PRMT2及其剪接体对雌激素受体ERα转录活性影响的相关研究

发布时间：2019-02-21 17:02

【摘要】： 目的:比较不同条件下,PRMT2及其新的剪接体PRMT2α、PRMT2β、PRMT2γ对ERα转录活性的影响;检测PRMT2及PRMT2α、PRMT2β、PRMT2γ与ERa在细胞外的相互作用。方法:提取pcDNA3.0-PRMT2、pcDNA3.0-PRMT2α、pcDNA3.0 -PRMT2β、pcDNA 3.0-PRMT2γ及pcDNA3.0空载体质粒,分别与pcDNA3.0-ERα瞬时共转染(采用脂质体介导法)293T细胞,建立萤火虫荧光素酶双报告基因检测系统,选用含受雌激素反应元件(ERE, estrogen response elements)一致序列调控的荧光素酶基因(ERE-Luc)作为报告基因,通过荧光测定仪检测ERE-luc荧光素酶基因活性值,进而观察PRMT2及其剪接体对ERα转录活性的影响。该部分包括两个内容,其一观察在有无雌激素条件下, PRMT2及PRMT2α、PRMT2β、PRMT2γ质粒分别对ERα转录活性的影响。其二是观察雌激素抑制剂4-羟基他莫西芬(4-OHT)、氟维司群(ICI182,780)条件下,PRMT2及其各剪接体PRMT2α、PRMT2β、PRMT2γ对ERα转录活性的影响。将表达融合蛋白GST-PRMT2及其剪接体的重组质粒和表达GST的空载体pGEX-5T转化到大肠杆菌BL21中,诱导融合蛋白的表达,将获得的蛋白样本进行SDS-PAGE电泳,考马斯亮蓝染色法验证蛋白表达水平。GST-Sepharose 4B亲和层析法纯化GST- PRMT2α、GST-PRMT2β、GST- PRMT2γ融合蛋白及GST蛋白,将pcDNA3.0-ERα瞬时转染293T细胞,收集细胞裂解液,运用GST pull-down技术检测PRMT2及其各剪接体PRMT2α、PRMT2β、PRMT2γ与ERα在细胞外相互作用情况,及其与雌激素的相关性。结果:1.通过双荧光素酶报告基因检测系统检测发现,在雌激素不存在时,PRMT2及其剪接体对ERα转录活性无明显提高。在雌激素存在时,与空载体相比,PRMT2及其剪接体PRMT2α、PRMT2β、PRMT2r分别使ERα的转录活性提高了约2.9倍、1.7倍、1.5倍及2.1倍。 2.分别加入雌激素及雌激素抑制剂(ICI182,780,4-OHT)条件下,雌激素抑制剂可降低PRMT2及其剪接体PRMT2α、PRMT2β、PRMT2γ对ERα转录活性的影响。 3.通过GST pull-down技术检测发现,无论雌激素是否存在, GST蛋白与ERα不能结合,而GST- PRMT2α、GST- PRMT2β、GST- PRMT2γ融合蛋白均能分别与ERα结合。结论:PRMT2α、PRMT2β、PRMT2γ与PRMT2一样能以雌激素依赖的形式提高ERα转录活性,雌激素抑制剂能降低PRMT2α、PRMT2β、PRMT2γ对ERα转录活性的影响。PRMT2及其各剪接体PRMT2α、PRMT2β、PRMT2γ在细胞外均能以不依赖于雌激素形式与ERα相结合,雌激素对其可能有增强效应。PRMT2及其各剪接体可能与PRMT2一样是ERα的共激活子,也可能通过ERα信号途径影响乳腺癌的发生发展,有望成为乳腺癌治疗的新靶标。
[Abstract]:Aim: to compare the effects of PRMT2 and its new splicing bodies, PRMT2 伪, PRMT2 尾, PRMT2 纬, on the transcriptional activity of ER 伪, and to detect the extracellular interaction of PRMT2, PRMT2 伪, PRMT2 尾, PRMT2 纬 and ERa. Methods: pcDNA3.0-PRMT2,pcDNA3.0-PRMT2 伪, pcDNA3.0-PRMT2 尾, pcDNA 3.0-PRMT2 纬 and pcDNA3.0 were extracted and cotransfected with pcDNA3.0-ER 伪 in 293T cells. A double reporter gene detection system for luciferase of firefly was established. Luciferase gene (ERE-Luc), which was regulated by the consistent sequence of estrogen response element (ERE, estrogen response elements), was selected as the reporter gene. The activity of ERE-luc luciferase gene was detected by fluorescence analyzer, and the effect of PRMT2 and its splicing on the transcriptional activity of ER 伪 was observed. This part includes two parts. One is to observe the effects of PRMT2 and PRMT2 伪, PRMT2 尾, PRMT2 纬 plasmids on the transcriptional activity of ER 伪 in the presence or absence of estrogen. The second was to observe the effects of PRMT2 and its splicing bodies, PRMT2 伪, PRMT2 尾, PRMT2 纬, on the transcriptional activity of ER 伪 under the condition of estrogen inhibitor 4-hydroxytamoxifen (4-OHT) and fluviz group (ICI182780). The recombinant plasmid expressing fusion protein GST-PRMT2 and its splice and the empty vector pGEX-5T expressing GST were transformed into Escherichia coli BL21 to induce the expression of fusion protein. The obtained protein samples were analyzed by SDS-PAGE electrophoresis. Coomassie brilliant blue staining was used to verify the protein expression. GST- PRMT2 伪, GST-PRMT2 尾, GST- PRMT2 纬 fusion protein and GST protein were purified by GST-Sepharose 4B affinity chromatography. PcDNA3.0-ER 伪 was transiently transfected into 293T cells, and cell lysate was collected. The extracellular interaction of PRMT2 and its splicing bodies PRMT2 伪, PRMT2 尾, PRMT2 纬 and ER 伪 was detected by GST pull-down technique. Results: 1. The double luciferase reporter gene detection system showed that PRMT2 and its splice did not increase the transcriptional activity of ER 伪 in the absence of estrogen. In the presence of estrogen, PRMT2 and its splice PRMT2 伪, PRMT2 尾 and PRMT2r increased the transcriptional activity of ER 伪 by about 2.9 times, 1.7 times, 1.5 times and 2.1 times, respectively. 2. The effects of estrogen inhibitor and estrogen inhibitor (ICI182,780,4-OHT) on the transcriptional activity of ER 伪 in PRMT2 and its splice PRMT2 伪, PRMT2 尾, PRMT2 纬 were decreased. 3. GST pull-down assay showed that GST protein could not bind to ER 伪, but GST- PRMT2 伪, GST- PRMT2 尾, GST- PRMT2 纬 fusion protein could bind to ER 伪, regardless of whether estrogen was present or not. Conclusion: PRMT2 伪, PRMT2 尾, PRMT2 纬 can increase the transcriptional activity of ER 伪 in an estrogen-dependent manner, and estrogen inhibitors can decrease the effects of PRMT2 伪, PRMT2 尾, PRMT2 纬 on the transcription activity of ER 伪. PRMT2 and its splices PRMT2 伪, PRMT2 尾, PRMT2 and their splices can increase the transcriptional activity of ER 伪. PRMT2 纬 can bind to ER 伪 in the form of estrogen independent, and estrogen may enhance it. PRMT2 and its splicing bodies may be coactivators of ER 伪 just like PRMT2. It may also affect the occurrence and development of breast cancer through ER 伪 signal pathway, and may become a new target for breast cancer treatment.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

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