|关键词||HCV NS5A 自噬 凋亡 Beclin 1 NS5ATP9|
|其他题名||The mechanism of HCV non-structural protein 5A induced autophagy and inhibited apoptosis|
研究目的：丙型肝炎病毒（hepatitis C virus，HCV）是世界范围内引起急性和慢性肝病的主要因素之一。慢性HCV感染者有发展为肝硬化和肝癌的高风险。丙型肝炎病毒的致病性除了病毒本身因素外，病毒蛋白与宿主细胞蛋白的相互作用对HCV的致病发挥重要作用。HCV非结构蛋白5A（NS5A）通过与细胞蛋白的相互作用在信号转导、胞内物质转运、抗细胞凋亡方面发挥了重要作用。多项研究报道HCV感染细胞后会出现自噬泡的累积，HCV需要自噬蛋白以前病毒因子的角色参与其复制，自噬对HCV的生活周期发挥着不可或缺的作用。文献报道NS5A可以独立诱导细胞自噬的发生，但是机制还不清楚；NS5ATP9是本课题组利用抑制性消减杂交技术发现的NS5A反式激活的基因，它在NS5A致病机制中发挥什么样的作用还尚未研究清楚，而NS5A也可以抑制细胞凋亡的发生，但尚无文献报道与细胞自噬相关。本课题在第一部分探索了HCV NS5A诱导细胞自噬的机制，探索NS5ATP9在NS5A诱导细胞自噬过程中发挥的作用。第二部分探索了NS5A抑制饥饿诱导细胞凋亡的机制，探索 NS5A介导的细胞自噬对饥饿诱导的细胞凋亡所起的作用。
1. 为了研究NS5A对细胞自噬的诱导机制，构建了自噬重要基因Beclin 1双荧光素酶报告基因载体，检测NS5A对Beclin 1启动子活性的影响。
2. 为了验证NS5A可以诱导细胞自噬的发生，建立瞬时表达NS5A蛋白的细胞模型，利用Western blot，激光共聚焦荧光成像技术检测自噬相关蛋白的表达和自噬泡形成情况。
3. 为了验证NS5ATP9可以诱导细胞自噬的发生，建立瞬时表达NS5ATP9蛋白的细胞模型，利用Western blot，激光共聚焦荧光成像技术检测自噬相关蛋白的表达和自噬泡形成情况。
4. 为了研究NS5ATP9对细胞自噬诱导的作用机制，检测了NS5ATP9对Beclin 1启动子活性的调控。
5. 为了验证NS5ATP9作为NS5A反式上调的基因参与了NS5A对细胞自噬的诱导作用，在NS5ATP9蛋白表达缺失的细胞内表达NS5A，利用Western blot，激光共聚焦荧光成像技术检测自噬相关蛋白的表达和自噬泡形成情况。同时，也检测Beclin 1启动子活性变化及mRNA水平的变化。
6. 为了验证NS5A及NS5ATP9对细胞自噬的诱导作用是Beclin 1依赖性的，用siRNA干扰掉Beclin 1蛋白的表达，利用Western blot，激光共聚焦荧光成像技术检测自噬相关蛋白的表达和自噬泡形成情况。
1. 为了验证NS5A可以抑制饥饿诱导的细胞凋亡及促进细胞增殖，建立瞬时表达NS5A蛋白的细胞模型，利用Annexin-V/7-AAD 染色，流式细胞仪检测细胞早期凋亡和晚期凋亡的发生情况，并利用Caspase 3/7活性检测试剂盒检测Caspase 3/7活性，利用CCK-8试剂盒检测细胞增殖情况。
2. 为了探索细胞自噬对饥饿诱导细胞凋亡的影响，使用自噬抑制剂3-MA或CQ抑制细胞自噬，通过Annexin-V/7-AAD染色和Caspase 3/7活性检测试剂盒检测细胞凋亡情况，利用CCK-8试剂盒检测细胞增殖情况。
3. 为了探索NS5A诱导的细胞自噬对其抑制细胞凋亡发挥什么样的作用，对表达NS5A的细胞用3-MA或CQ处理后，通过Annexin-V/7-AAD染色和Caspase 3/7活性检测试剂盒检测细胞凋亡情况，利用CCK-8试剂盒检测细胞增殖情况。
4. 为了多重验证NS5A对细胞自噬的诱导参与了其对细胞凋亡的抑制作用，在Beclin 1蛋白表达被干扰掉的细胞内表达NS5A，通过Annexin-V/7-AAD染色和Caspase 3/7活性检测试剂盒检测细胞凋亡情况，利用CCK-8试剂盒检测细胞增殖情况。
1. HCV NS5A显著上调Beclin 1启动子活性，mRNA和蛋白的表达，诱导自噬泡形成。
2. NS5ATP9显著上调Beclin 1启动子活性，mRNA和蛋白的表达，诱导自噬泡形成。
3. NS5ATP9蛋白表达缺失的情况下，NS5A对Beclin 1启动子活性，mRNA和蛋白的表达上调作用减弱，诱导自噬泡形成受抑。
Hepatitis C virus (HCV) infection is a worldwide health problem because of its
incidence and pathogenicity. It might evolve into chronic disease, cirrhosis, and/or
hepatocellular carcinoma (HCC). HCV is a positive-strand RNA virus that replicates out-side of the nucleus and does not have any potential to integrateits genetic information into the host cell’s genome. HCV, however, has been found to be able to hijack a number of normal molecular pathways that control cell cycle. Most attention has been devoted to the interaction of various HCV non-structural proteins with cellular proteins that control proliferation. HCV nonstructural protein 5A (NS5A) has been shown to modulate multiple cellular processes, including apoptosis and autophagy. Autophagy has been shown to facilitate replication or production of HCV; nevertheless, how HCV induces autophagy remains unclear. NS5ATP9, a gene up-regulated by NS5A was first identified by suppression subtraction hybridization (SSH) technique by our group. However, whether and how NS5ATP9 participates in the regulation of the biological effects mediated by NS5A is not clearly understood. Whether NS5ATP9 plays an important role in NS5A-mediated autophagy is one of the aim of this study; another aim of this study was to assess the effects of HCV NS5A-mediated autophagy on apoptosis induced by starvation .
