miR-100-5p对甲状腺癌细胞增殖与凋亡调控作用的实验研究  

Experimental Study on the Regulatory Effects of miR-100-5p on Proliferation and Apoptosis of Thyroid Cancer Cells

作　　者：张廷华[1] 胡友元 袁博 ZHANG Tinghua;HU Youyuan;YUAN Bo(Department of Clinical Laboratory,the Second People’s Hospital of Huaihua City,Hunan Huaihua 418000,China;Department of Pathology Laboratory,the Second People’s Hospital of Huaihua City,Hunan Huaihua 418000,China;Department of Clinical Laboratory,Southern University of Science and Technology Hospital,Guangdong Shenzhen 518055,China)

机构地区：[1]怀化市第二人民医院检验科,湖南怀化418000 [2]怀化市第二人民医院病理科,湖南怀化418000 [3]南方科技大学医院检验科,广东深圳518055

出　　处：《现代检验医学杂志》2024年第4期56-62,共7页Journal of Modern Laboratory Medicine

基　　金：怀化市科技计划项目(2021R3113)。

摘　　要：目的通过实验探讨微小核糖核酸(microRNA,miR)-100-5p在甲状腺癌细胞中的表达情况及其对细胞增殖与凋亡的调控作用。方法使用荧光定量PCR检测miR-100-5p在甲状腺癌细胞系(TPC-1,KTC-1)与甲状腺正常细胞系(Nthy-ori3-1)中的相对表达情况。TPC-1细胞分别转染miR-100-5p模拟物(miR-100-5p mimic)、抑制物(miR-100-5p inhibitor)及相应阴性对照(miR-mimic NC,miR-inhibitor NC)后,用CCK-8检测TPC-1细胞增殖情况,流式细胞仪检测TPC-1细胞凋亡情况。通过miRTarBase和TargetScan7.2数据库对miR-100-5p的靶基因进行预测和功能富集分析,用蛋白印迹实验与双荧光素酶报告基因实验验证miR-100-5p对成纤维细胞生长因子受体3(fibroblast growth factor receptor 3,FGFR3)的靶向调控作用。结果与Nthy-ori3-1细胞相比,miR-100-5p在TPC-1细胞中表达水平(1.87±0.03 vs 1.00±0.03)与KTC-1细胞中表达水平(6.33±0.47 vs 1.00±0.03)均上调,差异具有统计学意义(t=-34.220,-19.588,均P<0.05)。转染miR-100-5p mimic组在24,48,72h细胞450nm吸光度(A_(450nm))均高于miR-mimic NC组,差异具有统计学意义(t=-7.516,-17.828,-8.445,均P<0.05);转染miR-100-5p inhibitor组在24,48,72h A_(450nm)均低于miR-inhibitor NC组,差异具有统计学意义(t=6.720,6.782,6.073,均P<0.05)。与miR-mimic NC组相比,转染miR-100-5p mimic后凋亡率(7.43%±0.49%vs 10.55%±0.80%)下降(t=5.767,P=0.004),与miR-inhibitor NC组相比,转染miR-100-5p inhibitor后凋亡率(3.19%±0.22%vs 2.64%±0.15%)上升(t=-3.606,P=0.023),差异均有统计学意义。蛋白印迹实验显示,与miR-mimic NC组相比,FGFR3在miR-100-5p mimic组蛋白表达水平(0.78±0.12 vs 1.00±0.00)下调(t=3.071,P=0.037),与miR-inhibitor NC组相比,FGFR3在miR-100-5p inhibitor组蛋白表达水平(1.17±0.07 vs 1.00±0.00)上升(t=-4.509,P=0.046),差异均有统计学意义。与miR-mimic NC相比,miR-100-5p mimic没有降低FGFR33’UTR野生型组荧光素酶活性(1.01±0.17 vs 1.00±0.00)与突变型组荧光素酶活性(Objective To explore the expression of microRNA(miR)-100-5p in thyroid cancer cells and its regulatory effects on cell proliferation and apoptosis through experiments.Methods The relative expressions of miR-100-5p in thyroid cancer cell lines(TPC-1 and KTC-1)and normal thyroid cell lines(Nthy ori3-1)were detected using fluorescence quantitative PCR.After transfection of miR-100-5p mimic,miR-100-5p inhibitor,and corresponding negative controls(miR-mimic NC,miR-inhibitor NC)into TPC-1 cells,the proliferation condition of TPC-1 cells was detected using CCK-8,and the apoptosis condition of TPC-1 cells was detected using flow cytometry.Prediction and functional enrichment analysis of target genes of miR-100-5p were performed using the miRTarBase and TargetScan7.2 databases.The targeted regulatory effect of miR-100-5p on fibroblast growth factor receptor 3(FGFR3)was validated using Western blot and dual luciferase reporter gene experiments.Results Compared with Nthy-ori3-1 cells,the expression levels of miR-100-5p in TPC-1 cells(1.87±0.03 vs 1.00±0.03)and KTC-1 cells(6.33±0.47 vs 1.00±0.03)were both up-regulated,with significant differences(t=-34.220,-19.588,all P<0.05).The 450nm absorbance(A_(450nm))of cells transfected with miR-100-5p mimic at 24,48 and 72 h were higher than the miR-mimic NC group,with significant differences(t=-7.516,-17.828,-8.445,all P<0.05).Conversely,the A_(450nm) values of cells transfected with miR-100-5p inhibitor at 24,48 and 72 h were lower than the miR-inhibitor NC group,with significant differences(t=6.720,6.782,6.073,all P<0.05).The apoptosis rate after transfection with miR-100-5p mimic was decreased compared to miR-mimic NC group(7.43%±0.49%vs 10.55%±0.80%),with significant differences(t=5.767,P=0.004).Compared to miR-inhibitor NC group,the apoptosis rate after transfection with miR-100-5p inhibitor was increased(3.19%±0.22%vs 2.64%±0.15%),with significant differences(t=-3.606,P=0.023).Western blot experiments showed that FGFR3 protein expression levels in the miR-100-5p mimic

关 键 词：微小核糖核酸-100-5p 甲状腺癌 细胞增殖 细胞凋亡 

分 类 号：R736.1[医药卫生—肿瘤] R730.43[医药卫生—临床医学]