ERCC1和BRCA2基因共沉默显著增强顺铂对耐药肺癌细胞的细胞毒性

发布时间：2018-04-05 09:32

  本文选题：肺癌细胞　切入点：ERCC1　出处：《江苏大学》2017年硕士论文

【摘要】：目的探讨分别单沉默和共沉默ERCC1和BRCA2基因后顺铂(DDP)对耐药肺腺癌细胞(A549/DDP)的细胞毒性变化及其可能的机制?方法运用细胞脂质体转染技术将靶向于ERCC1和BRCA2基因的siRNAs(ERCC1-siRNA和BRCA2-siRNA)分别和同时转染于体外培养的A549/DDP细胞。Western Blot检测该细胞转染前后ERCC1和BRCA2蛋白表达水平以确认转染成功。用不同浓度DDP处理A549/DDP细胞,测定ERCC1基因沉默前后FANCD2单泛素化水平(以FANCD2-L/S比值表示),以及BRCA2基因沉默前后BRCA1和RAD51蛋白的表达水平。同时测定DDP敏感肺腺癌细胞(A549细胞)经不同浓度的DDP诱导后FANCD2单泛素化、BRCA1和RAD51蛋白的表达水平。应用CCK-8法和Annexin V/PI流式细胞术分别测定ERCC1和BRCA2基因单沉默及共沉默前后经不同浓度DDP处理的A549/DDP细胞增殖率与凋亡率变化。免疫荧光染色法分别测定ERCC1基因沉默前后的FANCD2核聚小体表达及BRCA2基因沉默前后RAD51小体表达。结果(1)Western Blot结果显示,应用ERCC1-siRNA和BRCA2-siRNA分别转染A549/DDP细胞后ERCC1和BRCA2蛋白的表达水平均明显低于转染前(P均0.05),表明ERCC1-siRNA和BRCA2-siRNA转染有效,ERCC1和BRCA2基因被沉默。(2)Western Blot和免疫荧光法结果显示,A549/DDP细胞的FANCD2单泛素化水平、BRCA1和RAD51蛋白的表达水平随着DDP浓度的增加总体上呈上升趋势,而A549细胞并未显示出这一趋势,提示范可尼贫血(FA)通路和同源重组修复(HRR)通路参与了A549/DDP细胞对DDP的耐药机制。ERCC1基因沉默后经DDP处理的FANCD2蛋白单泛素化水平和核聚小体表达较沉默前均显著下降(P均0.05);BRCA2基因沉默后经DDP处理的BRCA1和RAD51蛋白表达及RAD51核聚小体表达水平亦较沉默前显著下降(P均0.05),表明FA通路和HRR通路DNA修复功能受到抑制。(3)CCK-8法和流式细胞术测定结果示,分别沉默ERCC1基因和BRCA2基因后均能增强DDP对A549/DDP细胞的增殖抑制作用和促凋亡作用(P均0.05),这两个基因共沉默较单基因沉默可进一步增强DDP对A549/DDP的细胞毒性作用(P均0.05)?这一结果表明同时沉默ERCC1和BRCA2基因在增强DDP对耐药肺癌细胞的杀瘤效应方面具有协同作用。结论沉默ERCC1与BRCA2基因均可逆转耐药肺癌细胞A549/DDP对DDP的耐药性,且两个基因共沉默这种逆转DDP耐药的作用更明显。这种逆转耐药作用主要是通过抑制FA通路和HRR通路的DNA损伤修复功能功能来实现的。
[Abstract]:Objective to investigate the cytotoxicity of cisplatin (DDP) after single silencing and co-silencing of ERCC1 and BRCA2 genes to drug-resistant lung adenocarcinoma cell line A549 / DDP and its possible mechanism.Methods siRNAs(ERCC1-siRNA and BRCA2-siRNAs targeting ERCC1 and BRCA2 genes were detected by cell liposome transfection technique, and the expression levels of ERCC1 and BRCA2 protein were detected by Western Blot before and after transfection.A549/DDP cells were treated with different concentrations of DDP. The level of FANCD2 monoubiquitin (expressed as FANCD2-L/S ratio) before and after ERCC1 gene silencing and the expression of BRCA1 and RAD51 protein before and after BRCA2 gene silencing were measured.At the same time, the expression levels of FANCD2 monoubiquitin B BRCA1 and RAD51 protein were detected in DDP sensitive lung adenocarcinoma cell line (A549) induced by different concentrations of DDP.CCK-8 assay and Annexin V/PI flow cytometry were used to detect the proliferation and apoptosis of A549/DDP cells treated with different concentrations of DDP before and after ERCC1 and BRCA2 gene single silencing and co-silencing.The expression of FANCD2 nucleopolymer before and after ERCC1 gene silencing and the expression of RAD51 corpuscle before and after BRCA2 gene silencing were detected by immunofluorescence staining.Results Western Blot showed that,搴旂敤ERCC1-siRNA鍜孊RCA2-siRNA鍒嗗埆杞�鏌揂549/DDP缁嗚優鍚嶦RCC1鍜孊RCA2铔嬬櫧鐨勮〃杈炬按骞冲潎鏄庢樉浣庝簬杞�鏌撳墸�

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