The effect of methylated oligonucleotide targeting Ki-67 gene in human 786-0 renal carcinoma cells
The effect of methylated oligonucleotide targeting Ki-67 gene in human 786-0 renal carcinoma cells
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DOI：
10.1007/s13277-011-0187-0
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发表时间：
2011-05
期刊：
影响因子：
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通讯作者：
Xue-qin Li;D. Pei;G. Qian;Xiao‐xing Yin;Qian Cheng;Liantao Li;Huizhong Li;Junnian Zheng
中科院分区：
文献类型：
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作者：
Xue-qin Li;D. Pei;G. Qian;Xiao‐xing Yin;Qian Cheng;Liantao Li;Huizhong Li;Junnian Zheng
为研究Ki-67启动子甲基化寡核苷酸（methylated oligonucleotide， MON）对人786-0肾癌细胞Ki-67基因表达及增殖和凋亡的影响，将MON转染人786-0细胞，采用双荧光素酶报告基因检测系统检测Ki-67启动子活性。在5个甲基化寡核苷酸（MON1-MON5）中，mon4对Ki-67启动子活性的抑制效果最好。与0-nM组相比，40-nM组Ki-67启动子活性分别为77.88%、50.07%、35.63%、26.09%和16.98%。与pGLBK235组相比，MON组Ki-67启动子活性在8 h降至61.96%，12 h降至48.93%，24 h降至15.97%，36 h降至26.00%，48 h降至35.01%，72 h降至46.08%，96 h降至66.12%。这些结果表明，MON的作用具有时间和剂量依赖性。与pGLBK235组相比，MON组Ki-67活性降低至16.73%，而对照组无显著差异。分别用RT-PCR和免疫组织化学方法检测786-0细胞中Ki-67基因的表达。与空白组相比，MON组Ki-67 mRNA表达量下降至61.04%，Ki-67蛋白表达量下降至32.07%。WST-8检测786-0细胞的增殖情况。与空白组相比，MON组24 h、48 h、72 h和96 h的细胞增殖率分别为61.02%、73.78%、79.72%和91.53%。膜联蛋白V和碘化丙啶检测细胞凋亡。MON组早期凋亡细胞数为空白组的2.42倍，中期凋亡细胞数为空白组的2.57倍。Western blot检测MON对bax和p53表达的影响。与空白组相比，MON组bax蛋白的表达增加了66.12%，而p53的表达减少了67.31%。我们的研究表明，靶向Ki-67启动子的甲基化寡核苷酸对人786-0肾癌细胞的Ki-67表达和增殖有显著的抑制作用，并能诱导786-0细胞凋亡。
To investigate the effect of methylated oligonucleotide (MON) targeting Ki-67 promoter on the expression of Ki-67 gene and the proliferation and apoptosis of the human 786-0 renal carcinoma cells, human 786-0 cells were transfected with MON. The activity of Ki-67 promoter was detected by dual-luciferase reporter assay system. Among the five methylated oligonucleotides (MON1–MON5), MON4is the best excellent one in the inhibition of the Ki-67 promoter activity. The activity of Ki-67 promoter is decreased to 77.88% in 40-nM group, 50.07% in 80-nM group, 35.63% in 120-nM group, 26.09% in 160-nM group, and 16.98% in 200-nM group compared with 0-nM group. The activity of Ki-67 promoter in MON group is decreased to 61.96% at 8 h, 48.93% at 12 h, 15.97% at 24 h, 26.00% at 36 h, 35.01% at 48 h, 46.08% at 72 h, and 66.12% at 96 h compared with pGLBK235 group. These results show that the effect of MON is time- and dose-dependent. The activity of Ki-67 in MON group is decreased to 16.73% compared with pGLBK235 group, while the control groups have no significant difference. The expression of Ki-67 gene in 786-0 cells was detected by RT-PCR and immunohistochemistry, respectively. The expression of Ki-67 mRNA is decreased to 61.04% and that of Ki-67 protein is decreased to 32.07% in MON group compared with the blank group. The proliferation of 786-0 cells was determined by WST-8. The cell proliferation in MON group is decreased to 61.02% at 24 h, 73.78% at 48 h, 79.72% at 72 h, and 91.53% at 96 h compared with the blank group. The cell apoptosis was measured by annexin V and propidium iodide. The number of apoptosis cells in MON group is 2.42 times of that in the blank group at earlier period and 2.57 times at mid-anaphase. We detected the effect of MON on the expression of bax and p53 by Western blot. Compared with the blank group, the expression of bax protein in MON group is increased by 66.12%, while the expression of p53 is decreased to 67.31%. Our study demonstrates that the methylated oligonucleotide targeting Ki-67 promoter has a remarkable effect on the inhibition of Ki-67 expression and the proliferation of the human 786-0 renal carcinoma cells and can induce apoptosis of the 786-0 cells.