基于微小RNA-135b-5p靶向调控瓣状核酸内切酶1探究姜黄素对人卵巢癌细胞顺铂耐药的改善机制

Mechanism of curcumin improving cisplatin resistance of human ovarian cancer cells based on the targeted regulation of flap-like endonuclidene 1 by microRNA-135b-5p

目的 基于微小RNA-135b-5p(miR-135b-5p)靶向调控瓣状核酸内切酶1(FEN1)探究姜黄素对人卵巢癌细胞顺铂耐药的改善机制。方法 取对数生长期人卵巢癌顺铂耐药细胞株OVCAR-3/DDP,将细胞分为对照组、顺铂组、姜黄素组、顺铂+姜黄素组、顺铂+姜黄素+阴性对照抑制剂组、顺铂+姜黄素+miR-135b-5p抑制剂组、顺铂+姜黄素+miR-135b-5p模拟物组。对照组不予处理,其余各组分别予顺铂和/或姜黄素,后3组细胞采用脂质体转染法分别转染绿色荧光蛋白质粒(pGFP)-阴性对照抑制剂、pGFP-miR-135b-5p抑制剂、pGFP-miR-135b-5p模拟物载体。各组给药处理72 h后,采用实时荧光定量聚合酶链反应检测各组细胞miR-135b-5p及FEN1 mRNA表达量;蛋白质印迹法检测各组细胞FEN1蛋白表达;双荧光素酶靶标实验验证miR-135b-5p与FEN1之间的作用关系;噻唑盐比色法检测各组细胞增殖抑制率;流式细胞仪检测各组细胞凋亡率。结果 姜黄素组、顺铂+姜黄素组、顺铂+姜黄素+阴性对照抑制剂组、顺铂+姜黄素+miR-135b-5p抑制剂组、顺铂+姜黄素+miR-135b-5p模拟物组miR-135b-5p表达量、增殖抑制率及细胞凋亡率均高于对照组和顺铂组,FEN1 mRNA及蛋白表达量均低于对照组和顺铂组(均P<0.05)。脂质体转染的3组中,顺铂+姜黄素+miR-135b-5p抑制剂组miR-135b-5p表达量、增殖抑制率及细胞凋亡率最低[(0.75±0.10)、(34.399±6.055)%、(17.3±3.1)%],FEN1 mRNA和蛋白表达量最高[(0.77±0.11)、(0.69±0.09)];顺铂+姜黄素+miR-135b-5p模拟物组miR-135b-5p表达量、增殖抑制率及细胞凋亡率最高[(1.68±0.26)、(80.365±10.617)%、(48.7±8.4)%],FEN1 mRNA和蛋白表达量最低[(0.33±0.04)、(0.29±0.04)](均P<0.05)。双荧光素酶靶标实验提示miR-135b-5p靶向FEN1。结论 姜黄素可增强OVCAR-3/DDP对顺铂的敏感性,可能与促进miR-135b-5p的表达从而靶向抑制FEN1的表达相关。

Objective To investigate the mechanism of curcumin(CUR) improving cisplatin(DDP) resistance of human ovarian cancer cells based on the targeted regulation of flap-like endonuclidene 1(FEN1) by microRNA-135b-5p(miR-135b-5p). Methods Human ovarian cancer cisplatin-resistant cell lines OVCAR-3/DDP at logarithmic growth stage were divided into control group, DDP group, CUR group, DDP+CUR group, DDP+CUR+negative control(NC) inhibitor group, DDP+CUR+miR-135b-5p inhibitor group, and DDP+CUR+miR-135b-5p mimics group. The control group did not intervene, other groups were given DDP and/or CUR respectively, and the latter 3 groups were transfected with plasmid green fluorescent protein(pGFP)-NC inhibitor, pGFP-miR-135b-5p inhibitor and pGFP-miR-135b-5p mimics vectors by liposome transfection, respectively. After 72 h of administration, the expressions of miR-135b-5p and FEN1 mRNA in each group were detected by real-time quantitative polymerase chain reaction, the FEN1 protein expression was detected by western blotting, the interaction between miR-135b-5p and FEN1 was verified by dual luciferase target assay, the proliferation inhibition rate of cells in each group was detected by tetrazolium salt colorimetry assay, and the apoptosis rate of each group was detected by flow cytometry. Results Compared with the control group and DDP group, the miR-135b-5p expression, proliferation inhibition rate and apoptosis rate increased in CUR group, DDP+CUR group, DDP+CUR+NC inhibitor group, DDP+CUR+miR-135b-5p inhibitor group and DDP+CUR+miR-135b-5p mimics group, and FEN1 mRNA and protein expressions decreased(all P<0.05). Among the three liposome transfection groups, miR-135b-5p expression, proliferation inhibition rate and apoptosis rate in the DDP+CUR+miR-135b-5p inhibitor group were the lowest[(0.75±0.10),(34.399±6.055)%,(17.3±3.1)%], and FEN1 mRNA and protein expressions were the highest[(0.77±0.11),(0.69±0.09)];miR-135b-5p expression, proliferation inhibition rate and apoptosis rate in the DDP+CUR+miR-135b-5p mimics group were the highest [(1.68±0.26),(80.365±10.617)%,(48.7±8.4)%], and FEN1 mRNA and protein expressions were the lowest [(0.33±0.04),(0.29±0.04)](all P<0.05). Dual luciferase target assay showed that miR-135b-5p targeted FEN1. Conclusion CUR can enhance the sensitivity of OVCAR-3/DDP to DDP, which may be related to promoting the expression of miR-135b-5p and the targeted inhibition of FEN1 expression by miR-135b-5p.