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目的探讨组蛋白赖氨酸N端甲基转移酶SET结构域分支型1(SETDB1)对人肾癌细胞增殖、凋亡的影响。方法体外培养肾癌ACHN细胞及正常肾小管上皮HK-2细胞,qRT-PCR及Western blot分别检测两种细胞系中SETDB1 mRNA及蛋白的表达。采用转染SETDB1特异性干扰性小核糖核酸(siRNA)敲低ACHN细胞中SETDB1表达,并采用实时定量聚合酶链反应(qRT-PCR)、Western blot检测SETDB1的敲低效果,CCK-8法、溴脱氧尿嘧啶核苷(Brd U)结合试验检测细胞活力与增殖,流式细胞仪、caspase3/7活性分析检测细胞凋亡的变化。采用qRTPCR、Western blot检测沉默SETDB1后p53 mRNA及蛋白的表达水平。结果 SETDB1在肾癌ACHN细胞中表达水平明显高于正常肾小管上皮HK-2细胞(t=14.435,P<0.01);转染SETDB1特异性siRNA沉默SETDB1表达后,ACHN细胞中SETDB1 mRNA(t=13.091,P<0.01)及其蛋白表达水平明显降低;ACHN细胞活力显著降低(P<0.05);细胞内DNA复制活性明显降低(P<0.01);细胞凋亡率增加(t=6.765,P<0.01);Csapase3/7酶活性提高(t=5.763,P<0.01);同时p53 mRNA(t=9.241,P<0.01)及蛋白的表达水平显著提高。结论 SETDB1在肾癌细胞中表达升高,下调SETDB1能够通过促进p53表达而发挥抗肿瘤生长作用。
Objective To investigate the effect of SETDB1 on the proliferation and apoptosis of human renal cell carcinoma cells. Methods The human renal cell carcinoma ACHN cells and normal renal tubular epithelial HK-2 cells were cultured in vitro. The expressions of SETDB1 mRNA and protein in the two cell lines were detected by qRT-PCR and Western blot respectively. SETDB1 expression in ACHN cells was knocked down by transfected with SETDB1-specific siRNA, and the knockdown effect of SETDB1 was detected by real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot. CCK-8, BrdU binding assay was used to detect cell viability and proliferation. Flow cytometry and caspase3 / 7 activity assay were used to detect the changes of apoptosis. The qRTPCR and Western blot were used to detect the expression of p53 mRNA and protein after SETDB1 silencing. Results The expression of SETDB1 in ACHN cells was significantly higher than that in normal renal tubular epithelial HK-2 cells (t = 14.435, P <0.01). After SETDB1 siRNA was silenced by SETDB1, the expression of SETDB1 mRNA (t = 13.091, P <0.01). The ACHN cell viability was significantly decreased (P <0.05), the DNA replication activity was significantly decreased (P <0.01), and the apoptosis rate was increased (t = 6.765, P < 0.01). The activity of Csapase 3/7 was increased (t = 5.763, P <0.01), while p53 mRNA (t = 9.241, P <0.01) and protein expression were significantly increased. Conclusion The expression of SETDB1 is up-regulated in renal cell carcinoma. Down-regulation of SETDB1 can exert anti-tumor growth by promoting p53 expression.