【摘要】：目的賴氨酸特異性去甲基化酶1(LSD1)是黃素腺嘌呤二核苷酸依賴的胺氧化酶,其參與細(xì)胞的增殖、分化以及介導(dǎo)基因激活和抑制等多種生物學(xué)過程。文中旨在探討組蛋白去乙�；敢种苿┣乓志谹(TSA)對(duì)LSD1的乙�；�,以及LSD1在TSA誘導(dǎo)卵巢癌細(xì)胞凋亡中的作用。方法利用RNA干擾方法建立誘導(dǎo)型穩(wěn)定敲低LSD1基因表達(dá)的卵巢癌HO8910和SKOV3細(xì)胞株;實(shí)驗(yàn)設(shè)置p LKO對(duì)照組(感染空載體慢病毒)、p LKO+Dox(100 ng/m L)組、LSD1-KD組(感染LSD1-shRNA慢病毒)和LSD1-KD+Dox(100 ng/m L)組。用免疫沉淀(IP)和Western blot檢測(cè)LSD1乙酰化程度,及其底物組蛋白H3第4位賴氨酸的二甲基化(H3K4me2)水平;AnnexinⅤ/PI和流式細(xì)胞術(shù)分析細(xì)胞凋亡的變化;RT-PCR檢測(cè)凋亡相關(guān)基因Bax和p21 mRNA表達(dá)水平;染色質(zhì)免疫沉淀(Ch IP)分析Bax和p21基因啟動(dòng)子區(qū)H3K4me2程度。實(shí)驗(yàn)設(shè)置methanol對(duì)照組(2 mg/m L)、TSA組(200 nmol/L)、TCP組(抑制LSD1活性;100μmol/L)和TSA+TCP組,處理細(xì)胞48 h。在RNA干擾LSD1表達(dá)實(shí)驗(yàn)中,設(shè)置溶劑對(duì)照組(2 mg/m L methanol)、TSA處理組(200 nmol/L)、Dox組(干擾LSD1表達(dá);100 ng/m L)和聯(lián)合處理組(200 nmol/L TSA與100ng/m L Dox)聯(lián)合處理細(xì)胞48h。結(jié)果免疫沉淀結(jié)合Western blot檢測(cè)結(jié)果顯示,200nmol/L TSA處理后LSD1蛋白的乙�；虷3K9的乙酰化水平較methanol溶劑處理顯著增加(P0.01)。與methanol溶劑對(duì)照處理比較,200nmol/L TSA處理HO8910和SKOV3細(xì)胞后H3K4me2水平明顯升高(P0.01)。AnnexinⅤ/PI結(jié)果顯示,與methanol對(duì)照組比較,TSA組、TCP組和TSA+TCP組HO8910和SKOV3細(xì)胞的總凋亡率均顯著升高(P0.05);且TSA+TCP組較TSA組和TCP組細(xì)胞凋亡率明顯升高(P0.05)。LSD1-KD+Dox組細(xì)胞中LSD1蛋白水平(HO8910:0.21±0.16;SKOV3:0.26±0.11)較p LKO對(duì)照組(HO8910:1.15±0.16;SKOV3:0.97±0.31)和LSD1-KD組(HO8910:1.07±0.19;SKOV3:0.98±0.21)顯著減少(P0.01)。與溶劑對(duì)照組比較,TSA處理組、Dox組和聯(lián)合處理組細(xì)胞的總凋亡率均顯著增高(P0.05);其中聯(lián)合處理組較TSA或Dox組亦顯著增高(P0.05)。RT-PCR結(jié)果表明,在HO8910細(xì)胞中,與溶劑對(duì)照組Bax和p21 mRNA水平比較,TSA處理組、Dox組及聯(lián)合處理組均顯著上調(diào)(P0.05);且聯(lián)合處理組較TSA處理組或Dox組亦顯著上調(diào)(P0.05)。TSA處理組細(xì)胞中Bax和p21基因啟動(dòng)子區(qū)H3K4me2水平較methanol對(duì)照組顯著升高(Bax:2.92±0.26 vs 0.86±0.19;p21:3.07±0.29 vs 0.93±0.17,P0.01)。結(jié)論 TSA誘導(dǎo)了LSD1蛋白的乙酰化,抑制LSD1表達(dá)或活性可增強(qiáng)TSA對(duì)卵巢癌細(xì)胞的殺傷作用。
[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，

本文編號(hào)：2137130