Experimental Study on the Regulatory Effects of miR-100-5p on Proliferation and Apoptosis of Thyroid Cancer Cells
张廷华 1胡友元 2袁博3
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作者信息
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
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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(A450nm)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 A450nm 均低于 miR-inhibitorNC 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(0.99±0.11 vs 1.00±0.00),差异无统计学意义(t=-0.057,0.181,P=0.96,0.873).结论 miR-100-5p在甲状腺癌细胞中表达上调,可促进甲状腺癌细胞增殖、抑制细胞凋亡,其可能成为甲状腺癌诊疗中新的生物标志物与调控靶点.
Abstract
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(A450nm)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 A450nm 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 group were down-regulated compared to the miR-mimic NC group(0.78±0.12 vs 1.00±0.00),with significant differences(t=3.071,P=0.037).Compared to the miR-inhibitor NC group,FGFR3 protein expression levels in the miR-100-5p inhibitor group were up-regulated(1.17±0.07 vs 1.00±0.00),with significant differences(t=-4.509,P=0.046).There was no significant difference in the luciferase activity of the FGFR3 wild-type(1.01±0.17 vs 1.00±0.00)and mutant groups(0.99±0.11 vs 1.00±0.00)between miR-100-5p mimic and miR-mimic NC,and the differences were statistically significant(f=-0.057,0.181,P=0.96,0.873).Conclusion MiR-100-5p in thyroid cancer cells was up-regulated,which may promote cell proliferation and inhibit apoptosis.It may become a new biomarker and regulatory target in the diagnosis and treatment of thyroid cancer.
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