| Background:In the world today, tumor is the second cause of death. With the characteristics of high degree of malignancy, rapid development, and easy to recurrent, most of the HCC patients in the hospital is already late to have the best opportunity to operation, and hepatocellular carcinoma to chemotherapy is also not sensitive. It is only through continuous in-depth study of the occurrence and development mechanism of cancer, can we achieve more effective method to prevention and treatment tumor.In endoplasmic reticulum, the misfolded proteins and the accumulation of misfolded proteins which caused by various reasons, is called the endoplasmic reticulum stress(ERS). If the stimulation of Endoplasmic reticulum stress is too extend, which can not only lead to cell death by apoptosis pathway, but also by the type II programmed death way- autophagy. Autophagy is regulated by the genes and pathways in the evolutionarily conserved mediated cellular self digestion, acts as a dynamic regulatory mechanism of tumor. Autophagy on differentiation and development plays a vital role, it is the cell in response to a harsh environment and pressure, but the cell survival significance is not clear.The occurrence of ERS can lead to cell apoptosis. BCL-2 is apoptosis gene was identified from the district breakpoint human recurrent follicular lymphoma translocations. Bcl-2 family proteins are not only involved in apoptosis programmed cell death, but also involved in regulation of the form of autophagic cell death of. Autophagy can prevent apoptosis induced by antitumor drugs, promoting tumor drug resistance. However, autophagic cell death cell may be a way of death apoptosis resistance of tumor cells. This experiment mainly explored in the ERS state, the difference of autophagy in cell and liver Hep G2 of normal L-02 cell function, and through the study of Bcl-2 gene, to understand the relationship between apoptosis and autophagy, in order to improve the effect of the treatment of liver cancer to find new strategies.Part 1 The different function of autophagy inhibitors between Hep G2 liver cancer cells and normal liver cells L – 02 in the endoplasmic reticulum stress stateObjective: To investigate the difference in function of autophagy inhibitors between Hep G2 and L-02 cells in the state of endoplasmic reticulum stress.Methods: The Hep G2 cells and L-02 cells were routinely cultured in vitro and treated either with tunicamycin(TM) or combination with the autophagy inhibitors 3-methyladenine(3-MA) or chloroquine(CQ). The cell viability was detected by MTT assay. Cell apoptosis was detected by flow cytometry. The change of autophagy-related protein LC3 and the anti-apoptotic protein Bcl-2 was analysed by Western blot assay.Results:MTT assay demonstrated that TM could time-dependently induce the death of Hep G2 cells and L-02 cells, and the cell viability of Hep G2 cells was significantly restrained when it administrated in combination with 3-MA or CQ, the cell survival rate of 24 h were: 3-MA+TM(60%), CQ+TM(72%), TM(86%), respectively,(P<0.01). But, it has no significant effect with L-02 cells because of its cell survival rate of 24 h were: 83%, 84% and 83%. Annexin V/PI apoptosis experiment found that: TM+3-MA, CQ+TM and TM group on Hep G2 cell apoptosis rate were 15%, 11% and 7%, respectively, and the difference was statistically significant(P<0.01). But for L-02 cell group, the apoptosis rate were 16%, 17%, 16% which had no obvious difference. According to the results of Western blot, TM could cause increasedautophagy, and autophagy inhibitor CQ can lead to increased autophagy tide. With the function of inhibitors of autophagy, the expression of Bcl-2 protein was down-regulated in Hep G2 cells, not in the L-02 cells.Conclusion:The cell viability of Hep G2 cells, not the L-02 cells, can be signif icantly restrained by TM combined with different autolysosome inhibitor 3-MA or CQ. With the function of inhibitors of autophagy, the expression of Bcl-2 pr otein was down-regulated in Hep G2 cells, but not in the L-02 cells. So the aut ophagy can protect the hepatocarcinoma cell line Hep G2 under the state of End oplasmic Reticulum Stress, it can’t provide the same protection to L-02 cell line. While the Bcl-2 gene maybe one of the molecular mechanism, which lead to t his differency.Part 2 The study of Bcl-2 gene is one of the molecular mechanisms of autophagy which provide survival on liver cancer cells Hep G2Objective: Given that the different expression of Bcl-2 gene in hepatocellular carcinoma cells Hep G2 and normal hepatic cells L-02 during endoplasmic reticulum stress(ERS), this study was designed to investigate whether Bcl-2 invole the survival protection of autophagy on liver cancer cell Hep G2.Methods: The Hep G2 cells and L-02 cells were routinely cultured in vitro and treated either with PBS(control group), combination of negative control si RNA+ tunicamycin(NC+TM), combination of Bcl-2 si RNA+ tunicamycin(si RNA+TM), and tunicamycin(TM). After 24 hours, cell viability was measured by MTT assay, cell apoptosis was detected by flow cytometry, and the anti-apoptotic protein Bcl-2 expression was analyzed by Western blotting.Results: After si RNA-mediated knockdown of Bcl-2, cell survival rate of si RNA+TM was 70% in the Hep G2 group, which was significant lower than TM group(88%)(P<0.01), while in the L-02 group, it has no significant effect on survival rate of L-02 cells because of its cell survival rate of 24 h were 86% and 81%, respectively. Annexin V/PI apoptosis experiment found that in si RNA+TM and TM group Hep G2 cell apoptosis rate were 11.17% and 7.78%, respectively, and the difference wasstatistically significant(P<0.01). But in L-02 cells, the apoptosis rate was 19.45% in si RNA+TM group and 19.89% in TM group, which had no obvious difference. According to the results of Western blotting, the expression of Bcl-2 protein in Hep G2 cells was significantly decreased in si RNA+TM group, when compared with TM group(P < 0.01). however, there were no expression of Bcl-2 in the L-02 cells.Conclusion: Under the state of ERS induced by TM, si RNA-mediated knockdown of Bcl-2 significantly reduced the survival rate of Hep G2 cells, but had no effect on the growth rate of L-02 cells in vitro. Thus, we conclude that there is differencein the role of Bcl-2 between Hep G2 and L-02 during ERS, which is one of the molecular mechanism for promoting survival of Hep G2 cells by autophaga, but not the L-02 cells. |
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