Regulation Of Alpha-fetoprotein And MicroRNA-942 On Maligant Behavious Of The Human Hepatocellular Carcinoma Cells

[Objective]Hepatocellular carcinoma(HCC)is the fifth most prevalent cancer in the world,with an annual death rate exceeding 500,000.Alpha-fetoprotein(AFP),an endogenous protein which is closely related to tumor growth and immunity,has undoubtable value in the diagnosis of liver neoplasia.Recently,reports have showed that many MicroRNAs(miRNAs)play important roles in cancer initiation and progression by regulating their target genes negatively.Previous studies in our lab have predicted that miR-942 targets AFP through 39 UTR by bioinformatics analysis.The objective of this study is to validate miR-942 targets AFP protein and regulates its expression and to study the molecular mechanisms of miR-942 in the initiation and progression of human HCC.The study will provide theoretical basis for early HCC diagnosing and special cancer therapies.[Methods]1.The bioinformatics analysis was used in our study to predict the miRNAs that target AFP.The EGFP reporter assay revealed that miR-942 directly target AFP in QGY-7703 and PLC-PRF-5 cells.Furthermore,the influences of miR-942 on the mRNA or protein level of AFP was measured by Real-time PCR or Western blot respecively.2.After overexpressing or blocking miR-942 in QGY-7703,PLC-PRF-5 and HepG2 cells respectively,the changes of cell phenotypes on cell growth,migration and invasion were measured using MTT,colony formation and transwell assays.3.After overexpressing or repressing AFP in QGY-7703,PLC-PRF-5 and HepG2 cells respectively,the changes of cell phenotypes on cell growth,migration and invasion were measured using MTT,colony formation and transwell assays.4.Rescue experiments were performed to detect the effects of cell growth,migration and invasion tlirough cotransfection of AFP and miR-942.[Results]The bioinformatics analysis showed that miR-942 targets AFP mRNA 3’UTR.The EGFP reporter experiment confirmed that miR-942 could directly bind to mRNA 3’UTR and negatively regulated AFP.The results of the mRNA level and protein level of AFP both reduced after overexpression of miR-942 in hepatic carcinoma cells by using real-time PCR and Western blot.After overexpression or repression of miR-942,the viability,proliferation,migration and invasiveness were all dramatically inhibited or inhanced in QGY-7703,PLC-PRF-5 and HepG2 cells.Furthermore,we found that proliferation activity,migration and invasion capability were increased or decreased after overexpression or knockdown of AFP.Finally,overexpression of AFP can rescue the changes of cell phenotypes caused by ectopic expression of miR-942.[Conclusion]These results demonstrated that miR-942 may function as a tumor suppressor that suppressed the proliferation activity,migration and invasion capability of hepatic carcinoma cells through negative regulation of AFP.The elucidation of the mechanisms of miR-942 helps us to understand the mechanism of initiation and progression of hepatic cancer,and provide new theoretical basis for the clinical diagnosis and therapy of liver ceancer in the future.