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TRPM8通道介导的自噬调节可能通过JNK信号通路影响肾癌细胞的存活
作者:付婷1  熊毅2 
单位:1. 湖北医药学院附属十堰市人民医院肿瘤科, 湖北 十堰 442000;
2. 湖北医药学院附属十堰市人民医院手术室, 湖北 十堰 442000
关键词:瞬时受体电位M8 自噬 JNK信号通路 肾癌细胞 增殖 凋亡 
分类号:R737.11
出版年·卷·期(页码):2020·39·第三期(348-353)
摘要:

目的:探讨瞬时受体电位M8(TRPM8)与肾癌细胞增殖及凋亡的关系。方法:qRT-PCR检测肾癌细胞(A498、GRC-1、786-0)中TRPM8的表达水平。利用shRNA转染A498细胞来构建TRPM8低表达的稳定细胞株,qRT-PCR和Western blotting验证shRNA的干扰效率。细胞集落形成实验检测沉默TRPM8后A498细胞的增殖能力;流式细胞术检测沉默TRPM8后A498细胞的凋亡能力;自噬检测试剂盒检测沉默TRPM8后A498细胞的自噬通量的变化;Western blotting检测沉默TRPM8后自噬相关标志物(ATG3、ATG5、ATG6、ATG7和ATG12)的变化和c-Jun N末端激酶(JNK)通路相关蛋白的表达。结果:TRPM8在肾癌细胞中高表达;转染TRPM8 shRNA后,TRPM8的表达明显降低(P<0.01);沉默TRPM8后,A498细胞的增殖能力明显下降(P<0.01),凋亡率明显增高(P<0.01);转染TRPM8 shRNA,A498细胞的自噬通量也明显减少(P<0.05),且ATG3、ATG5、ATG6、ATG7和ATG12蛋白的表达也下降(均P<0.05);沉默TRPM8后,JNK的磷酸化水平降低(P<0.05);使用JNK抑制剂(SP600125)后,ATG5和ATG7蛋白水平明显降低(均P<0.05)。结论:沉默TRPM8通道介导的自噬调节通过JNK信号通路可抑制肾癌细胞的增殖,促进其凋亡。

Objective:To investigate the effect of TRPM8 on the proliferation and apoptosis of renal cancer cells through the JNK signaling pathway through the regulation of autophagy. Methods:qRT-PCR was used to detect the expression of TRPM8 in renal cancer cells (A498, GRC-1, 786-0); Transfection of A498 cells with shRNA to construct stable cell lines with low expression of TRPM8; qRT-PCR and Western blotting was used to verify the interference efficiency of shRNA; Cell colony formation assay was used to detect A498 cell proliferation after silencing TRPM8; Cell flow cytometry was used to detect the apoptotic capacity of A498 cells after silencing TRPM8; Autophagy detection kit was used to detect changes in autophagy flux of A498 cells after silencing TRPM8; Western blotting was used to detect changes in autophagy-related markers (ATG3, ATG5, ATG6, ATG7, and ATG12) and expression of JNK pathway-related proteins after silencing TRPM8. Results:TRPM8 was highly expressed in renal cancer cells, and TRPM8 expression was significantly reduced after transfection of TRPM8 shRNA (P<0.01); After TRPM8 was silenced, the proliferation ability of A498 cells decreased significantly (P<0.01), and the apoptosis rate increased significantly (P<0.01); Transfection of TRPM8 shRNA and A498 cells also significantly reduced autophagy flux (P<0.05), and ATG3, ATG5, ATG6, ATG7 and ATG12 protein expressions also decreased (all P<0.05); After TRPM8 silencing, the level of phosphorylation of JNK decreased (P<0.05). After using JNK inhibitor (SP600125), the levels of ATG5 and ATG7 protein decreased significantly (both P<0.05). Conclusion:Silent TRPM8 channel-mediated autophagy regulation inhibits the proliferation of renal cancer cells through the JNK signaling pathway and promotes their apoptosis.

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