林建贵+庄志明+林天旗+杨明根+刘洪杰+郑周达

[摘要] 目的 探討干扰沉默EZH2基因对肾癌ACHN细胞细胞株的增殖、凋亡及 mTOR信号通路的影响。 方法 设计针对EZH2基因干扰RNA（shRNA），经脂质体将转染EZH2 shRNA入肾癌ACHN细胞，应用RQ-PCR 及 Western blot 鉴定其干扰效果，MTT法绘制细胞生长曲线，TUNEL检测细胞凋亡的变化，Western blot 检测组蛋白H3K27三甲基化水平（H3K27me3），凋亡相关蛋白Bcl-2、Bax、Procaspase-3及mTOR信号通路蛋白mTOR、磷酸化mTOR蛋白（p-mTOR）、磷酸化P70核糖体蛋白S6激酶（phosphorylation P70 ribosomal protein S6 kinase，p-P70S6K）的变化。结果 EZH2 shRNA转染ACHN细胞48 h后，EZH2的 mRNA 和蛋白表达均明显下降，差异有统计学意义（P<0.05），EZH2 shRNA组细胞增殖率明显低于 Neg shRNA 组和Control组（P<0.05），EZH2 shRNA组细胞的凋亡率为（45.57±5.26）%，而Neg shRNA 组和Control组分别为（3.24±1.37）%、（1.65±0.94）%，差异有统计学意义（F=48.62，P<0.05）。促进凋亡蛋白Bax表达增强，凋亡抑制蛋白Bcl-2表达减弱，凋亡执行蛋白前体Procaspase3减少。H3K27me3水平较Control组明显下降，而检测mTOR通路蛋白发现mTOR总蛋白未见明显下降，但是活性形式的磷酸化p-mTOR、p-P70S6K表达下降。结论 干扰沉默EZH2基因可抑制肾癌ACHN细胞增殖，诱导细胞凋亡，其机制可能是调节组蛋白H3K27me3水平变化，特异性抑制mTOR信号通路的活性。

[关键词] EZH2；H3K27me3；肾癌；RNA干扰mTOR信号通路

[中图分类号] R5 [文献标识码] A [文章编号] 1674-0742（2017）12（c）-0001-05

[Abstract] Objective This paper tries to investigate the effect of silencing EZH2 geneon the proliferation， apoptosis andmTOR signalpathway in ACHN human renal cell carcinoma cells. Methods The interference of RNA （shRNA）aiming at EZH2 gene was designed， and EZH2 shRNA were transfected into ACHNhuman renal cell carcinoma cells through lipidosome. The interference effects were identified by RQ-PCR and Western blot， and cell growth curve was drawn by MTT method. Changes in apoptosiswere detected by TUNEL， and the histone H3K27 trimethylation level （H3K27me3）， changes in apoptosis related proteins Bcl-2， Bax and Procaspase-3， mTOR signalpathway proteins mTOR， phosphorylated mTOR protein （p-mTOR） and phosphorylation P70 ribosomal protein S6 kinase （p-P70S6K） were detected by Western blot. Results The mRNA and protein of EZH2 were markedly decreased at 48 h after EZH2 shRNA being transfected into ACHN human renal cell carcinoma cells（P<0.05）. The cell proliferation rate in EZH2 shRNA group was significantly lower than that in Neg shRNA group and Control group （P<0.05）. The cell apoptosis rate inEZH2shRNA group was （45.57±5.26）%， while the rate in Neg shRNA group and Control group were （3.24±1.37）% and （1.65±0.94）%， respectively （F=48.62， P<0.05）. The expression of protein Baxpromoting apoptosiswas increased， the expression of Bcl-2 inhibiting apoptosis was weakened， and the apoptosis precursor protein Procaspase3 was decreased. The level of H3K27me3 was significantly lower than that in control group while the detection of mTOR pathway protein showed that the total protein of mTOR was not decrease significantly. Besides， the expression of phosphorylated p-mTOR and p-P70S6K in active forms was decreased. Conclusion Silencing EZH2 gene can inhibit the proliferation and induce apoptosis of ACHN human renal cell carcinoma cells， and the mechanism may be regulatingchanges in the level of histone H3K27me3 and specifically inhibiting the activity of mTOR signaling pathway.endprint

[Key words] EZH2； H3K27me3； Renal cell carcinoma； RNA interference mTOR singalpathway

肾癌是泌尿系统常见的肿瘤之一，我国泌尿生殖系恶性肿瘤中占第2位，发病率仅次于膀胱癌。但对于肾癌的發生、发展、侵袭转移的确切的分子机制尚未完全清楚。目前多项研究证实，表观遗传学修饰异常是恶性肿瘤发生、发展、侵袭转移的重要原因之一[1]。EZH2（Enhancer of Zeste Homolog 2）基因是果蝇 E（z）在人类的同源基因，EZH2基因是PcG（Polycomb group）基因家族的重要成员之一，特异性完成H3K27三甲基化修饰。EZH2在多种肿瘤组织中高表达，与肿瘤的进展及预后相关[2-3]。该次实验通过shRNA干扰沉默人肾癌ACHN细胞EZH2基因，观察其对ACHN细胞和mTOR信号通路的影响，以寻求EZH2基因在肾癌靶向治疗中的可能性。

1 材料与方法

1.1 实验材料

实验细胞株来自科学院上海细胞库，RPMI-1640培养基、胎牛血清来自美国Gibico公司，RNA提取试剂盒、RQ-PCR试剂盒、TUNNEL试剂盒、Lipofectamine 2000均购自美国Invitrogen公司，MTT（浓度为5 mg/mL）及二甲基亚砜均购自美国Sigma公司，EZH2、三甲基化H3K27（H3K27me3）一抗购自美国Upstate公司。Bcl-2、Bax、Procaspase-3、mTOR、磷酸化mTOR蛋白（p-mTOR）、磷酸化P70核糖体蛋……