叉头框转录因子F2小发夹RNA对人眼小梁网细胞外基质蛋白表达的抑制作用

The inhibitory effect of FoxF2 shRNA on the expression of extracellular matrix of human trabecular meshwork

目的探讨叉头框转录因子F2(FoxF2)对小梁网细胞外基质表达的调控作用。方法将培养人眼小梁网细胞(HTMCs)分为Scramble对照组和FoxF2小发夹RNA(shRNA)组,构建FoxF2重组干扰载体FoxF2 shRNA,应用FoxF2 shRNA慢病毒颗粒感染HTMCs,采用Western blot法检测FoxF2 shRNA的敲低效果及细胞外基质(ECM)蛋白的表达变化,利用Transwell计数实验分析HTMCs的迁移能力。结果体外培养的HTMCs呈长梭形,生长状态良好,形态符合HTMCs形态学特征。FoxF2 shRNA组FoxF2蛋白相对表达量为0.72±0.02,显著低于Scramble对照组的1.27±0.05,差异有统计学意义(t=16.68,P<0.01)。FoxF2shRNA组纤连蛋白(FN)、Ⅰ型胶原蛋白(COLⅠ)、α平滑肌肌动蛋白(α-SMA)相对表达量分别为0.43±0.03、0.53±0.08和0.86±0.15,显著低于Scramble对照组的0.87±0.04、1.66±0.06和1.73±0.13,差异均有统计学意义(t=15.08、18.81、7.50,均P<0.01)。FoxF2 shRNA组HTMCs的迁移数量为117.30±11.41,显著低于Scramble对照组的251.00±10.37,差异有统计学意义(t=8.72,P<0.01)。结论成功制备可特异性敲低HTMCs内源性FoxF2表达的FoxF2 shRNA病毒,并证实FoxF2干扰对HTMCs中ECM的表达及细胞迁移具有抑制作用。

Objective To explore the role of forkhead box F2 (FoxF2) in the extracellular matrix of trabecular meshwork. Methods The cultured human trabecular meshwork cells (HTMCs) were divided into Scramble control group and FoxF2 small hairpin RNA (shRNA) group, then FoxF2 shRNA, the FoxF2 restructuring interference carrier was built, HTMCs were infected with FoxF2 shRNA lentivirus.Western blot assay was used to detect the expression of FoxF2 protein and extracellular matrix.Furthermore, Transwell counting experiment was used to analyze the migration ability of HTMCs. Results The cultured HTMCs grew well and showed a long spindle shape.The growth status of HTMCs was well, and their morphological characteristics were consistent with the HTMCs in vivo.The relative expression level of FoxF2 protein in the FoxF2 shRNA group was lower than that in the Scramble control group, with a significant difference between them (0.72±0.02 vs.1.27±0.05;t=16.68, P<0.01). The relative expression level of fibronectin (FN), collagen typeⅠ(COLⅠ) and α-smooth muscle actin (α-SMA) were 0.43±0.03, 0.53±0.08 and 0.86±0.15 in the FoxF2 shRNA group, and 0.87±0.04, 1.66±0.06 and 1.73±0.13 in the Scramble control group, respectively, the relative expression levels of FN, COLⅠ and α-SMA in the FoxF2 shRNA group were significantly lower than those in the Scramble control group (t=15.08, 18.81, 7.50, all at P<0.01). The migration number of HTMCs in the FoxF2 shRNA group was significantly lower than that in the Scramble control group (117.30±11.41 vs. 251.00±10.37;t=8.72, P<0.01). Conclusions The FoxF2 shRNA lentivirus are successfully constructed, which can decrease the expression of FoxF2 in HTMCs.Low expression of FoxF2 can reduce the expression level of extracellular matrix protein in HTMCs and inhibit the migration ability of HTMCs.