沉默信息调节因子3（SIRT3）对肝癌细胞化疗药物敏感性影响的机制研究
发布时间：2018-09-12 13:27
【摘要】：目的:探讨SIRT3对肝癌细胞化疗药物敏感性影响的分子机制。方法:1通过Western blot和q PCR检测不同化疗药物作用下,SIRT3在肝癌细胞和永生化肝细胞中的表达水平。2通过MTS实验检测在化疗药物不同浓度作用下,过表达SIRT3对肝癌细胞SMMC-7721、Huh-7和PLC/PRF/5细胞活率的影响。3通过流式细胞术检测过表达SIRT3后,化疗药物处理组和对照组中肝癌细胞的凋亡比例;通过Western blot检测凋亡相关蛋白PARP和Caspase9的表达水平。4通过MTS和流式细胞术分别检测在化疗药物的作用下,沉默SIRT3对肝癌细胞SMMC-7721和Huh-7细胞活率和凋亡比例的影响。5通过Western blot检测不同浓度索拉菲尼对SIRT3在肝癌细胞中表达水平的影响,MTS和流式细胞术分别检测过表达SIRT3对肝癌细胞SMMC-7721、Huh-7和PLC/PRF/5细胞活率和凋亡比例的影响。6通过Western blot和q PCR分别检测在化疗药物处理组和对照组中,过表达SIRT3对肝癌细胞中GSTP1蛋白水平和m RNA水平的影响;以及磷酸化蛋白p-JNK、总蛋白JNK、磷酸化蛋白p-c-Jun、总蛋白c-Jun蛋白的影响;进一步通过IP实验检测过表达SIRT3对肝癌细胞SMMC-7721中GSTP1与SIRT3结合的影响。7通过流式细胞术和Western blot检测在不同化疗药物作用下,过表达GSTP1或对稳定表达SIRT3的肝癌细胞凋亡比例和凋亡相关蛋白PARP的影响。通过流式细胞术检测JNK抑制剂SP600125对HCC细胞凋亡的影响。结果:1在三种肝癌细胞SMMC-7721、Huh-7和PLC/PRF/5中,SIRT3的蛋白水平和m RNA水平明显低于永生化肝细胞MIHA;并随着化疗药物浓度的增加,SIRT3在肝癌细胞中的表达逐渐降低。2过表达SIRT3可以促进肝癌细胞SMMC-7721、Huh-7和PLC/PRF/5对三种化疗药物的敏感性。3在不同化疗药物作用下,过表达SIRT3可以增加肝癌细胞SMMC-7721、Huh-7和PLC/PRF/5的凋亡比例,并促进凋亡相关蛋白PARP和Caspase9的剪切。4 SIRT3基因沉默可以降低肝癌细胞SMMC-7721和Huh-7对化疗药物的敏感性。5在索拉菲尼的作用下,不同药物浓度可以降低肝癌细胞中SIRT3的蛋白表达水平,并且SIRT3过表达可以促进肝癌细胞SMMC-7721和Huh-7对索拉菲尼的敏感性。6在不同化疗药物作用下,过表达SIRT3可以下调肝癌细胞中GSTP1蛋白水平和m RNA水平,Western blot证明JNK和c-Jun蛋白磷酸化水平增高;进一步,IP实验证实GSTP1与JNK的结合减少。7在不同化疗药物作用下,过表达GSTP1或JNK抑制剂SP600125可以拮抗SIRT3过表达对化疗药物处理下肝癌细胞凋亡的促进作用。结论:SIRT3通过GSTP1/JNK信号通路促进肝癌细胞对化疗药物的敏感性。
[Abstract]:Objective: to investigate the molecular mechanism of the effect of SIRT3 on chemotherapeutic drug sensitivity of hepatoma cells. Methods Western blot and Q PCR were used to detect the expression of SIRT3 in hepatoma cells and immortalized hepatocytes. 2. The expression of SIRT3 in hepatoma cells and immortalized hepatocytes was detected by MTS assay. The effect of overexpression of SIRT3 on the viability of SMMC-7721,Huh-7 and PLC/PRF/5 cells. 3 after overexpression of SIRT3 by flow cytometry, the apoptotic ratio of HCC cells in chemotherapeutic treatment group and control group was detected. Expression of apoptosis-related proteins PARP and Caspase9 were detected by Western blot. MTS and flow cytometry were used to detect the expression of apoptosis-related proteins under the action of chemotherapeutic drugs, respectively. Effects of silencing SIRT3 on the viability and apoptosis of SMMC-7721 and Huh-7 cells the effect of different concentrations of Solafenil on the expression of SIRT3 in Hepatocellular carcinoma cells by Western blot Assay and flow Cytometry The effect of SMMC-7721,Huh-7 and PLC/PRF/5 on cell viability and apoptosis in cancer cells. 6. Western blot and Q PCR were used to detect the cell viability and apoptosis in chemotherapeutic and control groups, respectively. The effects of overexpression of SIRT3 on the levels of GSTP1 protein and m RNA, and the effects of phosphorylated protein p-JNK, total protein JNK, phosphorylated protein p-c-Junand total protein c-Jun protein on the levels of GSTP1 protein and m RNA in hepatoma cells. Furthermore, the effect of overexpression of SIRT3 on the binding of GSTP1 to SIRT3 in hepatocellular carcinoma cell SMMC-7721 was detected by IP assay. 