis. Moreover, MicroRNAs can be used as a tumor suppressor genes, both cut target cancer gene activity; Cut target can also be used as a promote cancer gene, the activity of tumor suppressor genes.Emerging data show that miRNA-30a-5p is down-regulated and could serve as a potential tumor suppressor in several cancers. However, little is known about the role and underlying molecular mechanism of action of miR-30a-5p in HCC. In this study, we investigated the potential function of miR-30a-5p in the development and progression of HCC. We found that miR-30a-5p expression was downregulated in the majority of HCC tissues. Down-regulation of miRNA-30a-5p was significantly associated with tumor development.Methods1.RT-qPCR was applied to demonstrate the expression of miRNA-30a-5p in HCC surgical specimens(hepatocellular carcinoma and adjancent non-cancerous tissues);2. RT-qPCR was applied to verify the expression of miRNA-30a-5p in different hepatoma cell lines and liver cell strain L02;3.the expression of miRNA-30a-5p in hepatoma cells was rasied by infection of miRNA-30a-5p mimics, which were loaded with miRNA sequences;4.CCK-8 assay, flow cytometry, transwell invasion assay were applied to analyze the function of miRNA-30a-5p, observing their impact on the biological behavior of hepatocellular carcinoma cell in vitro;5. Effects of miR-30a-5p on tumorigenicity of SMCC-7721 cells in vivo;6.Bioinformatics techniques were used to predict target genes of miRNA-30a-5p, western Blot was applied to validate the role of miRNA-30a-5p regulation of target genes.Results1.To explore the relationship between miR-30a-5p and HCC, RT-qPCR analysis was performed to detect the expression level of miR-30a-5p in HCC cells line(SMCC-7721, HepG2, Huh7, MHCC-97H, MHCC-97L, HCCLM3, HCCLM6, L02) and HCC samples. A significant reduction in the expression of miR-30a-5p was observed in both all HCC cell lines examined. Down-regulation of miR-30a-5p expression was highly significant in 34 of 48 human hepatocellular carcinoma (HCC) tissues compared with adjacent non-tumorous liver tissues, the expression of miR-30a-5p in 7 liver cancer cell lines,7 cell lines expressed lower levels of miR-30a-5p than normal L02 liver cells.2.To clarify the biologic function of miR-30a-5p in HCC, its mimics was transfected into SMCC-7721 cells. HCC cell proliferation was examined by CCK-8 assay, the invasion and migration were investigated by transwell methods. Apoptosis assay and cell cycle analysis using a flow cytometer, nude mouse tumorigenicity assay also was used. These data show that enhanced miR-30a-5p expression in SMCC-7721 cells by transfection with miR-30a-5p mimics decreased tumor cell proliferation, invasion, migration, and the results of flow cytometry suggest that miR-30a-5p mimics induce the S phase cell cycle arrest and decrease cell apoptosis in SMCC-7721 cells. Meanwhile miR-30a-5p suppressed in vivo tumor growth in nude mice models bearing human HCC.3. Looking for the target genes of miR-30-5p with the application of bioinformatics tools. Potential target gene expression was assessed by western blot for proteins. Bioinformatics analysis deduced DNMT3A, as the targets of miRNA-30a-5p. Western blot results showed thatSMCC-7721 cells transfected with miR-30a-5p mimics have reduced expression of target genes DNMT3A.In summary, these findings suggest the involvement of miR-30a-5p in HCC. Thus, it may be provide a new target for diagnosis and treatment of liver cancer.