Characterization Of The Function And Molecular Mechanism Of MiR-95-3p In Hepatocarcinogenesis

Hepatocellular carcinoma(HCC)is one of the most malignant cancers and causes 662,000 deaths each year worldwide,but more than 50% of deaths occur in China.Despite achievements in improved diagnosis and surgical therapy in the past few decades,the prognosis of HCC is poor,and the 5 year survival rate of HCC patients is low.This may be due to incomplete understanding of the molecular pathogenic mechanisms of HCC.Dysregulation of cell cycle progression is a major characteristic of hepatocarcinogenesis,which leads to excessive HCC cell proliferation.Cell cycle is regulated by cyclin-dependent kinases(CDKs),whereas CDK activity is partly inhibited by CDK inhibitors including p21Waf/Cip1.The p21 protein is a well-known CDK inhibitor which belongs to the Cip/Kip family of CDK inhibitors and encoded by the CDKN1 A gene.The p21 protein inhibits the activity of cyclin-CDK2 or-CDK4 complexes and negatively modulates cell cycle progression at phase G1.It can also interact with PCNA(proliferating cell nuclear antigen)and is involved in regulation of S phase DNA replication and DNA damage repair.As an inhibitor of cell cycle and cell proliferation,p21 plays an important role in tumorigenesis.Because of the critical role of p21 in cell cycle and tumor cell proliferation,identification of new regulatory mechanisms for regulation of p21 is important for further understanding of HCC progression and for exploring new treatment and prevention for HCC.MicroRNAs(miRNAs)are a group of noncoding RNA molecules that are approximately 18~25 nucleotides in length and negatively regulate the expression of downstream target genes mainly through direct interaction with the 3'-untranslated regions(3'-UTR)of their corresponding mRNA targets.MiRNAs generally decrease target gene expression through mRNA degradation and/or translational suppression.Increasing evidence in the recent years indicates that many microRNAs play critical roles in tumorigenesis and progression.To elucidate new regulatory mechanisms for heptocarcinogenesis,we investigated the regulation of p21 in HCC.The expression level of p21 is decreased with the progression of HCC.Luciferase assays with a luciferase-p21-3'UTR reporter and its serial deletions identified a 15-bp repressor element at the 3'-UTR of CDKN1 A,which contains a binding site for miR-95-3p.Mutation of the binding site eliminated the regulatory effect of miR-95-3p on p21 expression.Posttranscriptional regulation of p21 expression by miR-95-3p is mainly on the protein level(suppression of translation).Overexpression of miR-95-3p in two different HCC cell lines,HepG2 and SMMC7721,promoted cell proliferation,cell cycle progression and cell migration,whereas a miR-95-3p specific inhibitor decreased cell proliferation,cell cycle progression and cell migration.The effects of miR-95-3p on cellular functions were rescued by overexpression of p21.Overexpression of miR-95-3p promoted cell proliferation and tumor growth in HCC xenograft mouse models.Expression of miR-95-3p was significantly higher in HCC samples than in adjacent non-cancerous samples.These results demonstrate that miR-95-3p is a potential new marker for HCC and regulates hepatocarcinogenesis by directly targeting CDKN1A/p21 expression.