微小RNA-375通过靶向PAX6对宫颈癌HeLa细胞顺铂敏感性的影响

Effect of microRNA-375 targeting PAX6 on cisplatin sensitivity of cervical cancer HeLa cells

目的探讨微小RNA-375(miR-375)和人类配对盒基因6(PAX6)在宫颈癌顺铂耐药株HeLa cisR中的表达及耐药逆转作用并初步探讨可能机制。方法采用实时定量PCR(QPCR)检测28例正常宫颈组织、30例宫颈癌组织(14例顺铂敏感和16例顺铂不敏感)和不同顺铂敏感性细胞HeLa、HeLa cisR中miR-375、PAX6 mRNA水平,采用脂质体法向HeLa和HeLa cisR细胞转染含miR-375模拟物(mimics)的寡核苷酸探针(过表达组)和阴性对照NC(阴性对照组),以不行任何转染的为空白对照组。采用QPCR检测转染后HeLa和HeLa cisR细胞中miR-375和PAX6 mRNA水平,采用MTT法检测不同浓度顺铂(1、3、10、30、100μmol/L)处理后的增殖情况并计算半数抑制浓度(IC_(50))以评价顺铂敏感性,Western blotting检测PAX6蛋白水平,双荧光素酶报告基因试验评价miR-375对PAX6的靶向调控作用。结果宫颈癌组织中的miR-375水平低于正常宫颈组织(0.714±0.341 vs.1.006±0.299),PAX6 mRNA水平高于正常宫颈组织(1.855±0.928 vs.1.011±0.257),miR-375水平与PAX6 mRNA水平呈负相关(r=-0.416,P<0.05)。与化疗敏感组相比,化疗不敏感组的miR-375水平降低,而PAX6 mRNA水平升高(P<0.05)。HeLa cisR细胞的miR-375水平低于HeLa细胞(0.365±0.098 vs.1.032±0.135),而PAX6 mRNA水平高于HeLa细胞(3.154±0.957 vs.1.067±0.168),差异均有统计学意义(P<0.05)。与其余两组相比,过表达组转染后的miR-375水平升高,PAX6蛋白和mRNA水平均降低,差异有统计学意义(P<0.05)。miR-375过表达可降低顺铂IC_(50)(P<0.05),先转染miR-375 mimics 24 h后再转染过表达PAX6的pCMV-PAX6质粒后细胞的IC_(50)与未转染细胞的差异无统计学意义(P>0.05)。结论 miR-375通过靶向PAX6参与了宫颈癌细胞的顺铂耐药,miR-375有望成为逆转宫颈癌顺铂耐药的靶点。

Objective To explore the expression of microRNA-375(miR-375)and human paired box gene 6(PAX6)in the cisplatin resistant strain HeLa cisR of cervical cancer and the reversal effect of drug resistance as well as the possible mechanism.Methods The level of miR-375 and PAX6 mRNA in 28 normal cervical tissues and 30 cervical cancer tissues(14 cases of cisplatin sensitive and 16 cases of cisplatin insensitive)and different cisplatin sensitive cells HeLa and HeLa cisR were measured by real-time quantitative PCR(QPCR).HeLa and HeLa cisR cells were transfected with miR-375 ana log(mimics)oligonucleotide probe(overexpressed group)and negative control(NC group)by liposome method,and cells without transfection was used as a blank control group.The MTT method was used to detect the proliferation of cells exposure to cisplatin(1,3,10,30,100μmol/L)and calculate the median inhibitory concentration(IC 50)as to evaluate the sensitivity of cisplatin.Western blotting was used to detect the level of PAX6 protein.The dual luciferase reporter test was used to evaluate the targeting effect of miR-375 on PAX6.Results The level of miR-375 in cervical cancer tissues was lower than that of normal cervical tissue(0.714±0.341 vs.1.006±0.299),and the level of PAX6 mRNA was higher than that of normal cervical tissue(1.855±0.928 vs.1.011±0.257).The level of miR-375 was negatively correlated with the level of PAX6 mRNA(r=-0.416,P<0.05).Compared with chemosensitivity group,the level of miR-375 in chemotherapy insensitive group decreased,while the level of PAX6 mRNA increased(P<0.05).The miR-375 level of HeLa cisR cells was lower than that of HeLa cells(0.365±0.098 vs.1.032±0.135)and PAX6 mRNA level was higher than that of HeLa cells(3.154±0.957 vs.1.067±0.168)with a statistical significant difference(P<0.05).Compared with other two groups,the miR-375 level of the overexpression group was increased after transfection,and the levels of PAX6 protein and mRNA were significantly different(P<0.05).MiR-375 overexpression can reduce IC 50 of cisplatin(P<0.05),and IC 50 of cells with 24 h-transfection of miR-375 mimics followed by pCMV-PAX6 was similar with untransfected cells(P>0.05).Conclusion miR-375 is involved in cisplatin resistance of cervical cancer cells through targeting PAX6.MiR-375 is expected to become a target for reversing cisplatin resistance in cervical cancer.