靶向PD-1基因的miRNA210对胆囊癌细胞增殖、凋亡的影响

Effects of miRNA210 Targeting PD-1 Gene on Proliferation and Apoptosis of Gallbladder Cancer Cells

目的探讨靶向PD-1基因的miRNA210对胆囊癌细胞增殖、凋亡的影响。方法电转染法将PD-1过表达PCDNA3.1载体转染至人胆囊癌GBC-SD细胞,CCK-8法、流式细胞仪检测细胞增殖、凋亡;miRNA Microarray分析miRNA表达谱变化。利用Lipofectamine^(TM)2000将miRNA210-mimic转染至GBC-SD细胞,双荧光报告基因验证PD-1是miRNA210的靶基因,CCK-8法、流式细胞仪检测miRNA210转染后细胞增殖、凋亡变化。结果PD-1转染后,细胞增殖活性升高,细胞凋亡率降低(F=25.90,P<0.0001);人GBC-SD细胞miRNA表达谱发生明显变化,其中上调miRNAs 33个,下调miRNA 22个,其中miRNA210表达差异最显著。miRNA210转染后,转染组荧光素酶活性降低(F=43.51,P<0.0001),细胞增殖活性降低(0.15±0.17)(F=4.134,P=0.0431),细胞凋亡率升高(F=47.83,P<0.0001)。结论miRNA210通过靶向下调PD-1基因表达,抑制GBC-SD细胞增殖,促进细胞凋亡,可能参与胆囊癌的发生发展过程。

Objective To investigate the effect of miRNA210 targeting PD-1 gene on proliferation and apoptosis of gallbladder cancer cells.Methods PD-1 over-expressed PCDNA 3.1 vector was transfected into human gallbladder cancer GBC-SD cells by electro-transfection.Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry.Microarray was used to analyze the changes of microRNA expression profile.miRNA210-mimic was transfected into GBC-SD cells by Lipofectamine TM2000.Double fluorescence report gene was confirmed that PD-1 was the target gene of miRNA210.CCK-8 and flow cytometry were used to detect the changes of cell proliferation and apoptosis after miRNA210 transfection.Results After PD-1 transfection,cell proliferation activity increased,apoptotic rate decreased(F=25.90,P<0.0001),33 microRNAs up-regulated,and 22 microRNAs down-regulated,and the expression of miRNA210 was the most significant difference.After miRNA210 transfection,luciferase activity decreased(F=43.51,P<0.0001),cell proliferation activity decreased(0.15+0.17)(F=4.134,P=0.0431),and apoptotic rate increased(F=47.83,P<0.0001).Conclusion miRNA210 can inhibit the proliferation of GBC-SD cells and promote cell apoptosis by targeting down-regulation of PD-1 gene expression,which may be involved in the occurrence and development of gallbladder cancer.