南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (08): 1090-1096.doi: 10.12122/j.issn.1673-4254.2020.08.04

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下调Pannexin2通道能增强顺铂诱导睾丸癌（I-10）细胞凋亡

姚言雪，董淑英，朱晨露，胡 淼，杜宝龙，童旭辉   

  • 出版日期:2020-08-20 发布日期:2020-08-20
  • 基金资助:

Down-regulation of pannexin 2 channel enhances cisplatin-induced apoptosis in testicular cancer I-10 cells

  

  • Online:2020-08-20 Published:2020-08-20

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摘要： 目的 探讨Pannexin2（Panx-2）蛋白在睾丸癌细胞中的表达水平以及干扰Panx-2表达对顺铂诱导睾丸癌（I-10）细胞凋亡的影响。方法 免疫印迹法Panx-2蛋白在睾丸癌细胞中的表达水平；以睾丸癌I-10细胞株为研究对象，实验分为转染试剂对照组（mork组）、阴性对照质粒组（NC组）、干扰Panx-2组（shRNA1质粒组和shRNA2质粒组），采用免疫印迹法验证Panx-2的表达；在转染细胞中添加16 μmol/L的顺铂诱导细胞死亡，通过MTT法分别检测细胞在24、48、72 h的存活率，集落克隆法检测细胞的集落形成能力；在顺铂（16 μmol/L）处理8 h后，采用AnnexinV/PI双染法检测细胞的早期凋亡；在顺铂（16 μmol/L）作用24 h后， 免疫印迹法检测细胞凋亡相关蛋白caspase 3、Bcl-2和Bax的表达水平。结果 与I-10细胞和Tcam-2细胞相比，I-10/DDP细胞（P< 0.001）和Tcam-2/DDP细胞（P<0.01）中Panx-2的表达显著增加；干扰Panx-2基因后，免疫印迹结果显示，与NC组相比，shRNA1和shRNA2组中Panx-2的表达量下降（P<0.05）；MTT法显示shRNA1和shRNA2组细胞的存活率均低于NC组（P<0.001）；在含顺铂培养基的培养下，集落克隆法显示shRNA1和shRNA2组细胞的集落形成能力低于NC组（P<0.001）；AnnexinV/PI双染法显示shRNA1和shRNA2组的早期凋亡率（P<0.01）均低于NC组；免疫印迹结果显示，与NC组相比，shRNA1和shRNA2组中凋亡相关蛋白caspase 3表达升高（P<0.05），Bcl-2表达降低（P<0.01），Bax表达升高（P<0.05）。结论 下调Pannexin2通道能增强顺铂诱导睾丸癌（I-10）细胞凋亡。

Abstract: Objective To investigate the effect of down-regulation of pannexin 2 (Panx-2) channels on cisplatin-induced apoptosis in I-10 cells. Methods The expression of Panx-2 protein in testicular cancer cells was detected with Western blotting. The testicular cancer cell line I-10 was transfected with two short hairpin RNA (shRNA1 and shRNA2) via Lipofectamine2000, the empty vector (NC group) or Lipofectamine2000 (blank control group), and the changes in the expression of Panx-2 was detected with Western blotting. The effects of transfection with a Panx-2 inhibitor on surviving fraction of the cells treated with cisplatin (16 μmol/L) for 24 h, 48 h and 72 h was assessed with MTT assay, and the clonogenic capacity of the cells was evaluated with colony-forming assay. At 8 h after incubation with 16 μmol/L cisplatin, AnnexinV/PI double staining was used to detect the early apoptosis of the cells. After 24 h of treatment with 16 μmol/L cisplatin, the cells were examined for expressions of caspase-3, Bcl-2 and Bax using Western blotting. Results The expression of Panx-2 was significantly increased in cisplatin-resistant I-10/DDP (P<0.001) cells and Tcam-2/DDP (P<0.01) cells as compared with I-10 cells and Tcam-2 cells. Transfection of I-10 cells with shRNA1 and shRNA2 resulted in significantly decreased Panx-2 expression (P<0.05) and significantly reduced cell surviving fraction (P<0.001). In the presence of cisplatin, the cells in NC group showed a higher clonogenic efficiency than those in shRNA1 and shRNA2 groups (P<0.001). The early-stage apoptosis rate of the cells in shRNA1 and shRNA2 groups were significantly higher than that in NC group (P<0.01). Panx-2 knockdown in I-10 cells significantly increased caspase-3 and Bax expressions (P<0.05) and significantly decreased the expression of Bcl-2 (P<0.01). Conclusion Down-regulation of Panx-2 channel enhances cisplatin-induced apoptosis in cultured testicular cancer cells.