miRNA-451通过MRP靶向调控胃癌细胞对5-Fu耐药性的机制研究

Mechanism of mi RNA-451 Regulating Resistance of Gastric Cancer Cells to 5-Fu Through MRP

目的探讨miR-451通过下调耐药基因MRP表达,调控胃癌对5-Fu耐药的相关机制。方法采用实时荧光定量PCR检测20对人胃癌组织及癌旁组织和胃癌细胞系中miR-451的相对表达量,根据患者对药物的反应进行分组并检测耐药组以及非耐药组miR-451的表达水平。构建plent-miR-451稳定过表达胃癌细胞系,通过荧光实时定量PCR检测胃癌细胞系miR-451过表达效率,CCK8法检测不同浓度5-Fu对细胞增殖活力的影响。生物信息学方法分析miR-451的靶基因,并通过荧光素酶实验验证。实时定量PCR以及Western blot检测miR-451对靶基因MRP mRNA和蛋白水平的影响。结果miR-451在正常组织中表达量高于胃癌组织,在非耐药胃癌组织中高于耐药胃癌组织。过表达miR-451降低了胃癌细胞对5-Fu的耐受能力(P=0.0006)。生物信息学分析显示MRP为miR-451的靶基因,荧光素酶实验同时也证实MRP为miR-451的潜在靶基因。过表达miR-451可导致耐药基因MRP mRNA以及蛋白水平均显著降低(P=0.00069)。结论过表达miR-451可下调耐药基因MRP表达,使肿瘤细胞对5-Fu耐药性降低。

Objective To investigate the mechanism of miR-451 regulating the resistance of gastric cancer to 5-Fu by down-regulating the expression of MRP.Methods Real-time quantitative PCR was used to detect the relative expression of miR-451 in 20 pairs of human gastric cancer tissues and adjacent tissues and gastric cancer cell lines.The expression of miR-451 in the resistance group and non-resistant group were detected.We constructed gastric cancer cell line with stable plent-miR-451 overexpression,and the overexpression efficiency of miR-451 in gastric cancer cell line was detected by real-time quantitative PCR.The proliferation activity of the cells was detected by CCK8.The miR-451 target gene was analyzed by bioinformatics method and verified by luciferase assay.The effect of miR-451 on the mRNA and protein of targeted gene MRP were detected by real-time quantitative PCR and Western blot.Results The expression of miR-451 in normal tissues was higher than that in cancer tissues,and miR-451 expression in the non-resistant gastric cancer tissues was higher than that in the drug-resistant gastric cancer tissues.The overexpression of miR-451 reduced the resistance of gastric cancer cells to 5-Fu treatment(P=0.0006).Bioinformatics analysis showed that MRP was the target gene of miR-451.Luciferase assay also confirmed that MRP was a potential target gene of miR-451.miR-451 overexpression decreased the mRNA and protein of the drug resistance gene MRP(P=0.00069).Conclusion Overexpression of miR451 could down-regulate MRP expression,which reduces the resistance of tumor cells to 5-Fu.