7 under different chemotherapeutic agents, flow cytometry and Western blot were used. Overexpression of GSTP1 or effect on apoptosis rate and apoptosis-related protein PARP of HCC cells stably expressing SIRT3. The effect of JNK inhibitor SP600125 on apoptosis of HCC cells was detected by flow cytometry. Results the protein level and m RNA level of sir3 in SMMC-7721,Huh-7 and PLC/PRF/5 were significantly lower than those in immortalized hepatocytes MIHA;. With the increase of chemotherapeutic drug concentration, the expression of SIRT3 in HCC cells decreased gradually, and the overexpression of SIRT3 could be promoted. The sensitivity of SMMC-7721,Huh-7 and PLC/PRF/5 to three kinds of chemotherapeutic drugs in hepatoma cells was different. Overexpression of SIRT3 could increase the apoptotic ratio of SMMC-7721,Huh-7 and PLC/PRF/5, and promote the silencing of apoptosis related protein PARP and Caspase9. 4 SIRT3 gene silencing could reduce the sensitivity of SMMC-7721 and Huh-7 to chemotherapeutic drugs. Different concentrations of drugs decreased the expression of SIRT3 protein in hepatoma cells, and the overexpression of SIRT3 could promote the sensitivity of SMMC-7721 and Huh-7 to Solafenib in different chemotherapeutic agents. Overexpression of SIRT3 could down-regulate the levels of GSTP1 protein and m RNA in hepatocellular carcinoma cells. Western blot showed that the phosphorylation of JNK and c-Jun protein was increased, and further IP assay showed that the combination of GSTP1 and JNK decreased by 7. 7 in different chemotherapeutic agents. Overexpression of GSTP1 or JNK inhibitor SP600125 could antagonize the effect of overexpression of SIRT3 on apoptosis of hepatoma cells treated with chemotherapeutic drugs. Conclusion: SIRT3 promotes the sensitivity of hepatocellular carcinoma cells to chemotherapeutic drugs through GSTP1/JNK signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2239144
[Abstract]:Objective: to investigate the molecular mechanism of the effect of SIRT3 on chemotherapeutic drug sensitivity of hepatoma cells. Methods Western blot and Q PCR were used to detect the expression of SIRT3 in hepatoma cells and immortalized hepatocytes. 2. The expression of SIRT3 in hepatoma cells and immortalized hepatocytes was detected by MTS assay. The effect of overexpression of SIRT3 on the viability of SMMC-7721,Huh-7 and PLC/PRF/5 cells. 3 after overexpression of SIRT3 by flow cytometry, the apoptotic ratio of HCC cells in chemotherapeutic treatment group and control group was detected. Expression of apoptosis-related proteins PARP and Caspase9 were detected by Western blot. MTS and flow cytometry were used to detect the expression of apoptosis-related proteins under the action of chemotherapeutic drugs, respectively. Effects of silencing SIRT3 on the viability and apoptosis of SMMC-7721 and Huh-7 cells the effect of different concentrations of Solafenil on the expression of SIRT3 in Hepatocellular carcinoma cells by Western blot Assay and flow Cytometry The effect of SMMC-7721,Huh-7 and PLC/PRF/5 on cell viability and apoptosis in cancer cells. 