TIGAR在肝癌细胞对表阿霉素化疗耐受中的作用及相关机制研究

发布时间：2018-06-19 21:59

本文选题：TIGAR + 凋亡　；参考：《苏州大学》2015年博士论文

【摘要】：目的:观察化疗药表阿霉素对肝癌细胞p53诱导的糖酵解和凋亡调节子(TIGAR)蛋白表达的影响以及TIGAR在肝癌细胞对表阿霉素化疗耐受中的作用;探讨TIGAR影响肝癌细胞对表阿霉素耐药的初步机制。方法:Western blot法检测表阿霉素对肝癌细胞TIGAR蛋白表达的影响;采用小干扰RNA技术干扰肝癌细胞TIGAR表达后通过MTT及克隆形成实验分别检测干扰TIGAR表达联合表阿霉素治疗对肝癌细胞生存的短期和长期影响;Annexin V-FITC/PI双染流式细胞术检测干扰TIGAR表达联合表阿霉素治疗对肝癌细胞凋亡的影响;采用慢病毒构建的TIGAR-sh RNA感染Hep G2肝癌细胞,经嘌呤霉素筛选,western blot及细胞免疫荧光法检测干扰效率,得到稳转株后通过裸鼠成瘤实验检测干扰TIGAR表达对肝癌肿瘤形成、生长及其对表阿霉素化疗疗效的影响;2’,7’-二氯二氢荧光素双醋酸盐(DCFH2-DA)标记流式细胞术、GSH检测试剂盒及NADPH检测试剂盒分别检测细胞内活性氧(ROS)水平、GSH/GSSG比值及NADPH水平;Western blot检测细胞内凋亡相关蛋白水平;细胞免疫荧光法、western blot及电镜技术检测干扰TIGAR表达对肝癌细胞自噬的影响;彗星实验检测细胞DNA损伤情况;采用细胞核质分离技术分离细胞核后western blot分别检测胞浆及胞核中TIGAR的表达;运用细胞免疫荧光及western blot技术检测DNA损伤修复中相关蛋白ATM、Cdk5等的表达。结果:为了研究表阿霉素对肝癌细胞TIGAR表达的影响,我们采用不同浓度的表阿霉素作用肝癌Hep G2细胞不同时间后,western blot检测TIGAR表达,结果显示,低浓度短时间的表阿霉素作用可诱导Hep G2肝癌细胞TIGAR表达,高浓度长时间的表阿霉素作用后TIGAR表达下调。且表阿霉素诱导的TIGAR表达是依赖p53变化的。为了进一步探讨表阿霉素诱导的肝癌细胞TIGAR高表达在肝癌细胞对表阿霉素疗效中的作用,我们采用小干扰RNA干扰肝癌Hep G2细胞TIGAR表达后通过MTT、克隆形成实验及流式等体外实验检测发现,干扰TIGAR表达联合表阿霉素作用后肝癌Hep G2细胞的存活明显下降,细胞凋亡显著增加;此外,通过体内实验即裸鼠成瘤实验检测干扰TIGAR表达对肝癌细胞成瘤的影响以及移植瘤对表阿霉素疗效的影响,结果显示,干扰TIGAR表达抑制肝癌的形成和生长,同时也增加了其对表阿霉素的化疗敏感性。研究还发现,不仅对于肝癌细胞,表阿霉素也增加了肺癌A549细胞的TIGAR表达且干扰TIGAR表达增加肺癌A549细胞对表阿霉素的化疗敏感性。肿瘤的化疗耐受与多种机制相关,其中ROS适应性反应、细胞自噬及细胞DNA损伤修复在肿瘤化疗耐药的形成中起着重要作用,TIGAR作为p53下游基因,通过抑制细胞糖酵解及增加磷酸戊糖通路产生NADPH降低细胞内ROS水平,从而降低ROS引起的细胞凋亡、ROS介导的细胞自噬激活及ROS诱导的DNA损伤修复,此外,磷酸戊糖通路的激活增加了DNA合成原料核苷酸的生成,有利于受损DNA的修复。因此,为了进一步探讨干扰TIGAR表达在肝癌细胞对表阿霉素化疗增敏中的机制,我们从细胞凋亡、细胞自噬及细胞DNA损伤修复三方面来进行研究。第一,干扰TIGAR表达显著增加表阿霉素诱导的肝癌细胞凋亡,并与ROS水平增加、Caspase3激活相关。采用NADPH或NAC清除细胞ROS水平或采用Z-VAD-FMK抑制Caspase3后可部分减轻细胞凋亡。提示,干扰TIGAR表达增加肝癌细胞对表阿霉素化疗敏感性与干扰TIGAR表达增加细胞凋亡有关。第二,干扰TIGAR表达显著增加表阿霉素诱导的肝癌细胞自噬激活,表现为LC3I向LC3 II转化增加、自噬体增多及自噬底物p62蛋白水平的下调。干扰TIGAR表达增加表阿霉素诱导的肝癌细胞自噬激活通过增加细胞ROS水平及抑制m TOR通路来实现,NADPH清除ROS后可部分抑制干扰TIGAR表达联合表阿霉素作用激活的自噬。采用3-MA或干扰Atg5抑制细胞自噬后进一步增加了干扰TIGAR表达联合表阿霉素作用所致的肝癌细胞凋亡。提示,干扰TIGAR表达增加表阿霉素诱导的肝癌细胞自噬激活对细胞的生存起保护作用,能够抵抗表阿霉素的毒性作用,在此基础上抑制细胞自噬能得到额外的抗肿瘤效果。即干扰TIGAR诱导的细胞自噬激活降低肝癌细胞对表阿霉素的化疗敏感性。第三,TIGAR增加磷酸戊糖通路产生NADPH减少细胞ROS从而减少DNA损伤,此外,产生的戊糖作为DNA合成原料有利于受损DNA的修复。表阿霉素除了诱导肝癌细胞TIGAR表达外还促进其入核,调控肿瘤细胞DNA损伤修复。干扰TIGAR表达显著增加表阿霉素诱导的肝癌细胞DNA损伤,当给予磷酸戊糖途径产生的戊糖、NADPH或ROS清除剂NAC后可部分缓解DNA损伤。提示干扰TIGAR表达除了通过抑制磷酸戊糖通路增加表阿霉素诱导的肝癌细胞DNA损伤外,还有其他机制的存在。进一步实验研究显示,TIGAR通过调节Cdk5来磷酸化DNA损伤修复关键蛋白ATM进而调控细胞DNA损伤修复。综上,TIGAR通过磷酸戊糖途径及Cdk5-AMT信号通路来调控表阿霉素对肝癌细胞DNA的损伤与修复进而影响肝癌细胞对表阿霉素的化疗敏感性。结论:表阿霉素诱导肝癌细胞TIGAR表达;干扰TIGAR表达增加肝癌细胞对表阿霉素的化疗敏感性,其机制涉及细胞凋亡增加、自噬水平激活以及细胞DNA的损伤与修复。TIGAR表达在肝癌细胞对表阿霉素耐受中的作用提示TIGAR成为抗肿瘤治疗作用靶点的可能,为临床抗肿瘤治疗尤其是对表阿霉素耐受的肝癌治疗提供理论基础及依据。
[Abstract]:Objective: To observe the effect of epirubicin on the expression of glycolysis and apoptosis regulator (TIGAR) induced by p53 in hepatoma cells and the role of TIGAR in the tolerance of liver cancer cells to adriamycin chemotherapy, and to explore the mechanism of TIGAR influence on the resistance of liver cancer cells to adriamycin. Methods: Western blot assay was used to detect epirubicin to liver cancer. The effect of the expression of TIGAR protein in cells; using small interference RNA technique to interfere with the expression of TIGAR in hepatoma cells, the short-term and long-term effects of TIGAR expression combined with epirubicin on the survival of hepatoma cells were detected by MTT and clonogenic experiments, and Annexin V-FITC/PI double dye flow cytometry interfered with TIGAR expression combined with epirubicin The effect of treatment on the apoptosis of hepatoma cells; TIGAR-sh RNA infected by lentivirus, Hep G2 hepatoma cells, the screening of