摘要
目的 研究下调激活转录因子3（ATF3）的表达对肾上腺皮质腺癌细胞增殖的影响及其作用机制.方法 利用免疫组化和Western印迹分别检测ATF3在人肾上腺皮质肿瘤组织和细胞中的表达;运用脂质体2000转染法将siATF3转染肾上腺皮质腺癌细胞Sw-13和NCI-H259R细胞,运用逆转录PCR检测ATF3 mRNA表达;Western印迹法检测ATF3、cleaved caspase 3、caspase 3、cleaved PARP、PARP蛋白表达;四甲基偶氮唑盐（MTT）和异硫氰酸胍标记磷脂结蛋白（AnnexinV-FITC）/碘化丙啶（ PI）分别测定细胞增殖率和细胞凋亡率;NCI-H259R细胞分别经 NVP-BEZ235、Perifosine、BKM120、IWP-2、PP2、KN93、Everolimus作用后,运用实时荧光定量PCR（ realtime PCR）测定ATF3 mRNA的表达;MTT比色法检测ATF3对抑制相关信号通路后细胞增殖的影响.结果 ATF3 在人肾上腺皮质腺肿瘤组织和细胞中呈高表达;而转染siATF3的Sw-13和NCI-F259R细胞ATF3 mRNA和蛋白表达显著降低;与阴性对照组（ NC siRNA）比较, siATF3转染显著抑制Sw-13和NCI-F259R细胞增殖（P＜0.05）,细胞凋亡率显著增加（P＜0.05）;Western印迹结果显示siATF3转染的细胞cleaved caspase 3和cleaved PARP蛋白水平显著增加;realtime PCR结果显示,与二甲基亚砜（DMSO）溶剂对照组相比,PP2、KN93、IWP-2信号通路抑制剂均显著抑制ATF3 mRNA的表达（P＜0.05）,而ATF3能够显著促进PP2、KN93、IWP-2信号抑制剂作用NCI-F259R细胞后的增殖活性.结论 ATF3在肾上腺皮质腺癌组织和细胞中高表达,下调ATF3的表达能抑制Sw-13和NCI-H259R细胞增殖,激活细胞凋亡通路诱导细胞凋亡;其机制与Wnt/β-catenin通路、CaMKI通路及SRC通路的激活相关.
Objective To investigate the effect of downregulated activating transcription factor 3 （ ATF3） expression on proliferation of adrenocortical carcinoma cells and its mechanisms. Methods Immunohistochemistry and Western blotting were used to detect the expression of ATF3 in human adrenocortical tumor tissues and cells. Adrenocortical carcinoma cells, Sw-13, and NCI-H259R cells, were transfected with siATF3 using lipidosome 2000, and expression of ATF3 mRNA was determined using RT-PCR; expression of ATF3, cleaved caspase 3, caspase 3, cleaved PARP, and PARP proteins were detected using Western blotting; cell growth inhibition rate and apoptosis rate were monitored using MTT and AnnexinV-FITC/PI, respectively. Sw-13 and NCI-H259R cells were treated with NVP-BEZ235, Perifosine, BKM120, IWP-2, PP2, KN93, Everolimus respectively followed by detected expression of ATF3 mRNA by realtime PCR. The effect of ATF3 on cell proliferation after inhibition of related signaling pathways were detected by MTT. Results The ATF3 in human adrenocortical gland tumor tissues and cells showed high expression. The levels of ATF3 mRNA and protein in Sw-13 and NCI-F259R cells transfected with siATF3 were significantly reduced. Compared with the negative control group （ NC siRNA）, siATF3 transfection significantly inhibited the proliferation of Sw-13 and NCI-F259R cells （ P＜0. 05 ）, and increased the apoptosis rate （ P＜0.05）. Western blotting shown that the levels of cleaved caspase 3 and cleaved PARP protein in siATF3 transfected cells increased significantly; and realtime PCR results indicated that the expression of ATF3 mRNA was dramatically inhibited by PP2, KN93, and IWP-2 in NCI-F259R cells compared with control group （ DMSO ）; but ATF3 significantly promoted the proliferation activity of NCI-F259R cells which treated by PP2, KN93, and IWP-2 signaling inhibitors. Conclusion High expression of ATF3 is existed in adrenocortical carcinoma cells. Downregulated ATF3 expression may inhibit cell proliferation and activate apoptosis pathway, resulting in apoptosis in Sw-13 and NCI-F259R cells, this mechanism of action is related to activating Wnt/β-catenin, CaMKI, and SRC pathway.
作者
魏光敏
陶海云
屈中玉
万里新
刘越
Wei Guangmin, Tao Haiyun, Qu Zhongyu, Wan Lixin, Liu Yue(Department of Internal Medical Oncology , Nanyang City Central Hospital, Nanyang 473009, China)
出处
《中华内分泌代谢杂志》
CAS
CSCD
北大核心
2018年第9期738-745,共8页
Chinese Journal of Endocrinology and Metabolism
基金
河南省科技厅支持项目（142300410034）
作者简介
Email：weiguangminsci@163．com;通信作者：万里新，Email：meiwen088@163.com
