反义miR-222上调PUMA基因表达对腺样囊性癌细胞生长的影响

The influence of antisense miR-222 upregulating PUMA gene expression on the growth of adenoid cystic carcinoma cells

目的探讨反义miR-222转染腺样囊性癌(adenoid cystic carcinoma,Acc)细胞SACC-83后p53上调凋亡调控因子(p53 up-regulated modulator of apoptosis,PUMA)基因表达的改变观察其对ACC细胞生长的影响。方法以SACC-83细胞作为实验对象,转染反义miR-222的细胞为实验组转染反义miR-222 NC的细胞为阴性对照组,并设置未转染的SACC-83细胞为空白对照组。应用RT-PCR与免疫印迹法,分别从mRNA水平与蛋白水平测定转染后备组细胞PUMA的表达,CCK-8法检测细胞增殖情况,Transwell法检测细胞迁移能力,流式细胞仪测定细胞凋亡情况。结果 RT-PCR与免疫印迹检测结果显示,实验组miR-222的表达水平较对照组明显降低(P<0.01),而PUMA的表达水平却显著上调(P<0.01)。实验组细胞生长受到抑制,细胞迁移能力明显降低。流式细胞仪分析结果显示,空白对照组、阴性对照组、实验组的细胞凋亡率分别为1.48%、1.76%和16.73%,实验组细胞凋亡率明显升高差异有统计学意义(P<0.01)。结论反义miR-222转染能上调PUMA的表达,降低SACC-83细胞的增殖与迁移能力,并促进SACC-83细胞凋亡。

Objective To explore the p53 up-regulated modulator of apoptosis（PUMA） gene expression changed after the transfection of antisense（As） miR-222 into the adenoid cystic carcinoma cell line SACC-83 in order to observe its effect on adenoid cystic carcinoma cells growth.Methods SACC-83 cells were transfected with As-miR-222 and AsmiR-222 NC respectively.The experiment was divided into 3 groups：the blank group（no trasnsfection group）,the negative control group（As-miR-222 NC group） and the experimental group（As-miR-222 group）.After transfection into SACC-83 cells with Lipofectamine^（TM） RNA iMAX,the expression of PUMA was analyzed by RT-PCR and Western Blot.The proliferation was detected by CCK-8 assay.The migratory ability was detected by Transwell assay.Apoptosis was examined by flow cytometry.Results In the experimental group,the expression of miR-222 was down-regulated significantly（P〈0.01）,while the expression of PUMA was up-regulated as confirmed by RT-PCR and Western blot（P 0.01）.After As-miR-222 transfection,CCK-8 assay showed that cell proliferation was inhibited,Transwell assay indicated that cell migratory ability was weakened,cell apoptosis ability was obviously enhanced.The apoptosis rates were1.48%,1.76%and 16.73%respectively.Apoptosis in experimental group was significantly increased,the difference was statistically significant（P〈0.01）.Conclusion Antisense miR-222 transfection can upregulate the expression of PUMA,reduce the proliferation and migration of SACC-83 cells and promote cell apoptosis.