6. Western blot and Q PCR were used to detect the cell viability and apoptosis in chemotherapeutic and control groups, respectively. The effects of overexpression of SIRT3 on the levels of GSTP1 protein and m RNA, and the effects of phosphorylated protein p-JNK, total protein JNK, phosphorylated protein p-c-Junand total protein c-Jun protein on the levels of GSTP1 protein and m RNA in hepatoma cells. Furthermore, the effect of overexpression of SIRT3 on the binding of GSTP1 to SIRT3 in hepatocellular carcinoma cell SMMC-7721 was detected by IP assay. 7 under different chemotherapeutic agents, flow cytometry and Western blot were used. Overexpression of GSTP1 or effect on apoptosis rate and apoptosis-related protein PARP of HCC cells stably expressing SIRT3. The effect of JNK inhibitor SP600125 on apoptosis of HCC cells was detected by flow cytometry. Results the protein level and m RNA level of sir3 in SMMC-7721,Huh-7 and PLC/PRF/5 were significantly lower than those in immortalized hepatocytes MIHA;. With the increase of chemotherapeutic drug concentration, the expression of SIRT3 in HCC cells decreased gradually, and the overexpression of SIRT3 could be promoted. The sensitivity of SMMC-7721,Huh-7 and PLC/PRF/5 to three kinds of chemotherapeutic drugs in hepatoma cells was different. Overexpression of SIRT3 could increase the apoptotic ratio of SMMC-7721,Huh-7 and PLC/PRF/5, and promote the silencing of apoptosis related protein PARP and Caspase9. 4 SIRT3 gene silencing could reduce the sensitivity of SMMC-7721 and Huh-7 to chemotherapeutic drugs. Different concentrations of drugs decreased the expression of SIRT3 protein in hepatoma cells, and the overexpression of SIRT3 could promote the sensitivity of SMMC-7721 and Huh-7 to Solafenib in different chemotherapeutic agents. Overexpression of SIRT3 could down-regulate the levels of GSTP1 protein and m RNA in hepatocellular carcinoma cells. Western blot showed that the phosphorylation of JNK and c-Jun protein was increased, and further IP assay showed that the combination of GSTP1 and JNK decreased by 7. 7 in different chemotherapeutic agents. Overexpression of GSTP1 or JNK inhibitor SP600125 could antagonize the effect of overexpression of SIRT3 on apoptosis of hepatoma cells treated with chemotherapeutic drugs. Conclusion: SIRT3 promotes the sensitivity of hepatocellular carcinoma cells to chemotherapeutic drugs through GSTP1/JNK signaling pathway.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
【参考文献】
相关期刊论文 前2条
1 陶颖;陈娟;;沉默信息调节因子3对肝癌细胞增殖的影响[J];南方医科大学学报;2016年02期
2 Bo Zhang;Lei Qin;Cui-Jie Zhou;Ye-Lu Liu;Hai-Xin Qian;Song-Bing He;;SIRT3 expression in hepatocellular carcinoma and its impact on proliferation and invasion of hepatoma cells[J];Asian Pacific Journal of Tropical Medicine;2013年08期
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