The HBX Protein-mediated Autophagy Effects And Mechanisms Of Multidrug Resistance

Hepatic carcinoma is one of the common and high malignancy malignant tumors inChina. Chemotherapy is an important treatment for liver cancer, but hepatic carcinoma isprone to be multidrug resistance (multidrug resistance, MDR). In the pathogenesis of HCC,hepatitis B virus (HBV) infection is an important factor in the occurrence of hepatocellularcarcinoma. Hepatitis B virus HBX gene integrated into the host cell as a function of theimportant gene, is the important factor that influences the biological characteristics ofhepatocellular carcinoma. Previous studies have shown that HBX-mediated and involvedin hepatic carcinoma multidrug resistance. But at the same time we also found thatapoptosis is not the only way of liver cancer cell death regulation of apoptosis level not fullintervention in hepatoma cell MDR. Therefore, we need to explore further from the pointof view of non-apoptotic to find new possible mechanism for HBX induced liver cancercells resistant to the comprehensive interpretation of the molecular mechanisms of HCCMDR. Autophagy as eukaryotic cells that are peculiar for the phenomenon of life, hasproved its participation in tolerance regulation of tumor cells to chemotherapeutic agents.The tumor cells by autophagy protective to exclude damaged proteins or organelles,isolation of hazardous substances, to speed up the circulation of macromolecules, leadingto tumor cells expressing the resistance phenotype. The body through this a special way tomaintain the stability of the internal environment. Signal pathway existence peripheralenvironment changes within cells, G interference (G α-interacting α protein protein, GAIP)as essential autophagy regulation upstream protein involved in the regulation of autophagy,changes of tumor cells. Therefore, we need to further explore the the HBX protein ismediated Gα interfere with protein expression changes the level of cellular autophagy,thereby affecting the change of the multidrug resistance phenotype of the tumor cells. Part1The HBX protein after transfection of HepG2humanhepatoma cell resistance to chemotherapeutic drugsObjective: to explore the impact of HBX protein multidrug resistance ofhepatocellular carcinoma cells, play a regulatory role on the expression of the multidrugresistance protein.Methods: Culture and identification of the HepG2human hepatoma cell lines stablyexpressing HBX protein. MTT assay was observed of HBX protein after transfection ofhepatoma cell line resistance to chemotherapy drugs. Western blotting was used to detectthe expression of the multidrug resistance protein p170.Results: HepG2human hepatoma cell lines stably expressing HBX proteinidentification and nurturing. HepG2cells stably transfected with HBX proteinhydrochloride doxorubicin Star and cisplatin chemotherapy sensitivity is low, there isobvious resistance, significantly higher expression of multidrug resistance protein andP170(expression of MDR-1transmembrane glycoprotein) in the control group, thedifference was statistically significant (P <0.01).Conclusions: The HBX stably expressing by protein HepG2cells impact thebiological behavior. HBX protein, stable transfection of HepG2cells is more sensitivethan ordinary HepG2cells for a variety of chemotherapy drugs reduce the upregulation ofthe multidrug resistance protein p170, suggesting that HBX protein is involved in andenhance the efflux of chemotherapy drugs, the experimental group cells appear resistantenhanced the phenomenon.Part2The HBX protein of the regulatory role of the level ofautophagy in hepatoma cellsObjective: Explore the role of the HBX protein on the regulation of the level ofhepatoma cell autophagy.Methods: Western blotting was used to detect the the HBX protein on the regulationof autophagy protein Becline1, LC3-Ⅱ expression.Results: The autophagy protein LC3-Ⅱ and Beclin1expression in HepG2cells stably transfected with HBX protein is more than the control. the difference was significant (P <0.01).Conclusions: The HBX protein is consideratd to regulate the levels of autophagy inliver cancer cells. HBX protein stably transfected HepG2cells as compared with ordinaryHepG2cells autophagy-related protein Beclin1and LC3-Ⅱ expression were increased,indicating that the upregulation of autophagy.Part3Change the level of autophagy impact hepatoma cellsensitivity to chemotherapeutic drugsObjective: To observe the effect of cell changes on the chemosensitivity of multidrugresistance protein p170expression in hepatocellular carcinoma cells after the change ofautophagy.Methods: Upregulation of autophagy rapamycin and down-regulation of3-MA,autophagy lysosomal inhibitor E64in hepatoma cells of two groups, Western blottingmethod and MTT method was used to detect the effects of different levels of autophagy onchemosensitivity of human hepatocellular carcinoma cells.Results: Autophagy up-regulation of the HepG2cells decreased sensitivity tochemotherapeutic drugs, upregulate the expression of multidrug resistance protein p170(P<0.05), the resistance has increased; and the inhibition of E-64,3-MA on autophagyhepatoma cells increased the sensitivity of HepG2cells to chemotherapy, multidrugresistance protein p170expression down-regulated (P<0.01), decreased drug resistance.Conclusions: The autophagy level change regulation of chemosensitivity ofhepatocellular carcinoma cells, demonstrated that the HBX protein mediated autophagy isinvolved in multidrug resistance. Part4THE GAIP induced autophagy in HCC cells in HBX proteinObjective: To explore HBX protein regulating effect on autophagy cell upstream G αinterference protein.Methods: Quantitative PCR and Western blotting are used to detected the expressionof GAIP in HBX protein induced liver cancer cell autophagy.Results: Stable transferred HBX protein of HepG2cells from macrophages regulatingprotein GAIP expression at by mRNA and protein levels were significantly increased,prompting HBX protein may be raised since macrophages regulating protein GAIP ofexpression, compared with control group differences in sexual significant significantly (P<0.01).Conclusions: HBX protein can upregulate autophagy mediated protein GAIPexpression.