氡致BEAS-2B细胞凋亡中miRNA-34a与YY1的作用
发布时间：2018-11-28 16:12
【摘要】：目的:明确miR-34a负性调控YY1在氡致BEAS-2B细胞凋亡中的作用,为研究氡致支气管上皮细胞凋亡的分子机制提供基础资料。方法:对BEAS-2B细胞进行常规培养,胰酶消化后接种于Transwell培养皿,12h后将培养皿移至染毒装置中进行氡染毒,氡暴露浓度为20,000Bq/m3,染毒时间30min,连续染毒2次后作为染毒1代(Rn1)。分别染氡5代(Rn5)、10代(Rn10)、15代(Rn15)、20代(Rn20)后,流式细胞术检测细胞凋亡率,Real-time PCR检测miRNA-34a基因的表达水平,Western blot检测YY1的蛋白表达水平。人工合成人源性miR-34a mimic、miR-34a inhibitor,分别瞬时转入BEAS-2B细胞中,观察对细胞凋亡率、凋亡相关蛋白Bcl-2、PARP-1和Bax表达的影响。生物信息学分析发现转录因子YY1的3’-UTR存在与miR-34a互补结合的位点,且符合miRNA靶基因的条件。在BEAS-2B细胞中分别瞬时转入miR-34a mimic、miR-34a inhibitor,观察YY1表达水平的改变。构建YY1过表达载体和沉默载体,观察对细胞凋亡、Bcl-2、PARP-1和Bax表达的影响。结果:(1)细胞凋亡率随氡染毒剂量的增加而升高,miRNA-34a表达水平随氡染毒剂量的增加而增加,而YY1表达水平随氡染毒剂量的增加而降低;(2)转染miR-34a mimic组细胞凋亡率增加,Bcl-2和PARP-1表达下调,Bax表达上调;(3)转染YY1过表达载体组细胞凋亡率减少,Bcl-2和PARP-1表达上调,Bax表达下调。结论:本次试验结果提示,在氡致支气管上皮细胞凋亡的过程中,mi R-34a通过负性调控YY1的表达,使Bax表达上调,Bcl-2和PARP-1表达下调,从而最终促进细胞的凋亡。但其确切的分子生物学机制还需更多的实验加以证实。
[Abstract]:Aim: to investigate the role of miR-34a in the negative regulation of YY1 in radon induced apoptosis of BEAS-2B cells, and to provide basic information for studying the molecular mechanism of radon induced apoptosis of bronchial epithelial cells. Methods: BEAS-2B cells were cultured routinely, trypsin was digested and inoculated into Transwell petri dish. After 12 hours, the culture dish was transferred to the device for radon exposure. The exposure concentration of radon was 20000Bq / m3, and the exposure time was 30 min. After continuous exposure for 2 times, it was used as the first generation (Rn1). After radon exposure to radon for 5 (Rn5), 10 (Rn10), 15 (Rn15) and 20 (Rn20), the apoptosis rate was detected by flow cytometry, and the expression level of miRNA-34a gene was detected by Real-time PCR., Western blot was used to detect the expression of YY1 protein. Synthetic human miR-34a mimic,miR-34a inhibitor, was transiently transferred into BEAS-2B cells to observe the effects on apoptosis rate, Bcl-2,PARP-1 and Bax expression of apoptosis-related proteins. Bioinformatics analysis showed that the 3'-UTR of transcription factor YY1 had complementary binding sites with miR-34a, and it was in line with the condition of miRNA target gene. The changes of YY1 expression were observed by transient transfer into miR-34a mimic,miR-34a inhibitor, in BEAS-2B cells. YY1 overexpression vector and silencing vector were constructed to observe the effect on apoptosis, Bcl-2,PARP-1 and Bax expression. Results: (1) the cell apoptosis rate increased with the increase of radon exposure dose, the expression of miRNA-34a increased with the increase of radon exposure dose, while the expression level of YY1 decreased with the increase of radon exposure dose. (2) the rate of apoptosis increased, the expression of Bcl-2 and PARP-1 down-regulated and the expression of Bax up-regulated in miR-34a mimic transfection group, (3) the rate of apoptosis decreased, the expression of Bcl-2 and PARP-1 increased, and the expression of Bax decreased in the group transfected with YY1 overexpression vector. Conclusion: in the process of radon induced apoptosis of bronchial epithelial cells, mi R-34a can up-regulate the expression of YY1, up-regulate the expression of Bax, and down-regulate the expression of Bcl-2 and PARP-1, thus promoting the apoptosis of cells. But more experiments are needed to confirm its exact molecular biological mechanism.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114
本文编号：2363434
[Abstract]:Aim: to investigate the role of miR-34a in the negative regulation of YY1 in radon induced apoptosis of BEAS-2B cells, and to provide basic information for studying the molecular mechanism of radon induced apoptosis of bronchial epithelial cells. Methods: BEAS-2B cells were cultured routinely, trypsin was digested and inoculated into Transwell petri dish. After 12 hours, the culture dish was transferred to the device for radon exposure. The exposure concentration of radon was 20000Bq / m3, and the exposure time was 30 min. After continuous exposure for 2 times, it was used as the first generation (Rn1). After radon exposure to radon for 5 (Rn5), 10 (Rn10), 15 (Rn15) and 20 (Rn20), the apoptosis rate was detected by flow cytometry, and the expression level of miRNA-34a gene was detected by Real-time PCR., Western blot was used to detect the expression of YY1 protein. Synthetic human miR-34a mimic,miR-34a inhibitor, was transiently transferred into BEAS-2B cells to observe the effects on apoptosis rate, Bcl-2,PARP-1 and Bax expression of apoptosis-related proteins. Bioinformatics analysis showed that the 3'-UTR of transcription factor YY1 had complementary binding sites with miR-34a, and it was in line with the condition of miRNA target gene. The changes of YY1 expression were observed by transient transfer into miR-34a mimic,miR-34a inhibitor, in BEAS-2B cells. YY1 overexpression vector and silencing vector were constructed to observe the effect on apoptosis, Bcl-2,PARP-1 and Bax expression. Results: (1) the cell apoptosis rate increased with the increase of radon exposure dose, the expression of miRNA-34a increased with the increase of radon exposure dose, while the expression level of YY1 decreased with the increase of radon exposure dose. (2) the rate of apoptosis increased, the expression of Bcl-2 and PARP-1 down-regulated and the expression of Bax up-regulated in miR-34a mimic transfection group, (3) the rate of apoptosis decreased, the expression of Bcl-2 and PARP-1 increased, and the expression of Bax decreased in the group transfected with YY1 overexpression vector. Conclusion: in the process of radon induced apoptosis of bronchial epithelial cells, mi R-34a can up-regulate the expression of YY1, up-regulate the expression of Bax, and down-regulate the expression of Bcl-2 and PARP-1, thus promoting the apoptosis of cells. But more experiments are needed to confirm its exact molecular biological mechanism.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114
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