SREBP1c调节人PERK蛋白表达并通过PERK信号通路影响骨肉瘤细胞增殖、凋亡和自噬

发布时间：2018-01-26 09:44

本文关键词： PERK SREBP1c PERK信号通路内质网应激骨肉瘤细胞自噬　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的探讨固醇调节元件结合蛋白1c(sterol regulating element binding protein 1c,SREBP1c)是否与人蛋白激酶样内质网激酶(protein kinase-like endoplasmic reticulum kinase,PERK)基因启动子结合,并通过调控PERK基因转录与表达,影响骨肉瘤细胞saos2和RCS发生增殖、凋亡和自噬的可能机制。方法构建人PERK基因启动子全长及一系列截短体报告基因载体,与内参质粒p RL-SV40和pc DNA3.1-SREBP1c、pc DNA3.1-SREBP1cm共转染骨肉瘤细胞后检测荧光素酶活性;运用Ch IP和EMSA方法检测SREBP1c与PERK启动子的结合作用;应用p WPT-GFP慢病毒载体系统,构建并包装慢病毒SREBP1c、SREBP1cm和PERK。感染细胞后,通过RT-PCR和蛋白质印迹法检测骨肉瘤细胞中PERK m RNA和蛋白的表达。应用衣霉素(Tunicamycin,TM)建立内质网应激(Endoplasmic reticulum stress,ERS)模型,检测和探讨ERS状态下,SREBP1c、SREBP1cm与PERK对骨肉瘤细胞的增殖、凋亡和自噬的调控作用及可能的分子机制。结果pc DNA3.1-SREBP1c、pc DNA3.1-SREBP1cm上调PERK启动子的活性;Ch IP和EMSA证明转录因子SREBP1c直接与PERK基因启动子区域结合并调节PERK的转录与表达;成功构建并获得慢病毒SREBP1c、SREBP1cm颗粒;成功建立内质网应激(Endoplasmic reticulum stress,ERS)模型;在ERS状态下,SREBP1c、SREBP1cm分组感染后,与对照组GFP相比较,SREBP1c、SREBP1cm可以不同程度的促进骨肉瘤细胞PERK及p-PERK的表达(P0.05);过表达SREBP1c、SREBP1cm及PERK在未经TM诱导的情况下同样可以诱导内质网应激的发生;在ERS状态下,过表达SREBP1c、SREBP1cm和PERK都可以抑制骨肉瘤细胞的增殖、促进其凋亡和自噬;其中SREBP1c/1cm可以通过上调PERK的表达及磷酸化增强PERK对细胞增殖的抑制作用、促进PERK介导的细胞凋亡和细胞自噬。而在加入PERK信号通路抑制剂GSK2606414后,可以有效逆转SREBP1cm对PERK各项生物学功能的促进作用,即:GSK2606414有效逆转SREBP1cm促进PERK对细胞增殖的抑制作用、促进PERK介导的细胞凋亡和细胞自噬,证明SREBP1c调节细胞增殖、凋亡和自噬均是通过上调PERK表达及磷酸化激活相应的信号通路来实现的。结论SREBP1c转录因子通过直接与PERK基因启动子结合调节PERK基因的转录与表达;SREBP1c/1cm增强PERK对细胞增殖的抑制作用,促进PERK介导的凋亡和自噬;SREBP1c/1cm的这一作用依赖于PERK信号通路。
[Abstract]:Objective to investigate sterol regulating element binding protein 1c. Whether SREBP1c is associated with human protein kinase-like endoplasmic reticulum kinase (SREBP1c). Protein kinase-like endoplasmic reticulum kinase. Perk) gene promoter binds and regulates the transcription and expression of PERK gene to affect the proliferation of saos2 and RCS in osteosarcoma cells. Methods the full-length promoter of human PERK gene and a series of truncated reporter gene vectors were constructed. The luciferase activity of osteosarcoma cells was detected after co-transfection with p#en0# and pcDNA3.1-SREBP1cpc DNA3.1-SREBP1cm. The interaction between SREBP1c and PERK promoter was detected by Ch IP and EMSA. Using p WPT-GFP lentivirus vector system, we constructed and packaged the cells infected with lentivirus SREBP1ctr SREBP1cm and PERK. The expression of PERK m RNA and protein in osteosarcoma cells was detected by RT-PCR and Western blotting. The endoplasmic reticulum stress reticulum stress (ERS) model was established to detect and study SREBP1c under ERS condition. Effects of SREBP1cm and PERK on proliferation, apoptosis and autophagy of osteosarcoma cells and their possible molecular mechanisms. Results PC DNA3.1-SREBP1c. PC DNA3.1-SREBP1cm upregulated the activity of PERK promoter. Ch IP and EMSA demonstrated that the transcription factor SREBP1c binds directly to the promoter region of PERK gene and regulates the transcription and expression of PERK. The lentivirus SREBP1ctr SREBP1cm particles were successfully constructed and obtained. The endoplasmic reticulum stress reticulum stress model was successfully established. Under the condition of ERS, SREBP1c was infected with SREBP1cm, and compared with control group (GFP). SREBP1cm could promote the expression of PERK and p-PERK in osteosarcoma cells in varying degrees. Overexpression of SREBP1cncnsSREBP1cm and PERK could also induce endoplasmic reticulum stress without TM induction. Under the condition of ERS, the overexpression of SREBP1cnsSREBP1cm and PERK could inhibit the proliferation of osteosarcoma cells and promote the apoptosis and autophagy of osteosarcoma cells. SREBP1c/1cm can up-regulate the expression of PERK and increase the inhibitory effect of PERK on cell proliferation by phosphorylation. Promote PERK mediated apoptosis and autophagy, but after adding PERK signaling pathway inhibitor GSK2606414. It can effectively reverse the role of SREBP1cm in promoting the biological functions of PERK. That is, GSK2606414 can effectively reverse the inhibitory effect of SREBP1cm on cell proliferation, promote apoptosis and autophagy mediated by PERK. It is proved that SREBP1c regulates cell proliferation. Apoptosis and autophagy are achieved by up-regulation of PERK expression and activation of corresponding signal pathways by phosphorylation. Conclusion SREBP1c transcription factors regulate PERK by directly binding to PERK gene promoter. Transcription and expression of genes; SREBP1c/1cm enhanced the inhibitory effect of PERK on cell proliferation and promoted PERK mediated apoptosis and autophagy. This role of SREBP1c/1cm depends on the PERK 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】：R738

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