purinomycin, Western blot and cell immunofluorescence method to detect the interference efficiency. After the stable transgenic mice were obtained, the interference of TIGAR expression to the formation of the cancer of the liver cancer, the growth and the chemotherapy of doxorubicin on the liver cancer were detected. Effect: 2 ', 7' - two chlorine two fluorescein diacetate (DCFH2-DA) labeled flow cytometry, GSH detection kit and NADPH detection kit to detect intracellular reactive oxygen species (ROS) level, GSH/GSSG ratio and NADPH level, Western blot detection of intracellular apoptotic phase protein level, cell immunofluorescence, Western blot and electron microscopy The effect of interfering TIGAR expression on autophagy of hepatoma cells was detected; Comet assay was used to detect DNA damage in cells; Western blot was used to detect the expression of TIGAR in cytoplasm and nucleus after cell nuclear separation technique, and cell immunofluorescence and Western blot were used to detect the associated protein ATM, Cdk5, etc. in DNA damage repair. Results: in order to study the effect of epirubicin on the expression of TIGAR in hepatoma cells, we used Western blot to detect TIGAR expression after different concentrations of epirubicin on the liver cancer Hep G2 cells. The results showed that the low concentration of epirubicin could induce the TIGAR expression of Hep G2 hepatoma cells, and the high concentration and long time table could be used. The expression of TIGAR was down regulated by doxorubicin, and the expression of epirubicin induced TIGAR was dependent on p53. In order to further explore the effect of TIGAR high expression of epirubicin induced hepatoma cells in the therapeutic effect of hepatoma cells on epuxorubicin, we used small interference RNA to interfere with the expression of TIGAR in Hep G2 cells to form solid clones through MTT. In vitro test and flow test, it was found that the survival of Hep G2 cells in HCC significantly decreased and apoptosis increased significantly after TIGAR expression combined with epirubicin. In addition, the effects of interfering TIGAR expression on the tumor formation of hepatoma cells and the effect of xenograft on adriamycin effect were detected by in vivo experiments. The results show that interfering TIGAR expression inhibits the formation and growth of liver cancer and also increases its sensitivity to epuxorubicin. The study also found that epirubicin also increased the TIGAR expression of lung cancer A549 cells and interfered with the expression of TIGAR in lung cancer cells and increased the chemosensitivity of A549 cells to epirubicin. Tolerance is related to a variety of mechanisms, in which ROS adaptive response, cell autophagy and cell DNA damage repair play an important role in the formation of chemotherapeutic drug resistance. TIGAR, as a downstream p53 gene, reduces cell apoptosis induced by ROS by inhibiting cell glycolysis and increasing NADPH in pentose phosphoric acid pathway to reduce the level of intracellular ROS induced by ROS, ROS Mediated cell autophagy activation and ROS induced DNA damage repair. In addition, the activation of pentose phosphate pathway increases the formation of DNA synthesis nucleotides and is beneficial to the repair of damaged DNA. Therefore, in order to further explore the mechanism of interference TIGAR expression in the chemosensitivity of hepatoma cells to adriamycin chemotherapy, we are from cell apoptosis and autophagy. Three