The Effects And Molecular Mechanism Of Silencing MIF Gene By RNA Interference On Proliferation And Apoptosis In Hepatic Carcinoma Cell Lines

Objective: To observe the expression levels of macrophage migration inhibitory factor(MIF)in hepatic carcinoma cell lines.To investigate the effect and potential molecular mechanism of silencing MIF gene by RNA interference on biological characteristics in hepatic carcinoma cell lines.Methods: We examined the mRNA and protein levels of MIF in liver cell line L-O2 and hepatic carcinoma cell lines Huh7,Hep3 B,BEL7402,PLC,HepG2 and SMMC-7721 by using RT-qPCR and Western blot.Liposome method was used to transfect specific MIF-siRNA which were synthesized by chemical methods into hepatic carcinoma cell lines SMMC-7721 and HepG2.In the experimental group,hepatic carcinoma cells were transfected with the MIF-siRNA,and in the control group,hepatic carcinoma cells were transfected with the Con-siRNA.CCK-8 and EdU fluorescence method were used to examine the cell proliferation.Flow cytometry was used to analyze cell cycle and cell apoptosis.Cell migration ability was detected by using Transwell method.Western blot were used to detect the expression levels of MIF,Bcl-2,Bax,p53 and ERK/RSK2 signal pathway related proteins.Results: The results of RT-qPCR and Western blot showed that MIF expressed in liver cell line L-O2 and hepatic carcinoma cell lines Huh7,Hep3 B,BEL7402,PLC,HepG2 and SMMC-7721,especially higher expressed in hepatic carcinoma cell lines HepG2 and SMMC-7721.The results of CCK-8 and EdU showed that,compared with the control group,specific MIF-siRNA could significantly inhibit cell proliferation of hepatic carcinoma cell lines HepG2 and SMMC-7721.Flow cytometry result showed that,cell apoptosis rate and percent of G0/G1 cells were increased after silencing of MIF.In addition,silencing of MIF could attenuate cell migration ability.Western blot results indicated that,proteins level of Bax,p53 and GSK-3? increased significantly,and the proteins level of Bcl-2,p-ERK,p-Bad,p-RSK2 and p-GSK-3? dramatically decreased.However,the proteins level of ERK,RSK2 and Bad had no significant difference between two groups.Conclusion: The higher expression of MIF was observed in six HCC cell lines.Silencing of MIF could inhibit the cell proliferation and the cell migration,block the cells in G0/G1 phase,increase the cell apoptosis rate,decrease the protein level of Bcl-2 and increase the level of p53 and Bax.The proteins expression levels of ERK/RSK2 signaling pathway were significantly changed after MIF silencing.It can be preliminary speculated that regulating of ERK/RSK2 signaling pathway might be the mechanism of silencing MIF could inhibit cell proliferation and promote cell apoptosis in HCC cell lines.