反义miR-222靶向上调PUMA基因表达促进口腔鳞状细胞癌凋亡

As-miR-222 transfection target PUMA to induce cell apoptosis in oral squamous cell carcinoma

目的探讨反义miR-222转染靶向调节p53上调凋亡调控因子(PUMA)基因表达对口腔鳞状细胞癌(OSCC)细胞Tca8113凋亡的影响。方法将反义miR-222和反义miR-222阴性对照(NC)分别转入人OSCC细胞Tca8113中,采用反转录聚合酶链反应(RT-PCR)以及Westernblot分别从mRNA和蛋白水平检测转染后各组细胞PUMA的表达,MTT法检测各组细胞的增殖,流式细胞仪检测细胞周期及细胞凋亡率。结果 RT-PCR及Westernblot检测显示,反义miR-222转染组miR-222的表达水平较空白对照组与反义miR-222NC转染组显著下调(P<0.01),而PUMA基因的mRNA转录水平以及蛋白水平的表达则明显升高(P<0.05);与空白对照组和反义miR-222NC转染组相比,反义miR-222转染组细胞生长明显抑制;流式细胞仪检测分析显示,三组细胞的凋亡率分别为2.65%、2.72%和13.58%,反义miR-222转染组与空白对照组和反义miR-222NC转染组相比较细胞凋亡率显著增加,差异有统计学意义(P<0.01),其中细胞早期凋亡尤为明显,处于G1期的细胞数目明显增多。结论反义miR-222基因转染可靶向上调PUMA基因表达,降低Tca8113细胞的增殖能力,促进细胞凋亡。

Objective To investigate the effect of overexpression of p53 upregulated modulator of apoptosis （PUMA） gene induced 155, As-miR-222 transfeetion on apoptosis of oral squamous cell carcinoma（OSCC）. Methods Tca8113 was treated with As-miR-222 and As-miR-222 NC respectively, the experiment was randomly divided into 3 groups： blank group, As-miR-222 transfection group and As-miR-222 NC transfection group. After the recombinants were transfected into Tca8113 with LipofectamineTM MAX, the expression of PUMA on mRNA and protein level was analyzed by RT-PCR and Western blot, respectively. The proliferation of Tca8113 cells was detected by MqT assay. Cell cycle and apoptosis were detected by flow cytometry. Results Compared with blank group and As-miR-222 NC transfection group, miR-222 expression was down-regulated obviously （P 〈 0.01） meanwhile the expression of PUMA at both mRNA level and protein level was up-regulated as validated by RT-PCR and Western blot （P 〈 0.05）. MTY assay indicated that cell proliferation was inhibited by As-miR-222 transfection. Cell apoptosis ability was significantly increased after treatment with As-miR-222. Conclusion As-miR-222 transfection could up-regulate the expression of PUMA to induce apoptosis in human oral squamous cell carcinoma,