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目的探讨肾细胞癌中线粒体促凋亡蛋白BNIP3表达下调机制。方法运用CCK-8法及流式细胞技术检测组蛋白去乙酰化酶抑制剂曲古霉素A(TSA)对肾癌细胞株786-O、ACHN、A498增殖、凋亡的影响;运用实时荧光定量PCR(Q-PCR)和蛋白质免疫印记(Western blot)技术检测TSA对肾癌细胞BNIP3表达的影响;运用染色质免疫沉淀(ChIP)技术检测TSA对肾癌细胞BNIP3基因启动子区组蛋白H3乙酰化状态的影响。结果经TSA处理后,3种肾癌细胞增殖均受到显著抑制(P<0.05),细胞早期凋亡明显增加,BNIP3mRNA(P<0.05)和蛋白表达水平上调。786-O、ACHN的BNIP3基因启动子区组蛋白H3呈去乙酰化状态,TSA恢复了其乙酰化状态;A498的BNIP3基因启动子区组蛋白H3呈乙酰化状态。结论 BNIP3基因启动子区组蛋白去乙酰化是其在肾癌中表达下调的主要机制,并可能参与了肾癌的发生发展。
Objective To investigate the down-regulation mechanism of mitochondrial apoptosis-promoting protein BNIP3 in renal cell carcinoma. Methods CCK-8 and flow cytometry were used to detect the effect of histone deacetylase inhibitor (TSA) on the proliferation and apoptosis of renal cell carcinoma 786-O, ACHN and A498 cells. Real-time fluorescence The effect of TSA on the expression of BNIP3 in kidney cancer cells was detected by quantitative PCR (Q-PCR) and Western blotting. ChIP was used to detect the effect of TSA on the expression of BNIP3 promoter region histone H3 The effect of acetylation status. Results After TSA treatment, the proliferation of all three cell lines was significantly inhibited (P <0.05). The apoptosis of the three cell lines was significantly increased, and the expression of BNIP3 mRNA (P <0.05) and protein were up-regulated. 786-O, ACHN BNIP3 gene promoter region histone H3 was deacetylated, TSA restored its acetylation status; A498 BNIP3 promoter region histone H3 acetylated state. Conclusion The histone deacetylation of BNIP3 gene promoter region is the main mechanism of its downregulation in renal cell carcinoma and may be involved in the development of renal cell carcinoma.