摘要
目的探讨miR－143靶向K－ras基因对宫颈癌细胞HeLa增殖的调控作用机制。方法选取宫颈癌细胞系HeLa，分别转染miR－143模拟物和模拟物对照，采用实时荧光PCR法检测HeLa细胞中miR－143的表达。将荧光素酶报告载体与miR－143模拟物共转染，采用荧光素酶活性实验验证miR－143对HeLa细胞的靶向作用。以miR－143模拟物、miR－143模拟物对照以及miR－143模拟物＋K－ras基因分别转染HeLa细胞，采用四甲基偶氮唑蓝（MTT）法检测各组细胞的增殖活性，采用Western blot法检测各组细胞中K－ras蛋白的表达。选取5例人宫颈癌组织标本和宫颈上皮内瘤变组织样本，采用实时荧光PCR法检测miR－143的表达，采用Western blot法检测K－ras蛋白的表达。结果实时荧光PCR检测结果显示，转染miR－143模拟物组宫颈癌HeLa细胞中miR－143的表达水平为3.31±0.45，明显高于阴性对照组（0.97±0.22, P〈0.05）。双荧光素酶活性检测结果显示，miR－143模拟物可明显抑制野生型K－ras报告载体的荧光素酶活性，但对突变型K－ras报告载体的荧光素酶活性并无明显的抑制作用。MTT法检测结果显示，转染miR－143模拟物后，宫颈癌HeLa细胞的增殖活性较阴性对照组明显降低（P〈0.05）；而转染miR－143模拟物＋K－ras基因后，宫颈癌HeLa细胞的增殖活性较单纯转染miR－143模拟物组明显提高（P〈0.05），但仍明显低于阴性对照组（P〈0.05）。Western blot法检测结果显示，阴性对照组、miR－143模拟物组、miR－143模拟物＋K－ras组K－ras蛋白的表达水平分别为521.36±41.89、115.27±34.08和706.52±89.44，miR－143模拟物组K－ras蛋白的表达较阴性对照组明显降低（P〈0.05）；miR－143模拟物＋K－ras组K－ras蛋白的表达较miR－143模拟物组和阴性对照组明显增高（P〈0.05）。实时荧光PCR检测结果显示，人宫颈癌组织中miR－143的表达水平（0.32±0.06
Objective To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods The lueiferase report carrier containing wild type 3＇-UTR of K-ras gene （K-ras-wt） or mutated 3＇-UTR of the K-ras （K-ras-mut） were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic （ miR-143 mimic group）, mimic control （ negative control group）, and miR-143 mimic plus K-ras gene （miR-143 mimic＋K-ras group） , respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR （RT-PCR）, and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples （ n = 5） and cervical intraepithelial neoplasia tissue samples （ n = 5） were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control （3.31±0.45 vs 0.97±0.22, P〈0.05）. The MTF assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group （P〈0.05）, and the cell proliferative activity of the miR-143 mimic＋K-ras group was also significantly lower than the control group （P〈0.05） but higher than the miR-143 mimic group significantly （P〈0.05）. The expression levels of K-ras protein in the miR-143 mimic group, the negative cont
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
2016年第12期893-897,共5页
Chinese Journal of Oncology
基金
河南省卫生厅科技攻关项目（200802009）
河南省教育厅自然科学研究计划（2008A320011）
