AR-V7在前列腺癌细胞耐药中的作用
发布时间：2018-08-26 06:57
【摘要】：目的:探讨雄激素受体剪接变异体7(AR-V7)在前列腺癌细胞耐药中的作用及分子机制。方法:运用Lipofectamine2000转染法将AR-V7 siRNA(siAR-V7)转染至4株前列腺癌细胞中,命名为PC3-siAR-V7、DU145-siAR-V7、LNCaP-siAR-V7和ArCaP-siAR-V7细胞,以转染无关序列(NC siRNA)为阴性对照。运用real-timePCR和Westernblot实验分别检测转染前后细胞中AR-V7的mRNA和蛋白表达水平;运用MTT法和Transwell法分别检测细胞活力和细胞迁移率;运用萤光素酶报告基因实验和Western blot实验分别检测AR的启动子活性及下游靶基因前列腺特异性抗原(PSA)和FK506结合蛋白5(FKBP5)的蛋白表达。构建耐比卡鲁胺的前列腺癌细胞系LNCaP-DR,运用免疫荧光观察LNCaP、LNCaP-siAR-V7和LNCaP-DR细胞中AR和AR-V7的亚细胞定位;运用蛋白质免疫共沉淀实验检测AR-V7与热休克蛋白90(HSP90)的相互作用。结果:4株前列腺癌细胞中的AR-V7 mRNA水平显著高于正常前列腺上皮细胞RWPE-1(P0.05);PC3-siAR-V7、DU145-siAR-V7、LNCaP-siAR-V7和ArCaPsiAR-V7细胞中AR-V7蛋白水平、细胞活性及细胞迁移率显著低于NCsiRNA转染的细胞(P0.05)。随着比卡鲁胺剂量的增加,所有细胞活力逐渐降低,而下调AR-V7表达显著增强前列腺癌细胞对比卡鲁胺的敏感性(P0.05)。Westernblot结果显示下调AR-V7表达显著抑制AR启动子活性,其下游PSA和FKBP5蛋白水平明显降低(P0.05)。免疫荧光结果发现AR和AR-V7主要存在于前列腺癌细胞核内,AR少量存在于细胞质中;下调AR-V7表达则抑制AR的核转运;AR存在于耐药细胞核中,AR-V7在耐药细胞中高表达。免疫共沉淀结果显示内源性AR-V7与HSP90相互作用。结论:AR-V7在前列腺癌细胞中高表达,下调AR-V7的表达显著抑制前列腺癌细胞活力和迁移;前列腺癌细胞耐药与AR-V7高表达相关,其机制可能通过AR-V7与HSP90相互作用介导AR-V7的核转运,激活AR信号通路调控下游靶基因的转录活性最终导致细胞耐药。
[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.
【作者单位】： 新疆医科大学第一附属医院泌尿外科;新疆医科大学第五附属医院泌尿外科;
【分类号】：R737.25
，
本文编号：2204081
[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.
【作者单位】： 新疆医科大学第一附属医院泌尿外科;新疆医科大学第五附属医院泌尿外科;
【分类号】：R737.25
，
本文编号：2204081
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