W Chen1, L Huang, H Liu, X-M Li, C-M Xu. 1. Department of Gastroenterology, Xiangyang No. 1 People's Hospital, Hubei University of Medicine, Xiangyang, China. chenwei4832@163.com.
Abstract
OBJECTIVE: To investigate the difference in expression level of hepatitis B virus (HBV) X gene, and to further study the difference of HBV X protein (HBx) at varied expression levels in apoptosis regulation of hepatocellular carcinoma cells. The recombinant plasmid rPX with HBV enhancer 1 (Enh1), X gene promoter, X gene and polyA tail were constructed, respectively. PATIENTS AND METHODS: HepG2 cells were transiently transfected with the recombinant plasmids and control vector pGEM®-7Zf (+) by virtue of Fugene®HD; reverse transcription PCR (RT-PCR) and Western blotting were applied to analyze the transcription and expression of HBV X gene as well as the difference in the expression level in multiple groups. The activity of the transfected cells in each group was detected by using the methyl thiazolyl tetrazolium (MTT) method for 6 consecutive days after transfection. A flow cytometer was utilized to measure the cell apoptosis rate. RESULTS: The RT-PCR results showed that messenger RNA expression of HBV X gene was detected in all HepG2 cells transfected by different recombinant plasmids, of which the relationships of the expression levels were rCX>rEX1 and rEX2> rPX (p<0.05). Only HepG2/rCX cells in each group of transfected cells showed HBx expression by Western blot. MTT method revealed that there were notable differences between HepG2/rCX, HepG2/rEX1, HepG2/rPX and HepG2/pGEM®-7Zf (+) (p<0.05). The apoptosis rates of HepG2/rCX, HepG2/rEX1 and HepG2/rPX were significantly higher than that of HepG2/pGEM®-7Zf (+) (p<0.05). CONCLUSIONS: HBx can promote cell apoptosis. Results of this research also indicate that there is a significant difference in the pro-apoptotic role of HBx when its expression is regulated by different promoters, and such a difference may be a part of the complex pathogenic mechanisms of HBV and hepatocellular carcinoma.
OBJECTIVE: To investigate the difference in expression level of hepatitis B virus (HBV) X gene, and to further study the difference of HBV X protein (HBx) at varied expression levels in apoptosis regulation of hepatocellular carcinoma cells. The recombinant plasmid rPX with HBV enhancer 1 (Enh1), X gene promoter, X gene and polyA tail were constructed, respectively. PATIENTS AND METHODS: HepG2 cells were transiently transfected with the recombinant plasmids and control vector pGEM®-7Zf (+) by virtue of Fugene®HD; reverse transcription PCR (RT-PCR) and Western blotting were applied to analyze the transcription and expression of HBV X gene as well as the difference in the expression level in multiple groups. The activity of the transfected cells in each group was detected by using the methyl thiazolyl tetrazolium (MTT) method for 6 consecutive days after transfection. A flow cytometer was utilized to measure the cell apoptosis rate. RESULTS: The RT-PCR results showed that messenger RNA expression of HBV X gene was detected in all HepG2 cells transfected by different recombinant plasmids, of which the relationships of the expression levels were rCX>rEX1 and rEX2> rPX (p<0.05). Only HepG2/rCX cells in each group of transfected cells showed HBx expression by Western blot. MTT method revealed that there were notable differences between HepG2/rCX, HepG2/rEX1, HepG2/rPX and HepG2/pGEM®-7Zf (+) (p<0.05). The apoptosis rates of HepG2/rCX, HepG2/rEX1 and HepG2/rPX were significantly higher than that of HepG2/pGEM®-7Zf (+) (p<0.05). CONCLUSIONS:HBx can promote cell apoptosis. Results of this research also indicate that there is a significant difference in the pro-apoptotic role of HBx when its expression is regulated by different promoters, and such a difference may be a part of the complex pathogenic mechanisms of HBV and hepatocellular carcinoma.