aspects of cell DNA damage repair were studied. First, interfering TIGAR expression significantly increased the apoptosis of HCC induced by epirubicin, and was related to the increase of ROS level and Caspase3 activation. NADPH or NAC was used to remove the cell ROS level or to reduce the apoptosis after the use of Z-VAD-FMK to inhibit Caspase3. It suggested that the expression of TIGAR increased in TIGAR. The chemosensitivity of hepatoma cells to epuxorubicin was associated with increased cell apoptosis by interfering TIGAR expression. Second, interfering TIGAR expression significantly increased autophagy induced by epirubicin induced hepatoma cells, showing an increase in LC3I to LC3 II, the increase of autophagosome and the downregulation of p62 protein levels of autophagic substrates. Interference with TIGAR expression increased epirubicin induced induction. Autophagy of hepatoma cells is activated by increasing cell ROS level and inhibiting the m TOR pathway. NADPH scavenging ROS can partially inhibit the interference of TIGAR expression and autophagy activated by the action of epirubicin. 3-MA or interference Atg5 inhibits autophagy of the cells to further increase the interference of the TIGAR table to the action of the combined adriamycin action. It is suggested that the interference of TIGAR expression to increase the autophagy induced by epirubicin induced activation of autophagy can protect the survival of the cells, and can resist the toxicity of epirubicin. On this basis, the inhibition of autophagy by cell autophagy can result in an additional anti-tumor effect. That is, interfering with autophagy induced by TIGAR to reduce hepatoma cells to opiophos Third, TIGAR increase the pentose phosphate pathway to produce NADPH to reduce cell ROS and reduce DNA damage. In addition, the pentose produced as a DNA synthetic material is beneficial to the repair of damaged DNA. Epirubicin also promotes its nucleation in addition to inducing the TIGAR expression of hepatoma cells and regulates the repair of DNA damage in tumor cells. Significantly increased DNA damage induced by epirubicin, when pentose pentose produced by pentose phosphate pathway was given, NADPH or ROS scavenger NAC could partially alleviate DNA damage. It suggests that there are other mechanisms in the presence of interference TIGAR expression in addition to increasing the DNA damage induced by epirubicin pathway by inhibiting the pentose phosphate pathway. The experimental studies have shown that TIGAR regulates the DNA damage and repair key protein ATM by regulating the phosphorylation of Cdk5 to regulate the repair of cell DNA damage. To sum up, TIGAR regulates the damage and repair of epirubicin on the liver cancer cell DNA by pentose phosphate pathway and Cdk5-AMT signaling pathway and then affects the chemosensitivity of hepatoma cells to adriamycin. Epirubicin induced the expression of TIGAR in hepatoma cells; interference with TIGAR expression increases the chemosensitivity of hepatoma cells to adriamycin, and its mechanism involves increased apoptosis, activation of autophagy, DNA damage and the role of.TIGAR expression in the tolerance of liver cancer cells to adriamycin tolerance, suggesting that TIGAR becomes an antitumor therapeutic target. It may provide a theoretical basis and basis for clinical antitumor therapy, especially for the treatment of epirubicin tolerant liver cancer.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

【共引文献】