miR-346在内质网应激诱导的细胞自噬中的作用及作用机制研究

发布时间：2018-03-18 14:58

本文选题：微小　切入点：RNA　出处：《天津医科大学》2016年博士论文　论文类型：学位论文

【摘要】：【目的】内质网是真核细胞内重要的细胞器,内质网腔内未折叠或者错误折叠蛋白的累积将导致内质网应激(Edoplasmic Reticulum Stress,ERS)。内质网应激将导致未折叠蛋白反应(Unfolded Protein Response,UPR),尝试恢复正常的内质网功能。ERS引起的UPR参与多种人类疾病的病理生理过程,包括神经退行性疾病、糖尿病以及肿瘤等。已有研究表明细胞自噬和微小RNA(micro RNA,miRNA)参与内质网应激过程中细胞命运的调节,但是在内质网应激过程中miRNA是否参与细胞自噬的调节还不是很清楚。我们发现在内质网应激的条件下miR-346的表达显著增加,并且miR-346可以显著提高内质网应激条件下细胞的活性。在此基础上我们探讨miR-346在内质网应激条件下促进细胞存活的机制,及内质网应激条件下miR-346在细胞自噬和线粒体自噬过程中的作用及其分子机制。【方法】首先,我们建立了毒胡萝卜素(Thapsigargin,Tg)诱导的HeLa细胞内质网应激模型,并通过western blot或者RT-q PCR的方法检测内质网伴侣蛋白GRP78或者XBP-1(U)与XBP-1(S)m RNA的表达情况来判断内质网应激是否诱导成功;通过RT-q PCR的方法检测内质网应激条件下miR-346的表达情况;通过MTT和Annexin V-FITC/PI法分别检测内质网应激条件下miR-346对细胞活性以及凋亡的影响。其次,我们通过GFP-LC3示踪实验、western blot检测LC3-I以及LC3-II表达等探讨了miR-346对内质网应激条件下细胞自噬的调节;通过GFP-LC3联合Mito Tracker?Red CMXRos染色探讨miR-346对线粒体自噬的调节;通过活性氧敏感探针DCFH-DA研究miR-346对内质网应激条件下活性氧的调节。而后,我们通过生物信息学预测,并通过western blot、RT-q PCR以及荧光报告载体实验验证内质网应激条件下miR-346靶基因,并进一步研究了靶基因的功能。最后我们通过蛋白质免疫共沉淀、蛋白质泛素化分析的方法探讨miR-346促进细胞自噬的可能机制。【结果】在内质网应激的条件下miR-346的表达水平显著增加,XBP-1参与了内质网应激条件下miR-346的表达调节,并且miR-346可以显著降低内质网应激条件下细胞的凋亡水平从而提高细胞的活性。miR-346可以显著增强内质网应激条件下细胞的自噬和线粒体自噬水平,降低细胞内的活性氧水平;miR-346通过自噬依赖的方式提高内质网应激条件下细胞的活性。miR-346直接靶定并上调GSK3B的表达;过表达GSK3B可以显著增强内质网应激条件下细胞的自噬,导致细胞内活性氧水平降低以及细胞活性增强,而敲降GSK3B则相反;功能挽救实验证实GSK3B是miR-346的直接功能靶基因。miR-346和GSK3B可以显著增加BCL2的泛素化水平,导致细胞内的BCL2蛋白水平显著降低,促进BCL2与BECN1的解离。【结论】在内质网应激条件下miR-346的表达水平显著增强;通过增强内质网应激条件下细胞自噬和线粒体自噬水平,miR-346可降低细胞内的活性氧,进而降低内质网应激条件下细胞的凋亡,提高细胞活性;同时在内质网应激条件下,miR-346可直接靶定并上调GSK3B的表达,通过促进BCL2的泛素化导致细胞内的BCL2水平显著降低,继而导致BECN1与BCL2的解离以及细胞自噬活性的增强。
[Abstract]:[Objective] the endoplasmic reticulum is important organelles in eukaryotic cells, endoplasmic reticulum unfolded or misfolded protein accumulation will lead to endoplasmic reticulum stress (Edoplasmic Reticulum, Stress, ERS). The endoplasmic reticulum stress will lead to the unfolded protein response (Unfolded Protein, Response, UPR), the pathophysiological process of ER function.ERS try return to normal by UPR in a variety of human diseases, including neurodegenerative diseases, diabetes and cancer. Studies have shown that autophagy and micro RNA (micro RNA miRNA) is involved in the regulation of cell fate during endoplasmic reticulum stress, but miRNA in the endoplasmic reticulum stress is involved in regulation of the process of autophagy is not very clear. We found that the expression of miR-346 in endoplasmic reticulum stress conditions increased significantly, and miR-346 can significantly improve the endoplasmic reticulum stress conditions in cell activity. On this basis, we explore the mechanism of miR-346 promoting cell survival in the endoplasmic reticulum stress, endoplasmic reticulum stress and under the condition of miR-346 in autophagy and the role of mitochondrial autophagy and its molecular mechanism. [Methods] first, we established the thapsigargin (Thapsigargin, Tg) HeLa cell model induced by endoplasmic reticulum stress. And through the method of Western blot or RT-q PCR detection of endoplasmic reticulum chaperone protein GRP78 or XBP-1 (U) and