Bao Xiu-li, Song Hui, Tang Xin
Clinical College of Ophthalmology of Tianjin Medical University, Tianjin 300020, China.
Zhonghua Yan Ke Za Zhi. 2012 Apr;48(4):312-7.
To investigate the effects of Wnt3a on epithelial-mesenchymal transition (EMT) in human lens epithelial cells and its molecular mechanisms.
Experimental research. The Wnt3a expression vector was designed and transiently transfected into SRA0104 cells and the PcDNA-HA expression vector was transfected into the controls. The expression of Wnt3a was detected by western blot analysis. The expression of β-catenin was detected by western blot analysis and the location of β-catenin was detected by immunofluorescence assay. The expression of E-cadherin protein as epithelial biomarker and fibronectin protein as mesenchymal biomarker was detected by Western blot analysis. The mobility of SRA0104 cells was assessed by scratch assay and transwell assay in vitro. Statistical significance was determined by Student's t-test.
The Wnt3a/SRA/01/04 cells were obtained by transfection of wnt3a cDNA expression vector in SRA01/04 cells and the pcDNA3-HA/SRA01/04 cells were used as the controls. The Wnt3a cDNA expression vector triggered β-catenin as a transcriptional activator accumulated and translocated into the nucleus, which induced the decreasing expression of E-cadherin proteins and increasing expression of fibronectin protein, and resulted in EMT in lens epithelial cells. Wound healing assay in vitro showed that the migration distance of Wnt3a/SRA01/04 cells was (0.36 ± 0.02) mm at 24 hours after scratch, significantly increased as compared to the control cells (t = 21.98, P < 0.01). After 48 hours of incubation in transwell migration assay, the number of migratory cells across polycarbonate membrane in Wnt3a/SRA01/04 cells was 92.25 ± 10.34, which was much more than the control cells (t = 10.40, P < 0.01). The mobility and migration of Wnt3a/SRA01/04 cells was increased.
Wnt3a activates Wnt/β-catenin signaling pathway, induces EMT in lens epithelial cells and then increases mobility and migration of lens epithelial cells.
探讨Wnt3a对人晶状体上皮细胞上皮-间质转化（EMT）的影响及其分子机制。
实验研究。设计Wnt3a表达载体并瞬时转染至SRA0104细胞，将PcDNA-HA表达载体转染至对照组细胞。采用蛋白质免疫印迹分析检测Wnt3a的表达。采用蛋白质免疫印迹分析检测β-连环蛋白的表达，采用免疫荧光测定法检测β-连环蛋白的定位。采用蛋白质免疫印迹分析检测作为上皮生物标志物的E-钙黏蛋白和作为间质生物标志物的纤连蛋白的表达。采用划痕试验和Transwell试验在体外评估SRA0104细胞的迁移能力。采用Student t检验确定统计学意义。
通过将wnt3a cDNA表达载体转染至SRA01/04细胞获得Wnt3a/SRA/01/04细胞，将pcDNA3-HA/SRA01/04细胞作为对照。Wnt3a cDNA表达载体促使作为转录激活因子的β-连环蛋白积累并转运至细胞核，这导致E-钙黏蛋白表达降低、纤连蛋白表达增加，并引起晶状体上皮细胞发生EMT。体外伤口愈合试验显示，划痕后24小时Wnt3a/SRA01/04细胞的迁移距离为（0.36±0.02）mm，与对照细胞相比显著增加（t=21.98，P<0.01）。在Transwell迁移试验中孵育48小时后，Wnt3a/SRA01/04细胞中穿过聚碳酸酯膜的迁移细胞数为92.25±10.34，远多于对照细胞（t=10.40，P<0.01）。Wnt3a/SRA01/04细胞的迁移能力和迁移率增加。
Wnt3a激活Wnt/β-连环蛋白信号通路，诱导晶状体上皮细胞发生EMT，进而增加晶状体上皮细胞的迁移能力和迁移率。
