目的:探讨活化T 细胞核因子5（nuclear factor 5 of activated T cells, NFAT5）对人胃癌MGC803 细胞增殖及凋亡能力的影响及其可能的机制。方法：设计并合成3 条靶向NFAT5 基因的siRNA（siRNA2567、siRNA2714 和siRNA4562）及1 条与NFAT5 基因无同源性的阴性对照siRNA（NC-siRNA），脂质体介导转染人胃癌MGC803 细胞后，采用Real-time PCR检测分析细胞中NFAT5 mRNA 表达水平的变化，进而筛选出有效抑制NFAT5 基因表达的siRNA（NFAT5-siRNA）。NFAT5-siRNA 转染MGC803 细胞48 h 后，进一步采用Real-time PCR和Western blotting 验证并检测细胞中NFAT5 和S100A4 mRNA及蛋白表达水平的变化，流式细胞术和CCK-8 法分析抑制NFAT5 表达对细胞增殖及凋亡的影响。结果: 转染siRNA2567 对NFAT5 mRNA的表达抑制最为明显（P<0.01），siRNA2567 被验证为NFAT5-siRNA。转染NFAT5-siRNA 48 h 后，NFAT5 和S100A4 mRNA及蛋白的表达水平均明显降低（P<0.05）;与NC-siRNA 组相比较，NFAT5-siRNA 组MGC803 细胞的增殖率在72 h 和96 h 均显著降低（P<0.01）；NFAT5 基因沉默48 h 后，MGC803 细胞的凋亡率由（2.7±0.2）%上升至（7.9±0.2）%（P<0.01）。结论: NFAT5-siRNA 能有效沉默人胃癌MGC803 细胞中NFAT5 基因表达，在抑制细胞增殖率的同时能够有效促进细胞凋亡，该作用可能通过调控S100A4 表达实现。

Objective: To investigate the effects of nuclear factor 5 of activated T cells (NFAT5) on proliferation and apoptosis of human gastric cancer MGC803 cells and to explore the possible mechanisms. Methods: Three siRNAs targeting NFAT5 gene (siRNA2567,siRNA2714 and siRNA4562) and one negative control siRNA were designed and chemically synthesized before transfected into human gastric cancer cell line MGC803 by liposome. Real-time PCR was used to detect the changes of NFAT5 mRNA expression in MGC803 cells to further pick out the siRNA that most effectively inhibit the expression of NFAT5. Further, Real-time PCR and Western blotting assay were carried out to test mRNA and protein levels of NFAT5 and S100A4 in cells 48 h after NFAT5-siRNA transfection.Then, CCK-8 assay and FCM assay were used to detect the influence of silencing NFAT5 on cell proliferation and apoptosis, respectively.Results: siRNA2567 was the most effective siRNA that significantly inhibited the expression of NFAT5 mRNA (P<0.01),and thus was validated as NFAT5-siRNA. Real-time PCR and Western blotting assay confirmed that both mRNA and protein levels of NFAT5 and S100A4 were down-regulated in cells 48 h after NFAT5-siRNA transfection. Compared with NC-siRNA group, the proliferation ability of MGC803 cells in the NFAT5-siRNA group was significantly down-regulated at 72 h and 96 h (P<0.01). And FCM assay showed that compared with NC-siRNA group, cell apoptosis rate of NFAT5-siRNA group was significantly increased from (2.7±0.2)% to (7.9±0.2)%, (P<0.01) 48 h after NFAT5-siRNA transfection. Conclusion: NFAT5-siRNA transfection can silence NFAT5 gene expression in gastric cancer MGC803 cells effectively. NFAT5 may inhibit proliferation and promote cell apoptosis of gastric cancer cells possibly through regulating S100A4 expression.

国家自然科学基金资助项目(No. 81803855); 辽宁省教委高等学校科研基金资助项目(No. L201614）