XBP-1 (S) expression of M RNA to determine whether endoplasmic reticulum stress induced by RT-q method successfully; the detection of PCR under endoplasmic reticulum stress miR-346 expression; effect of endoplasmic reticulum stress under the condition of miR-346 cells activity and apoptosis were detected by MTT and Annexin V-FITC/PI method. Secondly, we use GFP-LC3 Western blot tracer experiment, detection of LC3-I and LC3-II expression of miR-346 Regulation of endoplasmic reticulum stress under the condition of autophagy; by GFP-LC3 combined with Mito Tracker? To investigate the regulation of miR-346 on mitochondrial autophagy staining Red CMXRos; by adjusting the reactive oxygen sensitive probe DCFH-DA miR-346 on active oxygen under endoplasmic reticulum stress. Then, we through bioinformatics prediction, and by Western blot, miR-346 target RT-q PCR and EGFP gene experiments under endoplasmic reticulum stress, and further study the target gene function. Finally, we through the protein immunoprecipitation method, analysis of protein ubiquitination to explore possible mechanisms of miR-346 promoting autophagy. [results] the expression level of miR-346 in the endoplasmic reticulum stress conditions increased significantly XBP-1, involved in the regulation of expression of endoplasmic reticulum stress under the condition of miR-346, and miR-346 can significantly reduce the endoplasmic reticulum stress conditions. The dead level so as to improve the activity of.MiR-346 cells can significantly enhance the endoplasmic reticulum stress under the condition of the autophagy and mitochondrial autophagy, decrease intracellular ROS levels through autophagy dependent manner; miR-346 increase the activity of.MiR-346 cells under endoplasmic reticulum stress in the direct expression of target and upregulation of GSK3B; overexpression of GSK3B can significantly enhance the autophagy under endoplasmic reticulum stress, resulting in decreased intracellular ROS level and cell activity increased, while knockdown of GSK3B on the contrary; save function experiments show that GSK3B is a direct function of target gene.MiR-346 and GSK3B miR-346 can significantly increase the ubiquitination level of BCL2, resulting in BCL2 protein levels in cells decreased significantly, promote dissociation the BCL2 and BECN1. [Conclusion] the expression level of miR-346 in the endoplasmic reticulum stress condition was significantly enhanced by endoplasmic reticulum stress; enhance the fine Autophagy and mitochondrial autophagy, miR-346 can decrease the intracellular ROS, thereby reducing apoptosis under endoplasmic reticulum stress, increase cell activity; at the same time in the endoplasmic reticulum stress conditions, miR-346 expression can be directly targeted and the expression of GSK3B by promoting the ubiquitination of BCL2 leads to intracellular level of BCL2 was significantly decreased BECN1 and BCL2, and then lead to dissociation and autophagy activity enhancement.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R363

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