微小RNA-519靶向ATP结合盒转运体G1调控乳腺癌细胞化疗耐药性的机制

Mechanism of microRNA-519 on chemoresistance via targeting ATP-binding cassette transporter G1 in breast cancer

目的探讨微小RNA(miRNA,miR)-519靶向ATP结合盒转运体G1(ABCG1)对乳腺癌细胞吉西他滨耐药性的影响。方法选取2015年6月到2018年6月来焦作市人民医院接受治疗的乳腺癌患者148例。根据患者对吉西他滨化疗敏感性分为敏感组和耐药组。荧光定量聚合酶链反应(PCR)分析两组乳腺癌组织中miR-519表达水平。构建miR-519或对照miRNA过表达慢病毒载体,包装慢病毒,感染MCF-7乳腺癌细胞,构建miR-519(miR-519组)和对照组细胞株(miRNA对照组)。采用细胞计数试剂盒(CCK-8)法分析miR-519组和miRNA对照组细胞对吉西他滨的敏感性。采用生物信息学和双荧光素酶报告基因分析miR-519的靶基因;采用蛋白质印迹法(Western blot)分析miR-519对靶基因表达的影响。组间数据比较采用t检验,以P<0.05为差异有统计学意义。结果与吉西他滨敏感组(1.21±0.23)比较,耐药组患者肿瘤组织中miR-519 mRNA表达水平(0.30±0.12)明显下降,差异有统计学意义(t=3.091,P<0.05)。CCK-8结果显示,miR-519组和miRNA对照组细胞增殖能力(1.76±0.23比1.65±0.26)比较差异无统计学意义(t=0.691,P>0.05)。经吉西他滨处理后,miR-519组细胞增殖能力(0.68±0.19)较miRNA对照组细胞(1.12±0.22)明显下降,差异有统计学意义(t=2.819,P<0.05)。生物信息学和双荧光素酶报告基因显示ABCG1是miR-519靶基因。耐药组肿瘤组织中ABCG1蛋白表达水平(1.65±0.25)明显高于敏感组肿瘤组织(0.84±0.19),差异有统计学意义(t=2.619,P<0.05)。miR-519组细胞ABCG1蛋白表达水平(0.34±0.11)较miRNA对照组细胞(1.43±0.24)明显增加,差异有统计学意义(t=3.014,P<0.05)。结论 miR-519可能通过ABCG1调控乳腺癌对吉西他滨的敏感性。

Objective To investigate the mechanism of microRNA(miRNA,miR)-519 on chemoresistance via targeting ATP-binding cassette transporter G1(ABCG1)in breast cancer.Methods 148 patients with breast cancer from June 2015 to June 2018 were selected.According to the sensitivity of patients to gemcitabine chemotherapy,they were divided into sensitive group and resistant group.The expression of miR-519 in breast cancer tissues of the two groups was detected by fluorescence quantitative polymerase chain reaction(PCR).The miR-519 or control miRNA overexpression lentivirus vectors were constructed and lentiviruses were packaged to infect MCF-7 breast cancer cells.The stable cells were named miR-519 group and miRNA control group.The sensitivity of cells for gemcitabine in miR-519 group and miRNA control group was analyzed by cell counting kit-8(CCK-8)assay.The target gene of miR-519 was analyzed by bioinformatics and double luciferase reporter gene.The target gene expression of miR-519 was examined by Western blotting.T test was used to compare the data between groups,and P<0.05 was statistically significant.Results As compared with sensitive group(1.21±0.23),the expression level of miR-519 in tumor tissue of drug resistant group(0.30±0.12)significantly decreased(t=3.091,P<0.05).CCK-8 assay showed that there was no significant difference in cell proliferation between miR-519 group and miRNA control group(1.76±0.23 vs.1.65±0.26,t=0.691,P>0.05).After treatment with gemcitabine,the cell proliferation ability of miR-519 group(0.68±0.19)was significantly lower than that in miRNA control group(1.12±0.22,t=2.819,P<0.05).Bioinformatics and double luciferase reporter gene showed that ABCG1 was targeting gene of miR-519.The expression level of ABCG1 protein in the resistant group was significantly higher than that in the sensitive group(1.65±0.25,t=2.619,P<0.05).The expression level of ABCG1 protein in miR-519 group was significantly higher than that in miRNA control group(1.43±0.24,t=3.014,P<0.05).Conclusion MiR-519 may regulate the sensitivity of breast cancer to gemcitabine through ABCG1.