沉默ATG5及ATG7可抑制顺铂介导的耐顺铂睾丸癌细胞的自噬和增殖

Inhibiting autophagy by silencing ATG5 and ATG7 enhances inhibitory effect of DDP on DDP-resistant Ⅰ-10 testicular cancer cells

目的观察顺铂作用后,耐顺铂睾丸癌Ⅰ-10/DDP细胞自噬水平的变化;探讨沉默ATG5、ATG7对顺铂作用后的自噬及细胞增殖的影响。方法顺铂(15μmol/L,12 h)作用Ⅰ-10/DDP细胞后,免疫印迹法检测LC3、p62的表达水平;透射电子显微镜和mCherry-GFP-LC3B双标法检测自噬水平。实验分为转染试剂对照组(Mork组)、阴性对照质粒组(NC组)、转染shRNA-ATG5质粒组(shRNA-ATG5组)、转染shRNA-ATG7质粒组(shRNA-ATG7组),免疫印迹法检测ATG5、ATG7的表达。顺铂(15μmol/L,12 h)作用转染组细胞后,免疫印迹法检测LC3、p62的表达;透射电子显微镜、mCherry-GFP-LC3B双标法检测自噬水平;MTT法检测细胞存活率;集落克隆实验检测细胞增殖情况。结果顺铂处理后,Ⅰ-10/DDP细胞中LC3Ⅱ的表达水平明显增加(P=0.001),p62的表达水平降低(P=0.01),自噬体数量增加。与NC组相比,shRNA-ATG5组、shRNA-ATG5组中ATG5、ATG7的表达量明显降低(P=0.005,P<0.001)。与单用顺铂组相比,shRNA-ATG5组与shRNA-ATG7组细胞用顺铂处理后LC3Ⅱ表达水平明显降低(P<0.001),p62的表达水平明显增加(P<0.001),自噬体数量减少、细胞存活率(P<0.001)及克隆形成能力均降低。结论沉默ATG5、ATG7后抑制顺铂介导的自噬,增强顺铂抑制细胞增殖的作用。

Objective To observe the changes in autophagy of cisplatin-resistant Ⅰ-10 testicular cancer cells(Ⅰ-10/DDP cells) in response to cisplatin treatment and the effect of silencing ATG5 and ATG7 on autophagy and proliferation of cisplatin-treated cells. Methods Ⅰ-10/DDP cells treated with 15 μmol/L cisplatin for 12 h were examined for expressions of LC3 and p62 by Western blotting and for autophagy level through transmission electron microscopy and mCherry-GFP-LC3 B. Ⅰ-10/DDP cells were transfected with short hairpin RNAs shRNA-ATG5 or shRNA-ATG7 via Lipfectamine2000, the empty vector(NC group),or Lipfectamine2000 alone(blank control group), and the cellular expressions of ATG5 and ATG7 were detected with Western blotting. The transfected cells were treated with 15 μmol/L cisplatin for 12 h, after that the expressions of LC3 and p62 were detected with Western blotting. Transmission electron microscopy and mCherry-GFP-LC3B were used to detect autophagy level in the cells. MTT assay and colony-forming assay were performed to assess the cell survival fraction and colony formation ability of the treated cells, respectively. Results After cisplatin treatmert, the expression level of LC3Ⅱ increased significantly(P<0.001), the expression level of p62 decreased(P<0.05), and the number of autophagosomes increased in Ⅰ-10/DDP cells. The cells transfected with shRNA-ATG5 or shRNA-ATG7 showed significantly decreased expressions of ATG5 or ATG7(P=0.005 or P<0.001). Cisplatin treatment of the transfected cells obviously reduced the cellular expression of LC3 II(P<0.001), increased the expression of p62(P<0.001), and decreased the number of autophagosomes, cell survival fraction and colony formation ability of the cells(P<0.001). Conclusion Silencing ATG5 and ATG7 inhibits cisplatin-mediated autophagy and enhances the inhibitory effect of cisplatin on inhibiting cell proliferation.