MicroRNA-371 Regulates The Cell Cycle Of Human Hepatoma Cell Line QGY-7703

Aims:MicroRNAs(miRNAs)are a class of small non-coding RNAs that post-transcriptionally regulate gene expression.Recent evidences indicate that special miRNAs may involve in many physiological and pathological processes,such as cell proliferation,differentiation,apoptosis and cancer initiation and progression.Previous studies in our lab have showed that miR-373 is directly related with the growth of hepatocellular carcinoma cells.We have already known that miR-371,-372,-373 express together as a cluster,so we want to further study the expression level of miR-371 in hepatocellular carcinoma cells and its effects on cell growth and cell cycle.To explore which genes participate in miR-3 71-mediated tumor suppression,we identified its direct target genes.This study enriches our understanding of the molecular mechanisms of miR-371 in the initiation and progression of human hepatocellular carcinoma.Methods:First,we detected the differential expression of miR-371 in human hepatocellular carcinoma tissues and adjacent normal tissues by real-time RT-PCR assay.We overexpessed and/or blocked miR-371 in human hepatocellular carcinoma QGY-7703 cells,then used real-time PCR to validate the changes of miR-371.Then we used MTT assay,colony formation assay and growth curve to detect the changes of cell phenotypes of QGY-7703 cells.And the FACS was used to observe the effects of miR-371 on the cell cycle process.According to bioinformatics analysis and known gene functions,we chose the PRPF4B as the candidate target genes of miR-371.The reliability of the direct target gene was confirmed by fluorescent reporter experiment.Furthermore,the mRNA levels and protein levels of target gene in hepatocellular carcinoma cells or tissues with different expression of miR-371 were detected with real-time RT-PCR and Western blot.Finally,the function of target gene was inhibited in human hepatocellular carcinoma cells by RNA interference and the changes of cell phenotypes and cell cycle process were detected with the above assays.Also,we generated the plasmid(lacking the 3' UTR)to increase the protein expression of the target gene.The cells were transfected with the plasmid to rescue the effects of miR-371 overexpression,and the cell phenotypes were also tested.Results:Compared with normal hepatocellular tissues,miR-371 is up-expressed in hepatocellular carcinoma tissues.Blocking miR-371 decreased cell growth,while over-expression of miR-371 promoted hepatocellular carcinoma cells cell growth.FACS showed blocking miR-371 arrested cells cycle in G1 phase.The PRPF4B was identified to be a putative target gene,whose mRNA 3'-untranslated region(3'UTR)contains the potential binding site of miR-371.The fluorescent reporter experiment also confirmed that miR-371 can directly bind to the target genes mRNA 3'UTR.The mRNA and protein level of PRPF4B in in hepatocellular carcinoma cells or tissues gave the clue that miR-371 can negatively regulate the genes expression through mRNA decay.Knockdown of PRPF4B by RNA interference can promote the cell proliferation.Finally,when cells was transfected with PRPF4B and miR-371,the cell proliferation abiliby was significantly inhibit comparing to the cells transfected with pcDNA3 and miR-371,which was consistent with the results of RNA interference assays.Conclusions:Our results indicate that miR-371 functions as a tumor oncogene through altering the expression of its target gene PRPF4B in QGY-7703 cells.The elucidation of common mechanisms of miR-371 in hepatocellular carcinoma helps us to further understand cancer initiation and progression,and provides new evidences to cancer diagnosis and therapy.