The first part
1. The autophagy related gene Beclin 1 promoter reporter vector was constructed.
2. HepG2 and L02 cells were transfected with pNS5A transiently, and then the mRNA and protein expression of autophagy related genes were determined. The autophagy vacuoles carrying the autophagy marker LC3 were detected by confocal.
3. HepG2 and L02 cells were transfected with pNS5ATP9 transiently, and then the mRNA and protein expression of autophagy related genes were determined. The autophagy vacuoles carrying the autophagy marker LC3 were detected by confocal.
4. Beclin 1 promoter reporter vector were co-transfected with pNS5ATP9, and the relative luciferase activity was determined.
5. To determine whether NS5ATP9 plays an important role in NS5A up-regulation of Beclin 1 and autophagy induction, NS5A was overexpressed in NS5ATP9-silenced HepG2 and L02 cells, and Beclin 1 promoter activity, mRNA expression and protein levels were analysed by luciferase activity detection, Realtime-PCR and Western blot, respectively.
6. Beclin 1 siRNA was used to knock-down Beclin 1 expression in HepG2 cell line, and autophagy levels were determined by Western blot and confocal microscopy.
The second part:
1. HepG2 cells were transfected with pNS5A transiently, and then starved for 24 and 48 h, and then stained with Annexin-V and 7-AAD. The apoptosis were determined by flow cytometer; the Caspase 3/7 activity of the starved-cells were detected by microplate luminometer; cell viability was measured by CCK-8 kit for 24 h and 48 h.
2. HepG2 cells starved for 24 h with or without 3-MA or CQ treatment for 24 h, and then determined by flow cytometer; the Caspase 3/7 activity of the starved-cells were detected by microplate luminometer, cell viability was measured by CCK-8 kit for 24 h and 48 h.
3. To determine whether autophagy-induced by NS5A plays an important role in the suppression of starvation-induced apoptosis, the NS5A-expressed cells were starved with or without 3-MA or CQ treatment for 24 h, and then determined by flow cytometer; the Caspase 3/7 activity of the starved-cells were detected by microplate luminometer, cell viability was measured by CCK-8 kit for 24 h and 48 h.
4. NS5A were expressed in Beclin 1-silenced cells, and the apoptosis levels were determined by flow cytometer or Caspase 3/7 activity detection kit, cell viability was measured by CCK-8 kit for 24 h and 48 h.
The first part
1. Exogenous overexpression of NS5A up-regulates Beclin 1and induces autophagy.
2. Exogenous overexpression and knock-down of NS5ATP9 has a reverse effect on Beclin 1 regulation and autophagy induction.
3. NS5ATP9 knock-down attenuates Beclin 1 up-regulation and NS5A-induced autophagy.
4. NS5A and NS5ATP9 enhanced autophagy is Beclin 1 dependent.
The second part
1. Exogenous overexpression of NS5A suppresses the apoptosis induced by starvation, and cell viability of HepG2 was enhanced.
2. The apoptosis cells were significantly decressed and the cell viability of HepG2 was enhanced with 3-MA/CQ-mediated blockage of autophagy.
3. The inhibition of starvation-induced apoptosis and the cell viability of HepG2 mediated by NS5A were attenuated by3-MA/CQ-mediated blockage of autophagy.
4. The inhibition of starvation-induced apoptosis and the cell viability of HepG2 mediated by NS5A were attenuated in Beclin 1-silenced cells.
1. Our results showed that NS5ATP9 could induce autophagy; NS5ATP9 plays a functional role in the induction of HCV NS5A-mediated autophagy, furthermore, we demonstrated that NS5A and NS5ATP9 enhanced autophagy is Beclin 1 dependent.
2. Autophagy-induced by NS5A plays an important role in the suppression of starvation-induced apoptosis and the promotion of cell viability in HepG2 cells. Therefore, this study has important pathological significances and clinical implications for the understanding and management of HCV-induced autophagy.
|全敏. HCV非结构蛋白5A诱导细胞自噬及抑制细胞凋亡的机制研究[D]. 北京地坛医院教学医院. 北京大学,2014.|