赖氨酸特异性去甲基化酶1对曲古抑菌素A诱导卵巢癌细胞凋亡的作用
发布时间：2018-07-22 10:28
【摘要】：目的赖氨酸特异性去甲基化酶1(LSD1)是黄素腺嘌呤二核苷酸依赖的胺氧化酶,其参与细胞的增殖、分化以及介导基因激活和抑制等多种生物学过程。文中旨在探讨组蛋白去乙酰化酶抑制剂曲古抑菌素A(TSA)对LSD1的乙酰化,以及LSD1在TSA诱导卵巢癌细胞凋亡中的作用。方法利用RNA干扰方法建立诱导型稳定敲低LSD1基因表达的卵巢癌HO8910和SKOV3细胞株;实验设置p LKO对照组(感染空载体慢病毒)、p LKO+Dox(100 ng/m L)组、LSD1-KD组(感染LSD1-shRNA慢病毒)和LSD1-KD+Dox(100 ng/m L)组。用免疫沉淀(IP)和Western blot检测LSD1乙酰化程度,及其底物组蛋白H3第4位赖氨酸的二甲基化(H3K4me2)水平;AnnexinⅤ/PI和流式细胞术分析细胞凋亡的变化;RT-PCR检测凋亡相关基因Bax和p21 mRNA表达水平;染色质免疫沉淀(Ch IP)分析Bax和p21基因启动子区H3K4me2程度。实验设置methanol对照组(2 mg/m L)、TSA组(200 nmol/L)、TCP组(抑制LSD1活性;100μmol/L)和TSA+TCP组,处理细胞48 h。在RNA干扰LSD1表达实验中,设置溶剂对照组(2 mg/m L methanol)、TSA处理组(200 nmol/L)、Dox组(干扰LSD1表达;100 ng/m L)和联合处理组(200 nmol/L TSA与100ng/m L Dox)联合处理细胞48h。结果免疫沉淀结合Western blot检测结果显示,200nmol/L TSA处理后LSD1蛋白的乙酰化和H3K9的乙酰化水平较methanol溶剂处理显著增加(P0.01)。与methanol溶剂对照处理比较,200nmol/L TSA处理HO8910和SKOV3细胞后H3K4me2水平明显升高(P0.01)。AnnexinⅤ/PI结果显示,与methanol对照组比较,TSA组、TCP组和TSA+TCP组HO8910和SKOV3细胞的总凋亡率均显著升高(P0.05);且TSA+TCP组较TSA组和TCP组细胞凋亡率明显升高(P0.05)。LSD1-KD+Dox组细胞中LSD1蛋白水平(HO8910:0.21±0.16;SKOV3:0.26±0.11)较p LKO对照组(HO8910:1.15±0.16;SKOV3:0.97±0.31)和LSD1-KD组(HO8910:1.07±0.19;SKOV3:0.98±0.21)显著减少(P0.01)。与溶剂对照组比较,TSA处理组、Dox组和联合处理组细胞的总凋亡率均显著增高(P0.05);其中联合处理组较TSA或Dox组亦显著增高(P0.05)。RT-PCR结果表明,在HO8910细胞中,与溶剂对照组Bax和p21 mRNA水平比较,TSA处理组、Dox组及联合处理组均显著上调(P0.05);且联合处理组较TSA处理组或Dox组亦显著上调(P0.05)。TSA处理组细胞中Bax和p21基因启动子区H3K4me2水平较methanol对照组显著升高(Bax:2.92±0.26 vs 0.86±0.19;p21:3.07±0.29 vs 0.93±0.17,P0.01)。结论 TSA诱导了LSD1蛋白的乙酰化,抑制LSD1表达或活性可增强TSA对卵巢癌细胞的杀伤作用。
[Abstract]:Objective Lysine specific demethylase 1 (LSD1) is a kind of amine oxidase which is dependent on the adenine dinucleotide of Flavin. LSD1 is involved in many biological processes such as cell proliferation, differentiation, gene activation and inhibition. The aim of this study was to investigate the acetylation of LSD1 by Traguodendrine A (TSA), a histone deacetylase inhibitor, and the role of LSD1 in the apoptosis of ovarian cancer cells induced by TSA. Methods Ovarian cancer cell lines HO8910 and SKOV3 with low expression of LSD1 gene were established by RNAi method, and the LSD1-KD group (infected with LSD1-shRNA lentivirus) and LSD1-KD Dox group (100 ng/m L) were used to establish pLKO control group (infected with empty vector lentivirus). The acetylation of LSD1 was detected by immunoprecipitation (IP) and Western blot, and the level of lysine dimethylation (H3K4me2) at the fourth position of histone H3 was detected. Annexin 鈪，
本文编号：2137130
[Abstract]:Objective Lysine specific demethylase 1 (LSD1) is a kind of amine oxidase which is dependent on the adenine dinucleotide of Flavin. LSD1 is involved in many biological processes such as cell proliferation, differentiation, gene activation and inhibition. The aim of this study was to investigate the acetylation of LSD1 by Traguodendrine A (TSA), a histone deacetylase inhibitor, and the role of LSD1 in the apoptosis of ovarian cancer cells induced by TSA. Methods Ovarian cancer cell lines HO8910 and SKOV3 with low expression of LSD1 gene were established by RNAi method, and the LSD1-KD group (infected with LSD1-shRNA lentivirus) and LSD1-KD Dox group (100 ng/m L) were used to establish pLKO control group (infected with empty vector lentivirus). The acetylation of LSD1 was detected by immunoprecipitation (IP) and Western blot, and the level of lysine dimethylation (H3K4me2) at the fourth position of histone H3 was detected. Annexin 鈪，
本文编号：2137130
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