MIR-331-3P Was UP-Regulated By Hepatitis B Virus And Promotes Proliferation Of Hepatocellular Carcinoma Cells

ObjectiveTo investigate the function and molecule mechanism of miR-331-3p in HBV related HCC.Methods1. Different miRNA expressions in HepG2 and HepG2.2.15 cells were screened by miRNA array and verified by qRT-PCR. We chose miR-33 1-3p which was up-regulated in HepG2.2.15 cells for our next step reasch. MiR-33 1-3p expression was detected in different HBV transient or stably expression HCC cell lines.2. We constructed miR-33 1-3p promoter vector which contains the promoter of miR-33 1-3p and investigate the mechanism of up-regulation miR-331-3p by HBV.3. MTS and clone formation assay were used to verify the proliferation of SMMC7721 cells which over-expressed miR-331-3p.4. Bioinformatics website was used to search for target genes of miR-33 1-3p. Then we constructed target genes 3’-UTR vector and testified luciferase activities of 3’-UTR in miR-331-3p over-expression SMMC7721 cells by using luciferase reporter assay. qRT-PCR and Western blot analysis were used to testify the expression of target genes in miR-331-3p over-expression SMMC7721 cells.5. qRT-PCR and Western blot analysis were used to verify the expression of target genes in HBV expression HCC cells and find the association among HBV, miR-331-3p and target genes.6. ING5 stably expression cell line was constructed and investigated the potential role of ING5 in modulating the ability of HCC cells to proliferate by using MTS and clone formation assay.7. Tumor formation in nude mice and immunohistochemistry were used to testify the effect of miR-33 1-3p and ING5 on tumor growth.8. Western blot was used to detect ING5 expression in human HCC tissue accompanied with HBV infection.Results1. MiR-331-3p was up-regulated in HBV expression HCC cell lines.2. HBV promotes the expression of miR-331-3p by increasing its promoter activity.3. MiR-331-3p promotes proliferation of HCC cells.4. MiR-33l-3p inhibits VHL and ING5 expression by directly target the 3’-UTR, VHL and ING5 were the target genes of miR-33 1-3p. 5. HBV inhibits ING5 expression by up-regulate miR-33 1-3p.6. MiR-331-3p promotes tumor growth and ING5 inhibit tumor growth in nude mice.7. ING5 was down-regulated in human HCC tissue with HBV infection.ConclusionMiR-331-3p was up-regulated by HBV and promoted HCC development and progression by directly targeting ING5.