摘要
目的:应用小干扰RNA(small interferingRNA,siRNA)沉默TET1(ten-eleven translocation1)基因表达,探索其对人肾癌786-O细胞增殖的影响及其可能的机制。方法:采用脂质体转染法将靶向TET1基因的TET1-siRNA转染人肾癌786-O细胞,实验设空白对照组、阴性对照组和干扰组(TET1-siRNA组)。分别采用实时荧光定量PCR和蛋白质印迹法检测转染TET1-siRNA后,786-O细胞中TET1、SUZ12(suppressor of zeste12)、EZH2(enhancer of zestehomolog 2)和EED(embryonic ectoderm development)mRNA及其蛋白的表达;MTT法和克隆形成实验检测786-O细胞的增殖活性和克隆形成率;FCM检测786-O细胞周期。结果:与空白对照组比较,转染TET1-siRNA后,786-O细胞中TET1 mRNA及其蛋白的表达水平分别下降了(85.13±0.03)%和(56.41±0.09)%,差异有统计学意义(P<0.05);786-O细胞的增殖受到抑制,克隆形成能力明显下降,G0/G1期细胞百分比明显增加(P<0.05);SUZ12 mRNA及其蛋白表达水平下调(P<0.05),EED和EZH2蛋白表达水平变化不明显(P>0.05)。结论:TET1-siRNA可显著下调肾癌786-O细胞中TET1蛋白的表达水平,明显抑制786-O细胞的增殖,并使细胞阻滞于G1期,其机制可能与TET1调控SUZ12的表达,影响其靶基因对PRC2(polycomb repressive complex 2)的招募有关。
Objective： To silence the expression of TET1 （ten-eleven translocation 1） gene by siRNA （small interfering RNA） and to investigate its impact on the proliferation of human renal cancer 786-O cells and explore its possible mechanism. Methods： The TET1-siRNA targeting human TET1 gene was transfected into 786-O cells by LipofectAMINE 2000.Three groups were designed as blank control, negative control and siRNA interference （TET1-siRNA） in this study. The expression levels of TET1, SUZ12 （suppressor of zeste 12）, EZH2 （enhancer of zeste homolog 2） and EED （embryonic ectoderm development） mRNAs and proteins after transfection with TET1-siRNA were determined by real-time fluorescence quantitative-PCR and Western blotting, respectively. The MTT method, colony formation assay and flow cytometry were performed to detect the proliferation ability, cell colony formation rate and cell cycle distribution of 786-O cells, respectively. Results： As compared with the blank control, the expression levels of TET1 mRNA and protein in 786-O cells were reduced by （85.13±0.03）% and （56.41±0.09）% respectively after transfection with TET1-siRNA （P 0.05）. The proliferation of 786-O cells was inhibited and the ability of colony-formation was also weakened. The cell cycle of 786-O cells was arrested at G0/G1 phase （P 0.05）. The expression levels of SUZ12 mRNA and protein were reduced significantly （P 0.05）, meanwhile the expression levels of EED and SUZ12 proteins had no change （P 0.05）. Conclusion： TET1-siRNA can down-regulate the expression levels of TET1 mRNA and protein in human renal cancer 786-O cells, suppress the proliferation of 786-O cells, and block the cells at G1 phase. This mechanism may be associated with the recruitment of PRC2 （polycomb repressive complex 2） influenced by TET1 which regulates the expression of SUZ12.
出处
《肿瘤》
CAS
CSCD
北大核心
2012年第12期962-968,共7页
Tumor
基金
国家自然科学基金资助项目(编号:81171883)
关键词
肾肿瘤
细胞增殖
RNA干扰
TET
SUZ12
Kidney neoplasms
Cell proliferation
RNA interference
Ten-eleven translocation
Suppressor of zeste 12
