Hepatocyte Nuclear Factor4α Induces Apoptosis Of Hepatoma Cells Via P38MAPK Signal Transduction

ObjectiveTo research effects of hepatocyte nuclear factor4a(HNF4a) on apoptosis of hepatoma cells and the relationship with p38MAPK signal transduction pathways, reveal the anti-hepatoma mechanism of HNF4a.Methods1. HepG2(hepatocellular carcinoma) cells in the logarithmic phase were collected, which were then transfected with the exogenous gene pCMVHNF4a by using cationic polymer transfection technology, while the control group is transfected with pEGFP-N1, and the culture medium was then replaced as the10%fresh DMME fetal bovine serum after24h of transfection. Fluorescence microscope was used to observe the protein expression of green fluorescence in control group after cultivating another24h, and the number of cells within the visual field was calculated under three white-light visual fields, as well as the number of cells emitting green fluorescence at the same visual fields and transfection efficiency. Moreover, the protein expression of target gene in HepG2was detected with Western blot.2. We have found that SB203580, a specific inhibitor of p38-MAPK pathway blocked the transduction pathway of p38MAPK; after staining cells with AnnexinV-FITC-PI, the flow cytometer was used to detect the apoptosis rate of each group; HepG2apoptosis rate of HNF4a group was compared with those in pCMV group and HNF4a+SB203580group. Expression levels of Caspase-3and Fas were detected by RT-PCR, while the two gene expression levels of HNF4a group and those in pCMV group and HNF4a+SB203580group were compared.Results:1. HepG2cells were transfected with HNF4a by using cationic polymer transfection technology. Protein expression of green fluorescence in control group was observed under the fluorescence microscope, and the transfection efficiency of control group was calculated as57%at48h. When using Western blot to detect the protein expression of HNF4a, it was then found the expression level of experimental group was significantly higher than that of control group, and the difference of HNF4a expression levels of both groups was statistically significant (P<0.05).2. When using flow cytometer to detect the apoptosis rate of each group, it was then found the HepG2apoptosis rate of HNF4a group was significantly increased comparing with the pCMV group and HNF4α+SB203580group, showing the significant difference (P<0.01). Expression levels of Caspase-3and Fas were detected by RT-PCR, and the two gene expression levels of HNF4a group were found to be significantly higher than pCMV group and HNF4a+SB203580group, while the difference of expression levels of Caspase-3and Fas between HNF4a group and the other two groups were statistically significant (P<0.01).Conclusion:1. As the research shows, the HNF4α expression upregulation of HepG2cells may induce the increase of their apoptosis rates.2. Also, the research shows that the HNF4a expression upregulation of HepG2cells will significantly increase the expression levels of apoptosis-related genes Caspase-3and Fas, and the apoptotic genes will be reduced after p38MAPK path is blocked by the SB203580, which indicates that the functional mechanism of HNF4a against cancer is probably associated with the p38MAPK signal transduction pathway.