Effects of Silencing EZH2 Gene Expression on Proliferation and Apoptosis of Human Renal Carcinoma Cell Lines
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摘要: 目的 研究RNA干扰靶向沉默EZH2基因表达对ACHN肾癌细胞株增殖、细胞周期和凋亡的影响。方法 应用Western blot法检测EZH2蛋白在人正常近端肾小管上皮细胞株HK-2和肾癌细胞株786-0和ACHN中的表达。脂质体法介导化学合成EZH2 siRNA转染ACHN细胞株，Western blot法检测转染后ACHN细胞EZH2蛋白的表达情况，CCK-8法检测转染后ACHN细胞增殖率，流式细胞术检测转染后ACHN细胞周期分布和凋亡情况。结果 肾癌细胞株786-0和ACHN中EZH2蛋白的表达水平明显高于人正常近端肾小管上皮细胞HK-2。RNA干扰EZH2基因可成功地敲减ACHN细胞EZH2蛋白的表达。EZH2-siRNA干扰后，实验组ACHN细胞生长明显受抑制，在转染后48和72 h细胞增殖受抑制效应著（P＜0.05）。细胞周期分析显示：siEZH2转染组G1期ACHN细胞比例呈增加趋势，而S期和G2期细胞比例呈减少趋势，其中在转染后48h G1期ACHN细胞比例显著增加，而S期和G2期细胞比例明显下降（P＜0.05）；凋亡分析显示：siEZH2转染组ACHN细胞凋亡率随转染后时间的延长而呈增加趋势，在转染后48和72 h细胞凋亡率增加最显著（P＜0.05）。结论 沉默EZH2基因表达可靶向肾癌ACHN细胞周期中G1/S限制点而阻滞细胞周期进展，同时可有效抑制肾癌细胞增殖和促进细胞凋亡。Abstract: Objective To explore the effect of silencing EZH2 expression on the proliferation and apoptosis of human ACHN renal cell carcinoma(RCC) cell lines by RNA interference. Methods The expression profiles of EZH2 protein in normal human proximal kidney tubular epithelial cell line HK-2, RCC cell line 786-0 and ACHN were detected by Western blot. The small interfering RNA(siRNA) was synthesized and mtransfected into ACHN cell line with Lipofectamin2000 for silencing the expression of EZH2. Western blot was used to detect the validity of RNAi on downregulating protein expression of EZH2 in transfected cells. Then the CCK-8 assay was performed for assessing cell proliferation and flow cytometry assay was used to detect cell cycle and apoptosis. Results EZH2 protein expression in 786-0 and ACHN cell lines were in higher levels than those in normal human kidney HK-2 cell line. The protein expression level of EZH2 was effectively downregulated by siRNA. The proliferation rate of ACHN cell transfected with siEZH2 was significantly decreased, especially at 48 and 72h post-transfection(P＜0.05). The flow cytometry analysis showed that there was an increase of cell number at G1 phase and a decrease of cell number at S and G2 phases in ACHN cells transfected with siEZH2, especially at 48h after transfection(P＜0.05). Further apoptosis analysis showed that there was an increase of apoptosis rate of siEZH2-transfected ACHN cells in a timedependent manner, especially at 48 and 72h post-transfection(P＜0.05). Conclusion Knockdown of EZH2 expression by RNAi could inhibit cell cycle progress by arresting cell cycle at G1/S checkpoint, and led to reduced proliferation and increased apoptosis of ACHN RCC cells.
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Key words:
- Renal cell carcinoma(RCC) /
- Proliferation /
- Apoptosis /
- EHZ2
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