miR-1271对卵巢癌SKOV3/DDP细胞顺铂耐药的影响及其机制

Effect of miR-1271 on cisplatin resistance in human ovarian cancer cell line SKOV3/DDP and its potential mechanism

目的:研究miR-1271在卵巢癌SKOV3/DDP细胞顺铂耐药形成中的作用。方法:采用实时定量PCR法检测卵巢癌SKOV3/DDP细胞和母代SKOV3细胞中miR-1271的表达,并通过蛋白质印迹法检测上述细胞X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)的表达。构建XIAP 3'UTR荧光素酶报告质粒验证miR-1271的靶基因,在耐药细胞株中瞬时转染miR-1271模拟物以观察上调miR-1271对卵巢癌SKOV3/DDP细胞XIAP表达及顺铂耐药的影响,并运用流式细胞术检测转染miR-1271模拟物后SKOV3/DDP细胞的凋亡率。结果:miR-1271在卵巢癌SKOV3/DDP细胞中呈低表达,抗凋亡蛋白XIAP在卵巢癌SKOV3/DDP细胞中呈高表达,荧光素酶报告实验证实XIAP是直接受miR-1271调控的靶基因,在耐药株中上调miR-1271显著抑制XIAP蛋白表达水平,显著增加细胞对顺铂的敏感性,并显著增加细胞对顺铂诱导的凋亡。结论:miR-1271靶向抑制XIAP表达可增加卵巢癌SKOV3/DDP细胞对顺铂的敏感性。

Objective： To investigate the possible role of miR-1271 in the development of cisplatin re- sistance in human ovarian cancer cell line SKOV3/DDP. Methods： Using quantitative real-time PCR a- nalysis for miRNA and Western blotting to detect the expression difference of miR-1271 and X-linked in- hibitor of apoptosis protein （XIAP） between cisplatin-resistant human ovarian cancer cell line SKOV3/ DDP and its parental SKOV3 cell line, respectively. Constructing XIAP 3＇-untranslated region-based lu- ciferase reporter plasmids to verify the target gene of miR-1271. Transient transfection of miR-1271 mimic was used to upregulate the expression level of miR-1271 in SKOV3/DDP cells and observe the effect of miR-1271 on the expression level of XIAP and the cisplatin resistance phenotype. Flow cytometry was used to detect cisplatin-induced apoptosis of SKOV3/DDP cells after the transfection. Results： miR-1271 was downregulated in cisplatin-resistant human ovarian cancer cell line SKOV3/DDP, which was concur- rent with the overexpression of its target anti-apoptotic gene XIAP in SKOV3/DDP cells, compared with the parental SKOV3 cell line. The luciferase activity of XIAP 3＇-untranslated region-based reporters con-struct in SKOV3/DDP cells suggested that XIAP was the direct target gene of the miR-1271. Overexpres- sion of miR-1271 sensitized SKOV3/DDP cells to cisplatin, inhibited the expression level of XIAP and sensitized SKOV3/DDP cells to DDP-induced apoptosis. Conclusion： miR-1271 may play a role in the development of cisplatin resistance in human ovarian cancer cell line, at least in part, by modulation of apoptosis via targeting XIAP.