南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (09): 1089-.doi: 10.12122/j.issn.1673-4254.2019.09.14

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Connexin43 蛋白对耐顺铂睾丸癌I-10/DDP细胞自噬的抑制作用

苑敏，董淑英，姚言雪，门运政，毛凯锦，童旭辉   

  • 出版日期:2019-09-20 发布日期:2019-09-20

Inhibitory effect of connexin43 protein on autophagy in cisplatin-resistant testicular cancer I-10 cells

  • Online:2019-09-20 Published:2019-09-20

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摘要： 目的探讨缝隙连接蛋白（Cx43）蛋白对耐顺铂睾丸癌I-10/DDP细胞自噬的作用。方法免疫印迹法检测Cx43蛋白在睾 丸癌I-10细胞与耐顺铂睾丸癌I-10/DDP细胞中的表达水平；实验分为转染试剂对照组（Mock 组）、阴性对照组（NC组）、过表达 组（mCx43 组），过表达（mCx43）Cx43 基因后，通过免疫印迹法检测Cx43 蛋白确定转染效率，自噬相关蛋白LC3 和p62 水平。 mCherry-GFP-LC3B转染法和透射电镜分析Cx43基因过表达后耐顺铂睾丸癌I-10/DDP细胞自噬水平。结果与I-10细胞相 比，I-10/DDP细胞中Cx43的表达量明显降低（P<0.01）；过表达Cx43基因后，与NC组相比，mCx43组中自噬相关蛋白LC3-II表 达降低（P<0.01），p62表达升高（P<0.05）。使用mCherry-GFP-LC3B转染法和透射电镜观察自噬水平，与NC组相比，mCx43组 中细胞自噬体数量降低。结论Cx43抑制耐顺铂睾丸癌I-10/DDP细胞自噬。

Abstract: Objective To investigate the effect of connexin43 (Cx43) protein on autophagy in cisplatin (DDP)-resistant testicular cancer I-10 cells. Methods The expression of Cx43 proteins in testicular cancer I-10 cells and I-10/DDP cells were detected with Western blotting. I-10/DDP cells were transfected with a full- length mouse Cx43 vector (mCx43) via Lipofectamine2000, the empty vector or Lipofectamine2000 (blank control group), and the changes in the expressions of LC3 and p62 proteins were determined with Western blotting. mCherry-GFP-LC3B transfection and transmission electron microscopy were used to analyze the changes in autophagy of the cells with Cx43 overexpression. Results Cx43 was significantly decreased in I-10/DDP cells compared with I-10 cells (P<0.01). Transfection of the I-10/DDP cells with mCx43 vector resulted in significantly increased Cx43 expression in the cells (P<0.01) and caused significantly decreased expression of LC3-II (P<0.01) and increased expression of p62 (P<0.05) as compared with the negative control cells. Both transmission electron microscopy and mCherry-GFP-LC3B transfection showed that the number of autophagosomes was obviously reduced in mCx43-transfected cells as compared with the negative control cells. Conclusion Cx43 inhibits autophagy in cisplatin-resistant testicular cancer I-10 /DDP cells.