| Hepatitis b virus（HBV) infection is a global public health problem.About1million people die from HBV infection every year. China is one ofthe highest HBV epidemic areas in the world. Persistent HBV infection canlead to chronic liver damage, liver failure, hepatic fibrosis, hepaticcirrhosis and hepatocellular carcinoma (HCC). Recently, there are someantiviral treatment and immunotherapy work well in HBV infecteddiseases. However, a eradication cure is still lack. The current goal ofHBV infection treatment is to maximum long-term inhibit HBVreplication, reduce hepatocytes necrosis and inflammation, delay ordecease hepatic decompensation, heptatic cirrhosis, HCC andcomplications, and improve the life quality and prolong survival time.Prohibitin(PHB), as a new type of molecular partner mainly distributed inthe cell membrane, the mitochondrial membrane and the nucleus,participates in cell proliferation, apoptosis, transcription, metabolism,aging, cellular homeostasis, mitochondrial protein folding and other important cellular biological function. PHB is involved in the occurrenceand development process of cancers, aging and degenerative diseases,diabetes, obesity and inflammatory bowel disease. iTRAQ (isotoperelative mark and absolute quantitative) is the newest technology inproteomics research, and has been widely used in the study of liverdiseases. In our previous study, we used this technique to analyze theproteomics differences between normal and chronic hepatitis B (CHB)liver biopsy. We found that PHB in liver biopsy CHB patients has asignificantly decrease. We firstly used antiviral drugs to interfere HBVreplication and detected the PHB expression. Then we transfected PHBplasmid into HepG2.2.15cells and detected the viral load. We aimed tostudy the mechanism and physiological significance of PHB lowerexpression in HBV affected liver biopsy and HepG2.2.15cells.In our study, we firstly used Realtime PCR(RT-PCR) and WesternBlotting to verify mRNA level and protein expression of PHB in liverbiopsy of patients with HBV infection and HepG2.2.15cells. The resultsshowed that PHB mRNA level and protein expression decreasedsignificantly in liver biopsy of patients with HBV infection andHepG2.2.15cells. In order to observe the influence of HBV infection onPHB expression, we add antiviral drugs(lamivudine and tenofovir) into thesupernatant of HepG2.2.15cells culture, the results showed that HBVreplication were suppressed, PHB mRNA level and protein expression are increased. To study the mechanism of PHB expression down-regulation inHBV infected liver biopsy and cells, we transfectedPCMV-tag2B-HBss、PCMV-tag2B-HBe、PCMV-tag2B-HBc、PCMV-tag2B-HBx、PCMV-tag2B-HBls and PCMV-tag2B-HBms into theHepG2cells and found that the HepG2cells which transfected withPCMV-tag2B-HBx had lower PHB expression. To observe the influence ofphysiological function of PHB expression on hepatocytes, we transfectedPHB plasmid（PCMV6-AC-GFP-PHB） into HepG2.2.15cells anddetected the changes of cell cycle, proliferation and apoptosis. Resultsshowed that the cell cycle was blocked in G1/S phase, cell proliferationwas inhibited and cell apoptosis increased. Finally, Western blotting wasused to study the PHB expression before and after the effective antiviraltreatment in the liver biopsy with HBV infection. Results showed that PHBexpression was obviously higher after effective antiviral treatment thanbefore treatment.The study showed that HBV infection is one of the causes for thedown-regulation of PHB expression in hepatic biopsy with HBVinfection and HepG2.2.15cells. PHB played a role of resistance to cellproliferation and promotion to cell apoptosis in hepatocytes. Effectiveantiviral treatment could increase the PHB expression in hepatocytes. Weprovided the theoretical basis for the necessity of antiviral therapy toHBV infected patients and for the occurrence of HCC after hepatocytes HBV infection, and offered an important scientific basis for the treatmentof drug targets in HCC. |
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