發(fā)布時間：2018-08-26 06:57

【摘要】：目的:探討雄激素受體剪接變異體7(AR-V7)在前列腺癌細(xì)胞耐藥中的作用及分子機(jī)制。方法:運(yùn)用Lipofectamine2000轉(zhuǎn)染法將AR-V7 siRNA(siAR-V7)轉(zhuǎn)染至4株前列腺癌細(xì)胞中,命名為PC3-siAR-V7、DU145-siAR-V7、LNCaP-siAR-V7和ArCaP-siAR-V7細(xì)胞,以轉(zhuǎn)染無關(guān)序列(NC siRNA)為陰性對照。運(yùn)用real-timePCR和Westernblot實(shí)驗(yàn)分別檢測轉(zhuǎn)染前后細(xì)胞中AR-V7的mRNA和蛋白表達(dá)水平;運(yùn)用MTT法和Transwell法分別檢測細(xì)胞活力和細(xì)胞遷移率;運(yùn)用螢光素酶報(bào)告基因?qū)嶒?yàn)和Western blot實(shí)驗(yàn)分別檢測AR的啟動子活性及下游靶基因前列腺特異性抗原(PSA)和FK506結(jié)合蛋白5(FKBP5)的蛋白表達(dá)。構(gòu)建耐比卡魯胺的前列腺癌細(xì)胞系LNCaP-DR,運(yùn)用免疫熒光觀察LNCaP、LNCaP-siAR-V7和LNCaP-DR細(xì)胞中AR和AR-V7的亞細(xì)胞定位;運(yùn)用蛋白質(zhì)免疫共沉淀實(shí)驗(yàn)檢測AR-V7與熱休克蛋白90(HSP90)的相互作用。結(jié)果:4株前列腺癌細(xì)胞中的AR-V7 mRNA水平顯著高于正常前列腺上皮細(xì)胞RWPE-1(P0.05);PC3-siAR-V7、DU145-siAR-V7、LNCaP-siAR-V7和ArCaPsiAR-V7細(xì)胞中AR-V7蛋白水平、細(xì)胞活性及細(xì)胞遷移率顯著低于NCsiRNA轉(zhuǎn)染的細(xì)胞(P0.05)。隨著比卡魯胺劑量的增加,所有細(xì)胞活力逐漸降低,而下調(diào)AR-V7表達(dá)顯著增強(qiáng)前列腺癌細(xì)胞對比卡魯胺的敏感性(P0.05)。Westernblot結(jié)果顯示下調(diào)AR-V7表達(dá)顯著抑制AR啟動子活性,其下游PSA和FKBP5蛋白水平明顯降低(P0.05)。免疫熒光結(jié)果發(fā)現(xiàn)AR和AR-V7主要存在于前列腺癌細(xì)胞核內(nèi),AR少量存在于細(xì)胞質(zhì)中;下調(diào)AR-V7表達(dá)則抑制AR的核轉(zhuǎn)運(yùn);AR存在于耐藥細(xì)胞核中,AR-V7在耐藥細(xì)胞中高表達(dá)。免疫共沉淀結(jié)果顯示內(nèi)源性AR-V7與HSP90相互作用。結(jié)論:AR-V7在前列腺癌細(xì)胞中高表達(dá),下調(diào)AR-V7的表達(dá)顯著抑制前列腺癌細(xì)胞活力和遷移;前列腺癌細(xì)胞耐藥與AR-V7高表達(dá)相關(guān),其機(jī)制可能通過AR-V7與HSP90相互作用介導(dǎo)AR-V7的核轉(zhuǎn)運(yùn),激活A(yù)R信號通路調(diào)控下游靶基因的轉(zhuǎn)錄活性最終導(dǎo)致細(xì)胞耐藥。
[Abstract]:Aim: to investigate the role and molecular mechanism of androgen receptor splicing variant 7 (AR-V7) in drug resistance of prostate cancer cells. Methods: AR-V7 siRNA (siAR-V7) was transfected into 4 prostate cancer cells by Lipofectamine2000 transfection, named PC3-siAR-V7,DU145-siAR-V7,LNCaP-siAR-V7 and ArCaP-siAR-V7 cells, and the unrelated (NC siRNA) was used as negative control. Real-timePCR and Westernblot were used to detect the expression of mRNA and protein in the cells before and after transfection, and MTT and Transwell methods were used to detect the cell viability and cell migration. The promoter activity of AR and the expression of prostate-specific antigen (PSA) and FK506 binding protein 5 (FKBP5) of downstream target genes were detected by luciferase reporter gene assay and Western blot assay, respectively. The subcellular localization of AR and AR-V7 in LNCaP,LNCaP-siAR-V7 and LNCaP-DR cells was observed by immunofluorescence, and the interaction between AR-V7 and heat shock protein 90 (HSP90) was detected by protein-immunoprecipitation assay. Results the level of AR-V7 mRNA in the prostate cancer cell line was significantly higher than that in the normal prostatic epithelial cell line RWPE-1 (P0.05). The levels of AR-V7 protein in LNCaP-siAR-V7 and ArCaPsiAR-V7 cells were significantly lower than those in NCsiRNA transfected cells (P0.05). With the increase of the dose of BiCarous amine, the activity of all cells gradually decreased, while the down-regulation of AR-V7 expression significantly enhanced the sensitivity of prostate cancer cells to Carous amine (P0.05) .Western blot showed that down-regulation of AR-V7 expression significantly inhibited the activity of AR promoter. The downstream PSA and FKBP5 protein levels were significantly decreased (P0.05). The results of immunofluorescence showed that AR and AR-V7 mainly existed in the cytoplasm of prostate cancer, and that the down-regulated AR-V7 expression inhibited the expression of AR in the nucleus of the resistant cells, and the expression of AR-V7 was highly expressed in the resistant cells. The results of immunoprecipitation showed that endogenous AR-V7 interacted with HSP90. Conclusion the overexpression of AR-V7 in prostate cancer cells and down-regulation of the expression of AR-V7 significantly inhibit the viability and migration of prostate cancer cells, and the drug resistance of prostate cancer cells is associated with the high expression of AR-V7, which may mediate the nuclear transport of AR-V7 through the interaction of AR-V7 and HSP90. Activation of AR signaling pathway regulates the transcriptional activity of downstream target genes and ultimately leads to drug resistance.
【作者單位】： 新疆醫(yī)科大學(xué)第一附屬醫(yī)院泌尿外科;新疆醫(yī)科大學(xué)第五附屬醫(yī)院泌尿外科;
【分類號】：R737.25
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本文編號：2204081