【摘要】： 目的:比較不同條件下,PRMT2及其新的剪接體PRMT2α、PRMT2β、PRMT2γ對ERα轉(zhuǎn)錄活性的影響;檢測PRMT2及PRMT2α、PRMT2β、PRMT2γ與ERa在細(xì)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空載體質(zhì)粒,分別與pcDNA3.0-ERα瞬時共轉(zhuǎn)染(采用脂質(zhì)體介導(dǎo)法)293T細(xì)胞,建立螢火蟲熒光素酶雙報告基因檢測系統(tǒng),選用含受雌激素反應(yīng)元件(ERE, estrogen response elements)一致序列調(diào)控的熒光素酶基因(ERE-Luc)作為報告基因,通過熒光測定儀檢測ERE-luc熒光素酶基因活性值,進(jìn)而觀察PRMT2及其剪接體對ERα轉(zhuǎn)錄活性的影響。該部分包括兩個內(nèi)容,其一觀察在有無雌激素條件下, PRMT2及PRMT2α、PRMT2β、PRMT2γ質(zhì)粒分別對ERα轉(zhuǎn)錄活性的影響。其二是觀察雌激素抑制劑4-羥基他莫西芬(4-OHT)、氟維司群(ICI182,780)條件下,PRMT2及其各剪接體PRMT2α、PRMT2β、PRMT2γ對ERα轉(zhuǎn)錄活性的影響。將表達(dá)融合蛋白GST-PRMT2及其剪接體的重組質(zhì)粒和表達(dá)GST的空載體pGEX-5T轉(zhuǎn)化到大腸桿菌BL21中,誘導(dǎo)融合蛋白的表達(dá),將獲得的蛋白樣本進(jìn)行SDS-PAGE電泳,考馬斯亮藍(lán)染色法驗證蛋白表達(dá)水平。GST-Sepharose 4B親和層析法純化GST- PRMT2α、GST-PRMT2β、GST- PRMT2γ融合蛋白及GST蛋白,將pcDNA3.0-ERα瞬時轉(zhuǎn)染293T細(xì)胞,收集細(xì)胞裂解液,運用GST pull-down技術(shù)檢測PRMT2及其各剪接體PRMT2α、PRMT2β、PRMT2γ與ERα在細(xì)胞外相互作用情況,及其與雌激素的相關(guān)性。 結(jié)果:1.通過雙熒光素酶報告基因檢測系統(tǒng)檢測發(fā)現(xiàn),在雌激素不存在時,PRMT2及其剪接體對ERα轉(zhuǎn)錄活性無明顯提高。在雌激素存在時,與空載體相比,PRMT2及其剪接體PRMT2α、PRMT2β、PRMT2r分別使ERα的轉(zhuǎn)錄活性提高了約2.9倍、1.7倍、1.5倍及2.1倍。 2.分別加入雌激素及雌激素抑制劑(ICI182,780,4-OHT)條件下,雌激素抑制劑可降低PRMT2及其剪接體PRMT2α、PRMT2β、PRMT2γ對ERα轉(zhuǎn)錄活性的影響。 3.通過GST pull-down技術(shù)檢測發(fā)現(xiàn),無論雌激素是否存在, GST蛋白與ERα不能結(jié)合,而GST- PRMT2α、GST- PRMT2β、GST- PRMT2γ融合蛋白均能分別與ERα結(jié)合。 結(jié)論:PRMT2α、PRMT2β、PRMT2γ與PRMT2一樣能以雌激素依賴的形式提高ERα轉(zhuǎn)錄活性,雌激素抑制劑能降低PRMT2α、PRMT2β、PRMT2γ對ERα轉(zhuǎn)錄活性的影響。PRMT2及其各剪接體PRMT2α、PRMT2β、PRMT2γ在細(xì)胞外均能以不依賴于雌激素形式與ERα相結(jié)合,雌激素對其可能有增強(qiáng)效應(yīng)。PRMT2及其各剪接體可能與PRMT2一樣是ERα的共激活子,也可能通過ERα信號途徑影響乳腺癌的發(fā)生發(fā)展,有望成為乳腺癌治療的新靶標(biāo)。
[Abstract]:Aim: to compare the effects of PRMT2 and its new splicing bodies, PRMT2 偽, PRMT2 尾, PRMT2 緯, on the transcriptional activity of ER 偽, and to detect the extracellular interaction of PRMT2, PRMT2 偽, PRMT2 尾, PRMT2 緯 and ERa. Methods: pcDNA3.0-PRMT2,pcDNA3.0-PRMT2 偽, pcDNA3.0-PRMT2 尾, pcDNA 3.0-PRMT2 緯 and pcDNA3.0 were extracted and cotransfected with pcDNA3.0-ER 偽 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 偽 was observed. This part includes two parts. One is to observe the effects of PRMT2 and PRMT2 偽, PRMT2 尾, PRMT2 緯 plasmids on the transcriptional activity of ER 偽 in the presence or absence of estrogen. The second was to observe the effects of PRMT2 and its splicing bodies, PRMT2 偽, PRMT2 尾, PRMT2 緯, on the transcriptional activity of ER 偽 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 偽, GST-PRMT2 尾, GST- PRMT2 緯 fusion protein and GST protein were purified by GST-Sepharose 4B affinity chromatography. PcDNA3.0-ER 偽 was transiently transfected into 293T cells, and cell lysate was collected. The extracellular interaction of PRMT2 and its splicing bodies PRMT2 偽, PRMT2 尾, PRMT2 緯 and ER 偽 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 偽 in the absence of estrogen. In the presence of estrogen, PRMT2 and its splice PRMT2 偽, PRMT2 尾 and PRMT2r increased the transcriptional activity of ER 偽 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 偽 in PRMT2 and its splice PRMT2 偽, PRMT2 尾, PRMT2 緯 were decreased. 3. GST pull-down assay showed that GST protein could not bind to ER 偽, but GST- PRMT2 偽, GST- PRMT2 尾, GST- PRMT2 緯 fusion protein could bind to ER 偽, regardless of whether estrogen was present or not. Conclusion: PRMT2 偽, PRMT2 尾, PRMT2 緯 can increase the transcriptional activity of ER 偽 in an estrogen-dependent manner, and estrogen inhibitors can decrease the effects of PRMT2 偽, PRMT2 尾, PRMT2 緯 on the transcription activity of ER 偽. PRMT2 and its splices PRMT2 偽, PRMT2 尾, PRMT2 and their splices can increase the transcriptional activity of ER 偽. PRMT2 緯 can bind to ER 偽 in the form of estrogen independent, and estrogen may enhance it. PRMT2 and its splicing bodies may be coactivators of ER 偽 just like PRMT2. It may also affect the occurrence and development of breast cancer through ER 偽 signal pathway, and may become a new target for breast cancer treatment.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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