Brm对永生化胃黏膜上皮细胞hTERT选择性剪接模式调控的研究

发布时间：2018-02-25 06:09

  本文关键词： SWI/SNF Brm 人端粒酶逆转录酶 选择性剪接 肿瘤　出处：《天津医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究目的： SWI/SNF (switch/sucrose non-fermenting)是位于基因编码区的染色质重塑复合物,具有调控选择性剪接体的功能。Brm (Brahma)是染色质重塑复合物SWI/SNF的催化亚单位,具有DNA依赖的ATP酶活性,通过调节染色质结构影响基因表达；同时Brm也参与多种基因前体mRNA的剪接调控,可以诱导E-钙粘着蛋白,BIM,细胞周期蛋白D1,CD44等多种基因mRNA发生外显子融合作用。选择性剪接是真核生物基因中普遍存在的一种现象,它在真核基因表达调控中起着十分重要的作用。选择性剪接是人端粒酶逆转录酶(human telomerase reverse transcriptase, hTERT)基因表达调控的机制之一。本研究利用Brm-siRNA转染永生化人胃黏膜上皮细胞,观察RNA干扰前后Brm基因对hTERT选择性剪接调控的影响,进一步阐明端粒酶逆转录酶选择性剪接的分子机制,为肿瘤的临床诊断和治疗提供了新的实验基础和理论依据。 研究方法： 实验用永生化人胃黏膜上皮细胞系GES-1购自北京肿瘤医院遗传室。传代培养GES-1细胞4-5代后,随机选取对数生长期细胞并转染入Brm-siRNA.提取RNA干扰前后GES-1细胞总RNA,以β-actin为内参,利用RT-PCR分别检测目的基因Brm的表达情况；然后分别提取RNA干扰前后GES-1细胞总蛋白,以β-actin为内参,经Western blot检测BRM蛋白的表达情况,确定Brm是否已被干扰。最后用RNA干扰前后提取的GES-1细胞总RNA进行逆转录,以逆转后的cDNA为模板,检测hTERT选择性剪接变异体的类型及表达情况,进一步观察Brm基因对人端粒酶逆转录酶选择性剪接的调控作用。数据分析采用SPSS17.0统计软件进行处理,采用单因素方差分析,P≤0.05作为差异有统计学意义。 结果： 1. RT-PCR法：对于空白对照组和阴性对照组,提取细胞总RNA逆转录后可以扩增出Brm目的基因,并且Brm mRNA的表达量无明显差异(P0.05)；对于转染入Brm-siRNA的实验组,提取细胞总RNA逆转录后Brm mRNA表达量明显低于空白对照组和阴性对照组,差异具有统计学意义(P≤0.01)。 2. Western blot法：对于空白对照组和阴性对照组,提取细胞总蛋白进行Western blot实验后可以检测到BRM蛋白的表达,并且BRM蛋白的表达量无明显差异(P＞0.05)；对于转染入Brm.siRNA的实验组,提取总蛋白进行Westernblot实验检测到BRM蛋白的表达量明显低于空白对照组和阴性对照组,差异具有统计学意义(P0.01)。 3.RT.PCR扩增hTERT选择性剪接转录本的表达：将空白对照组,阴性对照组以及转染Brm.siRNA的实验组中提取得到的总RNA进行逆转录,以逆转后的cDNA为模板PCR扩增后可检测到三种hTERT选择性剪接变异体(hTERTASV),分别为的α+β+hTERT ASV,α-β+hTERT ASV和α+β-hTERT ASV.并且在转染入Brm-siRNA的实验组中可检测到α+β+hTERT ASV表达量较空白对照组和阴性对照组明显下降,α+β-hTERT ASV的表达量较空白对照组和阴性对照组明显升高,差异具有统计学意义(P0.05)。 结论： 转染Brm-siRNA后,细胞中Brm mRNA表达量以及BRM蛋白的表达量均明显低于空白对照组和阴性对照组,提示Brm-siRNA可以有效的沉默目的基因和蛋白的表达。永生化胃黏膜上皮细胞GES-1 hTERT mRNA可产生三种选择性剪接转录产物,分别为α+β+hTERT ASV,α+β-hTERT ASV和α-β+hTERT ASV,且沉默Brm基因后,α+β+hTERT ASV表达明显下降,α+β-hTERT ASV表达明显升高。Brm可以调控永生化人胃黏膜上皮细胞GES-1 hTERT选择性剪接变异体的表达,从而引起端粒酶活性的改变,为肿瘤的临床治疗提供新的思路和方法。
[Abstract]:The purpose of the study is:
SWI/SNF (switch/sucrose non-fermenting) is a chromatin remodeling complex located in the gene encoding function,.Brm can regulate alternative splicing (Brahma) catalyzes the chromatin remodeling complex SWI/SNF subunit, with ATP enzyme activity dependent DNA, by regulating chromatin structure influence gene expression; at the same time also participate in a variety of Brm splicing the precursor of mRNA gene can induce E-, E-cadherin, BIM, cyclin D1, mRNA gene CD44 exon fusion. Alternative splicing is a common phenomenon in eukaryotic gene expression, it plays an important role in regulation of eukaryotic genes selectively. Splicing is the human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) of the regulation of gene expression. This study used Brm-siRNA transfected immortalized human gastric mucosal epithelial cells. We observed the influence of Brm gene on hTERT alternative splicing regulation before and after RNA interference, and further elucidated the molecular mechanism of the alternative splicing of hTERT, which provided a new experimental basis and theoretical basis for the clinical diagnosis and treatment of tumors.
Research methods:
The experiment used immortalized human gastric epithelial cell line GES-1 was purchased from Beijing Cancer Hospital. Genetic laboratory cultured GES-1 cells were randomly selected after 4-5 generations, the logarithmic growth phase were extracted and transfected into Brm-siRNA. RNA interference in GES-1 cells before and after total RNA with beta -actin expression using RT-PCR as reference, respectively detection of Brm gene and then extract; RNA interference in GES-1 cells before and after total protein respectively, with beta -actin for reference, the expression of BRM protein was detected by Western blot, to determine whether Brm has been interference. Finally RNA interference before and after extraction of GES-1 cell RNA by reverse transcriptase, to reverse after cDNA as template to detect the expression and type hTERT splicing variants. Further, observe the regulation effect of Brm gene on human telomerase reverse transcriptase gene splicing. Data were analyzed by SPSS17.0 statistical software for processing, using single factor variance Analysis of P is less than or equal to 0.05 as the difference was statistically significant.
Result锛，

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