MiR-96Promotes Cell Proliferation And Clonogenicity Of HepG2Cells By Targeting FOXO1and FOXO3a

Objective Hepatocellular carcinoma (HCC), the major type of liver cancer, is the fifth mostfrequent neoplasm and the third most common cause of cancer-related death in the world,especially in Asia and sub-Sahara Africa. Epidemiological studies have demonstrated thathepatitis B virus (HBV) infection is one of the key risk factors for HCC. In westerncountries it commonly develops in cirrhotic liver. MicroRNAs (miRNAs) are evolutionarilyconserved, small, noncoding RNA molecules, approximately22nucleotides in length,which are cleaved from a70-to80-nt partially duplexed precursor (pre-miRNA). MiRNAsregulate post-transcriptional gene expression by typically interacting with sequences withinthe3`-untranslational region (3`-UTR) of the target mRNA and play important roles in avariety of biological processes, including cell cycle, cell differentiation, proliferation andapoptosis. MiR-96has been recognized as an oncogene that is upregulated in several typesof cancer. In HCC, the role of miR-96remains largely unknown. Recent results identifedthe deregulation of miR-96as signifcantly associated with different risk factors of HCC,HBV infection, and alcohol consumption. Moreover, it was found that miR-96wassignifcantly upregulated in HCC cell lines. FOXO transcription factors are key tumorsuppressors in mammalian cells. Recent evidence suggests that the FOXO family oftranscription factors is targets for regulation by miRNAs. The level of miR-96wassignificantly upregulated in endometrial cancer compared with normal endometrium andoverexpression of miR-96effectively downregulated FOXO1(Forkhead box O1) expression in endometrial cancer cells. The expression of miR-96was also markedlyupregulated in breast cancer cells and breast cancer tissues compared with normal breastepithelial cells (NBEC) and normal breast tissues. Further study demonstrated that miR-96downregulated FOXO3a (Forkhead box O3a) expression in breast cancer cells by directlytargeting the FOXO3a3`-untranslated region. On the basis of these fndings, miR-96wasselected for further investigation in the present study and we hypothesized that miR-96could influence the biological behaviors of HCC cells and affect the expression of FOXO1and FOXO3a in HCC cells. The present study was aimed to test this hypothesis.Methods We examined the expression of miR-96in HepG2cells and primary humanhepatocytes (PHHC) by using real-time quantitative PCR. To explore the function ofmiR-96we performed inhibition study of miR-96by transfecting HepG2cells with miR-96inhibitor (anti-miR-96). The inhibition effect of anti-miR-96was evaluated by TaqmanmiRNA RT-PCR assay. The biology behaviors of HepG2cells transfected withanti-miR-96and negative control were analysed by cell proliferation assay, cell migrationassay, colony formation analysis, anchorage-independent growth assay and flow cytometryanalysis of cell cycle and apoptosis. The expressions of FOXO1and FOXO3a in HepG2cells transfected with anti-miR-96and negative control were detected by SYBR GreenRealTime PCR and western blot. Finally, we assessed effect of FOXO1and FOXO3asilence by siRNAs on proliferation, migration, cell cycle, apoptosis and colony formationof HepG2cells.Results Compared with PHHC cells, much higher expression of miR-96was detected inHepG2cells. Anti-miR-96actually inhibited the level of miR-96in HepG2cells. Themalignancy potential of tumor cells is refected by proliferation, migration and the amountof colony formation. We found that inhibition of miR-96could inhibit the proliferation andmigration of HepG2cells. Moreover, our results revealed that colonies formed in soft agarwere markedly smaller and fewer in the anti-miR-96group than those formed in theanti-miR-NC group. The same result was observed in co lony formation assay. That means the malignancy potential of HepG2cells is strengthened by miR-96. Inhibition of miR-96could upregulate the expression of FOXO1and FOXO3a in HepG2cells. Inhibition ofFOXO3a and FOXO1by siRNAs could promote the proliferation and colony formation ofHepG2cells. Downregulation of FOXO3a leaded to the formation of larger and morecolonies of HepG2cells in soft agar.Conclusions MiR-96could suppress the expression of FOXO3a and FOXO1that areconsidered key tumor suppressors and promote the proliferation, migration andclonogenicity of liver cancer cells. Further research is needed to explore the precisemolecular mechanism underlying this process. However, These findings suggest thatupregulation of miR-96may play an important role in promoting carcinogenesis andprogression of liver cancer.