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

IL-17A/1L-17RA reduces cisplatin sensitivity of ovarian cancer SKOV3 cells by regulating autophagy

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、20μg/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。

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(1-20μg/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.