微RNA-524-5p通过靶向抑制SOX9基因表达增加胃癌细胞的顺铂敏感性

MicroRNA-524-5p increases cisplatin-sensitivity of gastric cancer cells by targeting SOX9 gene

目的:探讨微RNA-524-5p(microRNA-524-5p,miR-524-5p)介导顺铂治疗胃癌的作用机制。方法:采用实时荧光定量PCR法检测胃癌组织、癌旁组织以及顺铂耐药和亲本胃癌细胞系中miR-524-5p的表达水平。顺铂耐药性胃癌SGC-7901和MKN-45细胞经miR-524-5p模拟物或对照RNA转染后,采用CCK-8法检测细胞活力。采用荧光素酶报告基因实验和蛋白质印迹法鉴定miR-524-5p靶向蛋白SOX9(SRY-related high mobility group-box 9)的表达情况。采用'拯救'实验检测SOX9过表达后miR-524-5p诱导胃癌细胞对顺铂的耐药性变化。结果:与正常胃上皮黏膜细胞GES1相比,胃癌细胞SGC-7901和MKN-45中miR-524-5p水平明显下调(P值均<0.05);与癌旁组织相比,胃癌组织中miR-524-5p水平明显下调(P <0.05)。与敏感性亲代细胞相比,顺铂处理后的胃癌耐药细胞SGC-7901和MKN-45中miR-524-5p表达水平均降低(P值均<0.05)。miR-524-5p模拟物转染后,顺铂耐药性胃癌细胞SGC-7901和MKN-45的化疗敏感性均明显增强(P值均<0.05)。SOX9是miR-524-5p的功能靶蛋白;SOX9过表达可以抵消miR-524-5p的化疗增敏作用,即与单独miR-524-5p模拟物转染组相比,SOX9过表达质粒+miR-524-5p模拟物转染后的胃癌细胞SGC-7901和MKN-45对顺铂的敏感性均明显降低(P值均<0.05)。结论:miR-524-5p影响人胃癌细胞对顺铂作用的敏感性,提示其可能成为治疗化疗耐药性胃癌患者的新靶点。

Objective:To investigate the mechanism of microRNA(miR)-524-5p mediating the effect of cisplatin in the treatment of gastric cancer.Methods:The level of miR-524-5p in gastric cancer tissues,the matched non-tumor tissues,cisplatin-resistant and parental gastric cancer cell lines was measured by real-time fluorescent quantitative PCR.The viability of cisplatin-resistant gastric cancer SGC-7901 and MKN-45 cells transfected with miR-524-5p mimics or the control RNA was detected by CCK-8 method.The expression of SRY-related high mobility group-box gene 9(SOX9),a targeting protein of miR-524-5p,was identified by luciferase reporter assay and Western blotting.The'rescue'assay was used to explore the effect of SOX9 overexpression on the cisplatin-resistance of gastric cancer cells induced by miR-524-5p.Results:Compared with the normal gastric epithelial GES1 cells,the level of miR-524-5p in gastric cancer SGC-7901 and MKN-45 cells was significantly down-regulated(both P<0.05);while the level of miR-524-5p in gastric cancer tissues was significantly lower than that in the adjacent non-tumor tissues(P<0.05).After the treatment of cisplatin,the expression level of miR-524-5p in cisplatin-resistant gastric cancer SGC-7901 and MKN-45 cells was decreased as compared with the sensitive parental cells(both P<0.05).After the transfection of miR-524-5p mimics,the sensitivity of cisplatin-resistant gastric cancer SGC-7901 and MKN-45 cells was significantly enhanced(both P<0.05).Subsequently,SOX 9 was identified as a functional target gene of miR-524-5p.Overexpression of SOX9 could counteract the chemosensitizing effect of miR-524-5p,in other words,the cisplatinsensitivity of SGC-7901 and MKN-45 cells transfected with SOX9 overexpression plasmids and miR-524-5p mimics was significantly lower than that in single miR-524-5p mimics transfection group(both P<0.05).Conclusion:The expression of miR-524-5p influences the sensitivity of human gastric cancer cells to cisplatin,suggesting that miR-524-5p has the potential to be a novel target for the treatment of chemoresistant gastric cancer patients.