南方医科大学学报

南方医科大学学报 ›› 2020, Vol. 40 ›› Issue (11): 1550-1556.doi: 10.12122/j.issn.1673-4254.2020.11.03

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IL-17A/IL-17RA通过上调自噬降低卵巢癌SKOV3细胞对顺铂敏感性

王丽华，张 璇，王亮亮，王蓓蓓，张 競，李玉芝
  

  • 出版日期:2020-11-20 发布日期:2020-11-23

IL-17A/lL-17RA reduces cisplatin sensitivity of ovarian cancer SKOV3 cells by regulating autophagy

  • Online:2020-11-20 Published:2020-11-23

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摘要： 目的 探讨白细胞介素（IL）17A通过调控细胞自噬对卵巢癌细胞顺铂（DDP）化疗敏感性的影响。方法 体外培养卵巢癌SKOV3细胞并分别用不同浓度DDP（1、2、4、6、8、10、15、20 μg/mL）处理，MTT法测定细胞增殖活性以及IL-17A（100 ng/mL，处理24 h）对DDP 诱导SKOV3细胞凋亡的影响；流式细胞术检测人卵巢癌细胞SKOV3中IL-17A受体（IL-17RA）的表达水平；Annexin V-FITC/PI双染法检测细胞凋亡；线粒体膜电位检测试剂盒（JC-1）检测细胞早期凋亡情况；IL-17RA 中和抗体（IL-17RA mAb）进行IL-17RA阻断实验；合成针对IL-17RA基因的siRNA片段（siRNA-IL-17RA），转染SKOV3细胞，沉默IL-17RA表达；3-甲基腺嘌呤（3-MA）抑制细胞自噬水平；Western blot检测细胞凋亡相关蛋白（Bcl2、Bax、Cleaved Caspase3）、自噬相关蛋白（P62、Beclin-1）表达。结果 DDP可以增加卵巢癌SKOV3细胞IL-17RA表达水平；IL-17A降低SKOV3细胞对DDP的敏感性（P<0.05）；DDP诱导SKOV3细胞内自噬相关蛋白Beclin-表达增强，自噬降解底物P62蛋白减少；IL-17A/IL-17RA增强了DDP自噬诱导作用（P<0.05）；3-MA 阻断自噬后，SKOV3细胞凋亡率较干扰前明显升高，细胞抗凋亡蛋白Bcl2表达水平降低、凋亡蛋白Bax表达水平升高，差异有统计学意义（P<0.05）。结论 IL-17A/IL-17RA可能通过上调DDP诱导的自噬水平降低人卵巢癌SKOV3细胞化疗敏感性。

关键词: 卵巢癌, IL-17A, 顺铂, 自噬, 耐药

Abstract: Objective To investigate the effect of interleukin-17A (IL-17A) on chemosensitivity of ovarian cancer cells to cisplatin (DDP) and explore the mechanism in light of autophagy regulation. Methods Ovarian cancer SKOV3 cells cultured in vitro were treated with different concentrations of DDP (1-20 μg/mL). MTT assay was used to observe the changes in proliferation of the treated cells and the effect of treatment with 100 ng/mL IL-17A for 24 h on DDP-induced apoptosis of SKOV3 cells. We then examined the expression of IL-17A receptor (IL-17RA) in SKOV3 cells using flow cytometry. Annexin V-FITC/PI double staining was used to detect the cell apoptosis rate, and early apoptosis of the cells was detected with JC-1 assay. A neutralizing monoclonal antibody (mAb) against IL-17RA was used to block IL-17RA. We also observed the effects of IL-17RA silencing mediated by a siRNA targeting IL-17RA (siRNA-IL-17RA) and treatment with 3-methyladenine (3-MA) for inhibiting autophagy on DDP-induced apoptosis of SKOV3 cells. The expressions of apoptosis-related proteins (Bcl-2, Bax, and cleaved caspase-3) and autophagy-related proteins (P62 and Beclin-1) in the treated cells were detected using Western blotting. Results DDP increased the expression of IL-17RA in ovarian cancer SKOV3 cells. Treatment with IL-17A significantly reduced the susceptibility of SKOV3 cells to cisplatin-induced apoptosis (P<0.05). DDP obviously augmented the expression of Beclin-1 and reduced the autophagy degradation substrate P62 protein in the cells (P<0.05). IL-17A/IL-17RA strongly enhanced the DDPinducted autophagy of the cells (P<0.05). Blocking autophagy with 3-MA significantly increased DDP- induced apoptosis of SKOV3 cells with IL-17RA silencing, lowered the expression of Bcl-2 and enhanced Bax expression in the cells (P<0.05). Conclusion IL-17A/IL-17RA can decrease chemosensitivity of SKOV3 cells to DDP by upregulating DDP-induced autophagy

Key words: ovarian cancer, interleukin-17A, cisplatin, autophagy, drug resistance