HBX Inhibits HepG2 Cell Apoptosis By Up-regulation The Methylation Level Of PTEN And Regulation PI3K/Akt Pathway

Objective: Clarify the mechanism of HBX inhibiting the apoptosis of HepG2 cells from the perspective of PTEN methylation and PI3K/Akt pathway, lay the foundation for more in depth understanding of HBX gene in the development of HCC and to find a new target for treatment of hepatocellular carcinoma.Methods: According to whether transfect the HBX gene and adding 5-Aza-CdR treatment, the experiment was divided into five groups: transfected with pCMV(+)- HBX group,transfected with pCMV(+)-empty plasmid group, blank group(not plasmid transfection), transfected with pCMV(+)- HBX+5-Aza-CdR group,transfected with pCMV(+) empty plasmid +5-Aza-CdR group.HepG2 cells were cultured in vitro. The transient transfection of HBX gene into HepG2 cells was completed. The transfection efficiency of HepG2 cells was observated by fluorescence microscope. Flow cytometry was used to test the apoptosis of HepG2 cells.Methylation specific PCR(MSP) was used to detect the methylation level of PTEN gene. RT-PCR and Western Blot were used to detect the expression level of PTEN,DNMT3 A and p-Akt/Akt.Results: In this experiment, gene sequencing results showed that recombinant plasmid pCMV(+)-HBX was successfully constructed; Fluorescence microscopy results showed that the transfection efficiency was 80%. It could be used for subsequent experiments. FCM results showed that transfected with pCMV(+)-HBX group compared with transfected with pCMV(+)- empty plasmid group, the apoptosis rate of HepG2 cells was significantly down-regulated(P<0.05);transfected with pCMV(+)-HBX+ 5-Aza-Cd R group compared with transfected with PCMV(+)-HBX group, the apoptosis rate of HepG2 cells was significantly up-regulated(P < 0.05);transfected with pCMV(+)-empty plasmid group compared with blank group, the difference was not statistically significant(P>0.05).MSP results showed that transfected with pCMV(+)-HBX group compared with transfected with pCMV(+)- empty plasmid group, the methylation level of PTEN gene was significantly up-regulated(P<0.05), the unmethylation level of PTEN gene was significantly down-regulated(P<0.05);transfected with pCMV(+)-HBX+ 5-Aza-CdR group compared with transfected with PCMV(+)-HBX group, the methylation level of PTEN gene was down-regulated(P < 0.05), the unmethylation level of PTEN gene was up-regulated(P < 0.05); transfected with pCMV(+)-empty plasmid group compared with blank group, the difference of above were not statistically significant(P>0.05). RT-PCR and Western Blot results showed that transfected with pCMV(+)-HBX group compared with transfected with pCMV(+)- empty plasmid group, the expressions of PTEN mRNA and protein were significantly decreased, the expressions of DNMT3 A and p-Akt/Akt protein were significantly up-regulated(P<0.05); transfected with pCMV(+)-HBX+ 5-Aza-CdR group compared with transfected with PCMV(+)-HBX group, the expressions of PTEN mRNA and protein were significantly up-regulated, the expressions of DNMT3 A and p-Akt/Akt protein were significantly decreased(P < 0.05);transfected with pCMV(+)-empty plasmid group compared with blank group, the difference of above were not statistically significant(P>0.05).Conclusion: HBX inhibits the apoptosis of HepG2 cells, which the mechanism may be related with up-regulation of the methylation level of PTEN gene and regulation PI3K/Akt pathway.