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m Cherry-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。
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 andⅠ-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-LC3 B transfection and transmission electron microscopy were used to analyze the changes in autophagy of the cells with Cx43 overexpression. Results Cx43 was significantly decreased inⅠ-10/DDP cells compared withⅠ-10 cells(P<0.01). Transfection of theⅠ-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-LC3 B 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.
作者
苑敏
董淑英
姚言雪
门运政
毛凯锦
童旭辉
YUAN Min;DONG Shuying;YAO Yanxue;MEN Yunzheng;MAO Kaijin;TONG Xuhui(School of Pharmacy,Bengbu Medical College,Bengbu 233030,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2019年第9期1089-1093,共5页
Journal of Southern Medical University
基金
安徽优秀青年人才支持计划重点项目(gxyqZD2016158)
蚌埠医学院研究生科研创新计划项目(Byycxz1805)
蚌埠医学院大学生创新训练项目(201810367031)
关键词
缝隙连接
CX43
自噬
顺铂
睾丸癌
gap junction
connexin43
autophagy
cisplatinum
testis
