SATB1-shRNA下调Bc12表达诱导A549细胞凋亡
发布时间：2018-11-21 18:26
【摘要】：目的：应用RNAi干扰技术干扰肺癌A549细胞株中特异性AT序列结合蛋白-1(Special AT-rich sequence-binding protein-1，，SATB1)基因的表达，探讨其对A549细胞凋亡的影响。方法：设计合成针对SATB1基因的shRNA表达质粒(SATB1-shRNA)。采用阳离子脂质体法将SATB1-shRNA转染入肺癌A549细胞。分别采用RT-PCR、Western-Blot法检测SATB1-shRNA转染A549细胞中SATB1、Bcl2、BAX及Caspase-3的表达。FCM检测SATB1-shRNA转染的A549细胞凋亡率。结果：经凝胶电泳、 DNA测序鉴定分析，成功构建了SATB1-shRNA。 RT-PCR和Western-Blot结果显示SATB1-shRNA转染成功后可显著下调A549细胞中SATB1mRNA和蛋白的表达水平（P0.05）。RT-PCR和Western-Blot结果显示Bcl2表达水平下降（P0.05），SATB1-shRNA转染组BAX、Caspase-3表达水平上调（P0.05）。FCM检测结果示SATB1-shRNA转染组细胞凋亡率显著增加（P0.05）。结论：SATB1-shRNA经转染肺癌A549细胞能有效抑制细胞中SATB1mRNA和蛋白的表达水平，诱导细胞凋亡，其机制可能与下调Bcl2基因表达所引起的级联效应有关。
[Abstract]:Aim: to investigate the effect of RNAi interference on the expression of specific AT sequence binding protein-1 (Special AT-rich sequence-binding protein-1,SATB1) gene in A549 cell line. Methods: shRNA expression plasmid (SATB1-shRNA) targeting SATB1 gene was designed and synthesized. SATB1-shRNA was transfected into lung cancer A549 cells by cationic liposome method. RT-PCR,Western-Blot assay was used to detect the expression of SATB1,Bcl2,BAX and Caspase-3 in A549 cells transfected with SATB1-shRNA, and FCM was used to detect the apoptosis rate of A549 cells transfected with SATB1-shRNA. Results: SATB1-shRNA. was successfully constructed by gel electrophoresis and DNA sequencing. RT-PCR and Western-Blot showed that SATB1-shRNA transfection could significantly down-regulate the expression of SATB1mRNA and protein in A549 cells (P0.05). RT-PCR and Western-Blot showed a decrease in Bcl2 expression (P0.05), and SATB1-shRNA transfected BAX,. The expression of Caspase-3 was up-regulated (P0.05). FCM results showed that the apoptotic rate of SATB1-shRNA transfection group was significantly increased (P0.05). Conclusion: transfection of SATB1-shRNA into lung cancer A549 cells can effectively inhibit the expression of SATB1mRNA and protein and induce apoptosis. The mechanism may be related to the cascade effect caused by down-regulation of Bcl2 gene expression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2347891
[Abstract]:Aim: to investigate the effect of RNAi interference on the expression of specific AT sequence binding protein-1 (Special AT-rich sequence-binding protein-1,SATB1) gene in A549 cell line. Methods: shRNA expression plasmid (SATB1-shRNA) targeting SATB1 gene was designed and synthesized. SATB1-shRNA was transfected into lung cancer A549 cells by cationic liposome method. RT-PCR,Western-Blot assay was used to detect the expression of SATB1,Bcl2,BAX and Caspase-3 in A549 cells transfected with SATB1-shRNA, and FCM was used to detect the apoptosis rate of A549 cells transfected with SATB1-shRNA. Results: SATB1-shRNA. was successfully constructed by gel electrophoresis and DNA sequencing. RT-PCR and Western-Blot showed that SATB1-shRNA transfection could significantly down-regulate the expression of SATB1mRNA and protein in A549 cells (P0.05). RT-PCR and Western-Blot showed a decrease in Bcl2 expression (P0.05), and SATB1-shRNA transfected BAX,. The expression of Caspase-3 was up-regulated (P0.05). FCM results showed that the apoptotic rate of SATB1-shRNA transfection group was significantly increased (P0.05). Conclusion: transfection of SATB1-shRNA into lung cancer A549 cells can effectively inhibit the expression of SATB1mRNA and protein and induce apoptosis. The mechanism may be related to the cascade effect caused by down-regulation of Bcl2 gene expression.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
【参考文献】
相关期刊论文 前1条
1 周来勇;刘芳;童健;陈群请;张福伟;郭琳琅;;实时荧光定量RT-PCR分析非小细胞肺癌SATB1的表达和临床病理意义[J];南方医科大学学报;2009年03期
本文编号：2347891
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