本文選題：微小　切入點：RNA　出處：《天津醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：【目的】內(nèi)質(zhì)網(wǎng)是真核細胞內(nèi)重要的細胞器,內(nèi)質(zhì)網(wǎng)腔內(nèi)未折疊或者錯誤折疊蛋白的累積將導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激(Edoplasmic Reticulum Stress,ERS)。內(nèi)質(zhì)網(wǎng)應(yīng)激將導(dǎo)致未折疊蛋白反應(yīng)(Unfolded Protein Response,UPR),嘗試恢復(fù)正常的內(nèi)質(zhì)網(wǎng)功能。ERS引起的UPR參與多種人類疾病的病理生理過程,包括神經(jīng)退行性疾病、糖尿病以及腫瘤等。已有研究表明細胞自噬和微小RNA(micro RNA,miRNA)參與內(nèi)質(zhì)網(wǎng)應(yīng)激過程中細胞命運的調(diào)節(jié),但是在內(nèi)質(zhì)網(wǎng)應(yīng)激過程中miRNA是否參與細胞自噬的調(diào)節(jié)還不是很清楚。我們發(fā)現(xiàn)在內(nèi)質(zhì)網(wǎng)應(yīng)激的條件下miR-346的表達顯著增加,并且miR-346可以顯著提高內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞的活性。在此基礎(chǔ)上我們探討miR-346在內(nèi)質(zhì)網(wǎng)應(yīng)激條件下促進細胞存活的機制,及內(nèi)質(zhì)網(wǎng)應(yīng)激條件下miR-346在細胞自噬和線粒體自噬過程中的作用及其分子機制。【方法】首先,我們建立了毒胡蘿卜素(Thapsigargin,Tg)誘導(dǎo)的HeLa細胞內(nèi)質(zhì)網(wǎng)應(yīng)激模型,并通過western blot或者RT-q PCR的方法檢測內(nèi)質(zhì)網(wǎng)伴侶蛋白GRP78或者XBP-1(U)與XBP-1(S)m RNA的表達情況來判斷內(nèi)質(zhì)網(wǎng)應(yīng)激是否誘導(dǎo)成功;通過RT-q PCR的方法檢測內(nèi)質(zhì)網(wǎng)應(yīng)激條件下miR-346的表達情況;通過MTT和Annexin V-FITC/PI法分別檢測內(nèi)質(zhì)網(wǎng)應(yīng)激條件下miR-346對細胞活性以及凋亡的影響。其次,我們通過GFP-LC3示蹤實驗、western blot檢測LC3-I以及LC3-II表達等探討了miR-346對內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞自噬的調(diào)節(jié);通過GFP-LC3聯(lián)合Mito Tracker?Red CMXRos染色探討miR-346對線粒體自噬的調(diào)節(jié);通過活性氧敏感探針DCFH-DA研究miR-346對內(nèi)質(zhì)網(wǎng)應(yīng)激條件下活性氧的調(diào)節(jié)。而后,我們通過生物信息學(xué)預(yù)測,并通過western blot、RT-q PCR以及熒光報告載體實驗驗證內(nèi)質(zhì)網(wǎng)應(yīng)激條件下miR-346靶基因,并進一步研究了靶基因的功能。最后我們通過蛋白質(zhì)免疫共沉淀、蛋白質(zhì)泛素化分析的方法探討miR-346促進細胞自噬的可能機制�！窘Y(jié)果】在內(nèi)質(zhì)網(wǎng)應(yīng)激的條件下miR-346的表達水平顯著增加,XBP-1參與了內(nèi)質(zhì)網(wǎng)應(yīng)激條件下miR-346的表達調(diào)節(jié),并且miR-346可以顯著降低內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞的凋亡水平從而提高細胞的活性。miR-346可以顯著增強內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞的自噬和線粒體自噬水平,降低細胞內(nèi)的活性氧水平;miR-346通過自噬依賴的方式提高內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞的活性。miR-346直接靶定并上調(diào)GSK3B的表達;過表達GSK3B可以顯著增強內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞的自噬,導(dǎo)致細胞內(nèi)活性氧水平降低以及細胞活性增強,而敲降GSK3B則相反;功能挽救實驗證實GSK3B是miR-346的直接功能靶基因。miR-346和GSK3B可以顯著增加BCL2的泛素化水平,導(dǎo)致細胞內(nèi)的BCL2蛋白水平顯著降低,促進BCL2與BECN1的解離。【結(jié)論】在內(nèi)質(zhì)網(wǎng)應(yīng)激條件下miR-346的表達水平顯著增強;通過增強內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞自噬和線粒體自噬水平,miR-346可降低細胞內(nèi)的活性氧,進而降低內(nèi)質(zhì)網(wǎng)應(yīng)激條件下細胞的凋亡,提高細胞活性;同時在內(nèi)質(zhì)網(wǎng)應(yīng)激條件下,miR-346可直接靶定并上調(diào)GSK3B的表達,通過促進BCL2的泛素化導(dǎo)致細胞內(nèi)的BCL2水平顯著降低,繼而導(dǎo)致BECN1與BCL2的解離以及細胞自噬活性的增強。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R363

【相似文獻】

相關(guān)博士學(xué)位論文 前1條

1 郭軍飛;miR-346在內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)的細胞自噬中的作用及作用機制研究[D];天津醫(yī)科大學(xué);2016年

，

本文編號：1630077