微小RNA-149靶向P-糖蛋白对皮肤基底细胞癌顺铂耐药的影响

Effect of microRNA-149 targeting P-glycoprotein on cisplatin resistance of skin basal cell carcinoma

目的观察微小RNA(miRNA,miR)-149靶向P-糖蛋白(P-gp)对皮肤基底细胞癌阿霉素耐药的影响。方法选取2014年8月到2018年8月河南省人民医院皮肤科收治的74例皮肤基底细胞癌患者作为研究对象。根据患者对顺铂化疗的敏感性程度,分为敏感组和耐药组。采用荧光定量聚合酶链反应(PCR)分析两组肿瘤组织中miR-149表达水平。采用脂质体法转染miR-149模拟物和对照miRNA至皮肤基底细胞株癌细胞株A431,分别作为miR-149组和对照组。采用细胞计数试剂盒(CCK-8)和5-乙炔基-2’脱氧尿嘧啶核苷(EdU)染色分析顺铂对两组细胞增殖能力的影响。采用生物信息学和双荧光素酶报告基因分析miR-149的靶基因;采用蛋白质印迹法(Western blot)分析肿瘤组织中靶基因的表达和miR-149对靶基因表达的影响。组间数据比较采用t检验。结果耐药组患者肿瘤组织miR-149表达水平(0.48±0.18)较敏感组miR-149表达水平(1.21±0.23)显著下降,差异有统计学意义(t=3.126,P<0.05)。CCK-8和EdU染色结果显示,经顺铂处理后,miR-149组细胞增殖能力(0.59±0.17)较对照组细胞(1.27±0.21)明显下降,差异有统计学意义(t=3.010,P<0.05)。miR-149组细胞EdU染色阳性率[(35.56±6.90)%]较对照组细胞[(79.32±8.01)%]明显下降,差异有统计学意义(t=3.399,P<0.05)。生物信息学和双荧光素酶报告基因显示P-gp是miR-149靶基因。耐药组肿瘤组织中P-gp蛋白表达水平(1.24±0.19)明显高于敏感组肿瘤组织(0.61±0.18),差异有统计学意义(t=2.091,P<0.05)。miR-149组细胞P-gp蛋白表达水平(0.57±0.18)较对照组细胞(1.97±0.32)明显增加,差异有统计学意义(t=2.581,P<0.05)。结论 miR-149通过调节P-gp蛋白的表达,介导了皮肤基底细胞癌顺铂化疗耐药。

Objective To investigate the effect of microRNA(miRNA,miR)-149 targeting P-glycoprotein(P-gp)on cisplatin resistance in skin basal cell carcinoma.Methods A total of 74 patients with skin basal cell carcinoma from August 2014 to August 2018 Henan provincial people’s hospital were selected as research subjects,and divided into sensitive group and resistant group.The expression level of miR-149 in tumor tissues was analyzed by fluorescence quantitative PCR.The miR-149 mimics and control miRNA were transfected into skin basal cell line A431 by liposome method as miR-149 group and control group respectively.The sensitivity of cells to gemcitabine in miR-149 group and control group was analyzed by cell counting kit-8(CCK-8)assay and 5-Ethynyl-2’-deoxyuridine(EdU)staining.The target gene of miR-149 was analyzed by bioinformatics and double luciferase reporter gene.The target gene expression in tumor tissues and cells was analyzed by Western blotting.T test was used to compare the data between groups,and P<0.05 was statistically significant.Results The expression level of miR-149 in the resistant group(0.48±0.18)was significantly lower than that in the sensitive group(1.21±0.23,t=3.126,P<0.05).CCK-8 assay and EdU staining results showed that after cisplatin treatment,the cell proliferation ability in miR-149 group(0.59±0.17)was significantly lower than that in the control group(1.27±0.21,t=3.010,P<0.05).The positive rate of EdU staining in miR-149 group[(35.56±6.90)%]was significantly lower than that in control group[(79.32±8.01)%,t=3.399,P<0.05].Bioinformatics and double luciferase reporter gene showed that P-gp was targeting gene of miR-149.The expression level of P-gp protein in the resistant group(1.24±0.19)was significantly higher than that in the sensitive group(0.61±0.18,t=2.091,P<0.05).The expression level of P-gp protein in miR-149 group(0.57±0.18)was significantly higher than that in control group(1.97±0.32,t=2.581,P<0.05).Conclusion MiR-149 can regulate the expression of P-gp protein and mediate cisplatin resistance of skin basal cell carcinoma.