Zhou Meng-Yuan, Cai Li, Feng Xiao-Wen, Mu Yu-Rong, Meng Bo, Liu Fang-Yuan, Li Rong
Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, School of Pharmacy, Anhui Medical University, Hefei, 230032, Anhui Province, People's Republic of China.
Department of Pathology, School of Basic Medicine, Anhui Medical University, Hefei, 230032, Anhui Province, People's Republic of China.
J Inflamm Res. 2021 May 14;14:1945-1957. doi: 10.2147/JIR.S312783. eCollection 2021.
Previous studies have confirmed the pathologic role of synovial aquaporin 1 (AQP1) in rheumatoid arthritis (RA), but its associations with the abnormal biologic behaviors of fibroblast-like synoviocytes (FLS) remain unclear. Herein, we examined the roles of AQP1 in the proliferation, migration and invasion of TNF-α-stimulated RA FLS (MH7A cells) and explored the underlying mechanisms.
Lentivirus-mediated AQP1 overexpression or silencing MH7A cells was constructed. Assays of MTT, flow cytometry (PI staining and Annexin V-PE/7-AAD staining), TMRM staining, wound-healing, transwell and phalloidin staining were performed to detect cell proliferation, cycle distribution, apoptosis, migration and invasion. The involvement of Wnt/β-catenin pathway was revealed by Western blot and β-catenin immunofluorescence staining.
AQP1 overexpression promoted cell proliferation of TNF-α-stimulated MH7A by facilitating transformation from G0/G1 to S phase and inhibiting cell apoptosis (ie, reduced apoptosis rates, raised mitochondrial membrane potential, increased Bcl-2 protein level and decreased levels of Bax and cleaved caspase 3 protein). Also, AQP1 overexpression increased the migration index as well as the numbers of migrated and invasive cells. Furthermore, AQP1 overexpression promoted the activation of Wnt/β-catenin pathway, and XAV939, an inhibitor of Wnt/β-catenin, canceled the above effects of AQP1 overexpression on MH7A cells. As expected, AQP1 silencing exhibited the opposite effects on TNF-α-stimulated MH7A cells, which could be reversed by LiCl, an activator of Wnt/β-catenin.
AQP1 can affect the proliferation, migration and invasion of MH7A cells by Wnt/β-catenin signaling pathway, and AQP1 can be as a crucial determiner that can regulate RA FLS biologic behaviors.
既往研究已证实滑膜水通道蛋白1（AQP1）在类风湿关节炎（RA）中的病理作用，但其与成纤维样滑膜细胞（FLS）异常生物学行为的关联仍不清楚。在此，我们研究了AQP1在肿瘤坏死因子-α（TNF-α）刺激的RA FLS（MH7A细胞）增殖、迁移和侵袭中的作用，并探讨了其潜在机制。
构建慢病毒介导的AQP1过表达或沉默的MH7A细胞。进行MTT法、流式细胞术（PI染色和膜联蛋白V-PE/7-AAD染色）、TMRM染色、伤口愈合实验、Transwell实验和鬼笔环肽染色，以检测细胞增殖、细胞周期分布、细胞凋亡、迁移和侵袭。通过蛋白质免疫印迹法和β-连环蛋白免疫荧光染色揭示Wnt/β-连环蛋白信号通路的参与情况。
AQP1过表达通过促进细胞从G0/G1期向S期转化并抑制细胞凋亡（即降低凋亡率、提高线粒体膜电位、增加Bcl-2蛋白水平以及降低Bax和裂解的半胱天冬酶3蛋白水平），促进TNF-α刺激的MH7A细胞增殖。此外，AQP1过表达增加了迁移指数以及迁移和侵袭细胞的数量。此外，AQP1过表达促进了Wnt/β-连环蛋白信号通路激活，而Wnt/β-连环蛋白抑制剂XAV939消除了AQP1过表达对MH7A细胞的上述作用。正如预期的那样，AQP1沉默对TNF-α刺激的MH7A细胞表现出相反的作用，而Wnt/β-连环蛋白激活剂氯化锂可逆转这种作用。
AQP1可通过Wnt/β-连环蛋白信号通路影响MH7A细胞的增殖、迁移和侵袭，且AQP可作为调节RA FLS生物学行为的关键决定因素。
