AGR2通过调控TGF-β/SMAD信号通路对鼻咽癌细胞迁移和侵袭的影响

The effect of AGR2 on the migration and invasion of nasopharyngeal carcinoma cells by regulating the TGF-β/SMAD signaling pathway

摘要:

目的：探讨前梯度蛋白2（AGR2）调控转化生长因子-β（TGF-β）/SMAD信号通路对鼻咽癌（NPC）细胞迁移和侵袭的影响。方法：体外培养人正常鼻咽上皮细胞（NP69）和NPC细胞（5-8F、6-10B、CNE-1），qRT-PCR测定细胞中AGR2 mRNA表达。将5-8F细胞随机分为Control组、sh-NC组、sh-AGR2组、sh-AGR2+SRI-011381组（sh-AGR2+TGF-β信号通路激活剂SRI-011381），qRT-PCR及Western blot测定AGR2的转染效率。CCK8和克隆形成测定细胞增殖；划痕实验测定细胞迁移；Transwell实验测定细胞侵袭；流式细胞仪测定细胞凋亡；Western blot测定细胞中PCNA、MMP-2、MMP-9、TGF-β1、p-SMAD2/SMAD2蛋白表达。结果：与NP69细胞相比，NPC细胞中AGR2 mRNA表达升高。与Control组或sh-NC组相比，sh-AGR2组细胞的OD450、克隆数、划痕愈合率、侵袭数、PCNA、MMP-2、MMP-9、TGF-β1、p-SMAD2/SMAD2表达水平下降，细胞凋亡率升高（P＜0.05）；与sh-AGR2组相比，sh-AGR2+SRI-011381组细胞的OD450、克隆数、划痕愈合率、侵袭数、PCNA、MMP-2、MMP-9、TGF-β1、p-SMAD2/SMAD2表达水平升高，细胞凋亡率下降（P＜0.05）。结论：AGR2在NPC细胞中表达水平升高，进而促进NPC细胞的增殖、迁移及侵袭能力，抑制细胞凋亡。这可能是通过调控TGF-β/SMAD信号通路实现的。

Abstract:

Objective: To investigate the effect of anterior gradient 2 (AGR2) on the migration and invasion of nasopharyngeal carcinoma (NPC) cells by regulating the transforming growth factor-β (TGF-β)/SMAD signaling pathway. Methods: Human normal nasopharyngeal epithelial cells (NP69) and NPC cells (5-8F, 6-10B, CNE-1) were cultured in vitro, and qRT-PCR was used to detect the expression of AGR2 mRNA in the cells. 5-8F cells were randomly assigned into Control group, sh-NC group, sh-AGR2 group, and sh-AGR2+SRI-011381 group (sh-AGR2+TGF-β signaling pathway activator SRI-011381). QRT-PCR and Western blot were applied to detect the transfection efficiency of AGR2. Cell proliferation was measured by CCK8 assay and colony formation assay. Cell migration was measured by scratch test. Transwell assay was used to measure cell invasion. Cell apoptosis was determined by flow cytometry. The protein expressions of PCNA, MMP-2, MMP-9, TGF-β1 and p-SMAD2/SMAD2 were detected by Western blot. Results: Compared with NP69 cells, the AGR2 mRNA expression was elevated in NPC cells. Compared with the Control group or sh-NC group, the OD450, clone number, scratch healing rate, invasion number, PCNA, MMP-2, MMP-9, TGF-β1, and p-SMAD2/SMAD2 expression levels in the sh-AGR2 group decreased, and the apoptosis rate was increased (P<0.05). Compared with the sh-AGR2 group, the OD450, clone number, scratch healing rate, invasion number, PCNA, MMP-2, MMP-9, TGF-β1, and p-SMAD2/SMAD2 expression levels in the sh-AGR2+SRI-011381 group increased, and the apoptosis rate was decreased (P<0.05). Conclusion: AGR2 is highly expressed in NPC cells, which can promote proliferation, migration, invasion, and inhibit cell apoptosis of NPC cells, and it may be related to the regulation of TGF-β/SMAD signaling pathway.