MiR-1236 Targets AFP To Regulate The Growth Of Hepatocarcinoma Cells

Objective:Hepatocellular carcinoma(HCC)is a serious disease that ranks among the most common cancers worldwide,as a member of the leading causes of cancer-related death.The processes of HCC are cascade of linked sequential steps involving multiple regulated genes.the discovery of microRNAs(miRNAs)explores a new way to find the answer.miRNAs are the new family of small RNA molecules,which are proved to negatively regulate certain genes containing complementary sequences in 3'untranslated regions.MiRNAs are involved in variety of physiological and pathological progresses.Recent evidence indicates that many miRNAs function as oncogenes or tumor suppressors by regulating their target genes negatively.Alpha-fetoprotein(AFP)a major plasma protein produced by the yolk sac and the liver during fetal life,and high concentration of AFP in serum is found in connection with,primary liver cancer and teratocarcinoma.AFP has undoubtable value in the diagnosis of liver neoplasia.AFP is a endogenous protein which is closely related to tumor growth and immunity.Our lab had proved that AFP was highly related with the proliferation of HepG2 cells.The growth inhibitory effect of AFP does not result from cell apoptosis,but the delay in the G1/S transition of cell cycle.The objective of this paper is to find out the miRNAs contributing to HCC,and to reveal the mechanism through confirmation of target genes.Methods:In order to detect the role of miRNA in hepatic cancer.The bioinformatics analysis was used to predict the targets of miRNAs which targets AFP.By EGFP reporter assay the predicted gene was verified experimentally,by Real-time PCR and Western blot the mRNA and protein levels of target gene were tested.The cell was transfected pri-miR-1236 or ASO-1236 into HepG2 and QGY-7703 cells,respectively,and perform the MTT assay and the colony formation assay.After we confirmed that miR-1236 closely related to the hepatoma cells growth,we constructed a plasmid named pcDNA3/AFP which could overexpress the AFP protein in vitro to rescue the cells growth phenotpye of overexpressed miR-1236.Results:AFP was the putative target gene of miR-1236 predicted by computational target prediction algorithm.EGFP reporter carrying the 3' untranslated regions of AFP can be inhibited by miR-1236.The results of real-time PCR and Western blot shown that miR-1236 could impact the expression of AFP on mRNA and protein levels.The viability of HepG2 and QGY-7703 cells were reduced dramatically after miR-1236 was high-expressed.Ectopic expression of AFP restore the phenotype caused by miR-1236 in hepatoma cell linesConclusion:We confirmed that miR-1236 is a key regulator of in hepatic cancer.AFP plays a role in cell growth of hepatic cancer as the one of its target gene.