miRNA-5193对子宫颈癌Caski细胞顺铂敏感性的影响

Effect of miRNA-5193 on the sensitivity of cervical cancer Caski cells to cisplatin

目的探讨miRNA-5193(miR-5193)对子宫颈癌Caski细胞顺铂敏感性的影响和机制。方法采用实时荧光定量聚合酶链反应(qRT-PCR)测定子宫颈癌细胞株C33A、SiHa、Caski和正常子宫颈细胞株Ect1/E6E7中miR-5193的表达水平。将Caski细胞分成对照组(不转染,正常培养)、miR-5193阴性对照(miR-NC)组(转染miR-NC模拟物)、miR-5193组(转染miR-5193模拟物)、miR-NC+顺铂组(10μg/ml顺铂处理转染miR-NC模拟物的细胞)、miR-5193+顺铂组(10μg/ml顺铂处理转染miR-5193模拟物的细胞)、miR-5193+顺铂+NC组(10μg/ml顺铂处理共转染Foxp3阴性对照载体、miR-5193模拟物的细胞)、miR-5193+顺铂+Foxp3组(10μg/ml顺铂处理共转染Foxp3过表达载体和miR-5193模拟物的细胞)。四甲基偶氮唑盐(MTT)法检测细胞增殖情况;流式细胞术PI单染法检测细胞周期,Annexin V-FITC/PI双染法检测细胞凋亡;蛋白质印迹法检测细胞CDK2、p27、C-caspase-3蛋白表达。应用生物信息学软件预测miR-5193靶基因,采用荧光素酶报告系统鉴定靶向关系。结果子宫颈癌C33A、SiHa、Caski细胞中miR-5193相对表达量均低于正常子宫颈Ect1/E6E7细胞(0.56±0.06、0.41±0.03、0.23±0.02比1.00±0.10,均P<0.05)。与对照组、miR-NC组比较,miR-5193组、miR-NC+顺铂组、miR-5193+顺铂组细胞增殖活性(吸光度值)降低(0.58±0.06、0.59±0.07比0.38±0.04、0.40±0.05、0.23±0.02,均P<0.05),细胞凋亡率升高[(2.5±0.2)%、(2.7±0.3)%比(12.6±1.2)%、(11.9±1.5)%、(18.9±1.7)%,均P<0.05],G0/G1期细胞比例升高[(50.4±4.2)%、(51.3±6.3)%比(62.3±3.2)%、(61.9±5.8)%、(71.4±5.4)%,均P<0.05],p27、C-caspase-3蛋白表达水平升高,CDK2蛋白表达水平下降。软件预测miR-5193靶基因为Foxp3,并由荧光素酶报告系统确认。与miR-5193+顺铂+NC组相比,miR-5193+顺铂+Foxp3组细胞增殖活性(吸光度值)升高(0.24±0.03比0.65±0.05,t=21.094,P<0.01),G0/G1期细胞比例降低[(71.0±6.4)%比(60.3±4.1)%,t=4.196,P<0.01],细胞凋亡率降低[(19.6±1.6)%比(11.5±1.2)%,t=11.880,P<0.01],细胞中p27、C-caspase-3蛋白表达水平下降,CDK2、Foxp3蛋白表达水平升高。结论体外miR-5193可能通过靶向抑制Foxp3基因提高子宫颈癌Caski细胞对顺铂的敏感性。

Objective To investigate the effect and mechanism of miRNA-5193(miR-5193)on the sensitivity of cervical cancer Caski cells to cisplatin.Methods The expression of miR-5193 in cervical cancer cell lines C33A,SiHa,Caski and normal cervical cell line Ect1/E6E7 were determined by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR).Caski cells were divided into control group(no transfection,normally cultured),miR-5193-negative control(miR-NC)group(transfected with miR-NC mimic),miR-5193 group(transfected with miR-5193 mimic),miR-NC+cisplatin group(transfected with miR-NC mimic and treated with 10μg/ml cisplatin),miR-5193+cisplatin group(transfected with miR-5193 mimic and treated with 10μg/ml cisplatin),miR-5193+cisplatin+NC group(cotransfected with Foxp3-negative control vector and miR-5193 mimic,and treated with 10μg/ml cisplatin),and miR-5193+cisplatin+Foxp3 group(cotransfected with Foxp3 overexpression vector and miR-5193 mimic,and treated with 10μg/ml cisplatin).Proliferation was detected by methyl thiazolyl tetrazolium(MTT),cell cycle was detected by PI single staining method,cell apoptosis was detected by Annexin V-FITC/PI double staining method,and expressions of CDK2,p27 and C-caspase-3 proteins in cells were detected by Western blot.Bioinformatics software was used to predict miR-5193 target genes,and the luciferase reporting system was used to identify the targeting relationship.Results The relative expression of miR-5193 in cervical cancer C33A,SiHa and Caski cells was lower than that in normal cervical Ect1/E6E7 cells(0.56±0.06,0.41±0.03,0.23±0.02 vs.1.00±0.10,all P<0.05).Compared with the control group and miR-NC group,the cell proliferation activity(absorbance value)in miR-5193,miR-NC+cisplatin and miR-5193+cisplatin groups decreased(0.58±0.06,0.59±0.07 vs.0.38±0.04,0.40±0.05,0.23±0.02,all P<0.05),the cell apoptosis rate increased[(2.5±0.2)%,(2.7±0.3)%vs.(12.6±1.2)%,(11.9±1.5)%,(18.9±1.7)%,all P<0.05],and the proportion of cells in G0/G1 phase increased[(50.4±4.2)%,(51.3±6.3)%vs.(62.3±3.2)%,(61.9±5.8)%,(71.4±5.4)%,all P<0.05].The expression levels of p27 and C-caspase-3 proteins increased,and the expression level of CDK2 protein decreased.The software predicted that the target gene of miR-5193 was Foxp3,which was confirmed by the luciferase reporting system.Compared with the miR-5193+cisplatin+NC group,the cell proliferation activity(absorbance value)in miR-5193+cisplatin+Foxp3 group increased(0.24±0.03 vs.0.65±0.05,t=21.094,P<0.01),the proportion of cells in G0/G1 phase decreased[(71.0±6.4)%vs.(60.3±4.1)%,t=4.196,P<0.01],the apoptosis rate of cells decreased[(19.6±1.6)%vs.(11.5±1.2)%,t=11.880,P<0.01],the expression levels of p27 and C-caspase-3 proteins in cells decreased,and the expression levels of CDK2 and Foxp3 proteins increased.Conclusion The miR-5193 may increase the sensitivity of cervical cancer Caski cells to cisplatin in vitro by targeted inhibition of the Foxp3 gene.