Xing Yao, Cui Lijun, Kang Qianyan
Medical School of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Department of Ophthalmology, The First Affiliated Hospital of Medical School of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
Int J Mol Med. 2016 Aug;38(2):407-16. doi: 10.3892/ijmm.2016.2644. Epub 2016 Jun 16.
The cytokine, transforming growth factor-β (TGF‑β), plays a key role in wound healing and tissue repair. Integrin‑linked kinase (ILK) is a downstream factor of the TGF-β signaling pathway. Research on ILK has mainly focused on its role in the invasion and metastasis of cancer cells. However, little has been reported on the effects of ILK in human Tenon's capsule fibroblasts (HTFs). In this study, we investigated the role of ILK in the proliferation and migration of HTFs exposed to TGF-β2. A lentiviral vector targeting ILK was screened from three candidates and the experimental result indicated that RNA interference can be used to inhibit ILK expression at both the mRNA and protein level. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to assess ILK mRNA expression. Cell proliferation was quantified by MTT assay and cell cycle progression was detected by flow cytometric analysis. Migration was measured by wound healing assay. It was observed that the silencing of ILK suppressed the TGF-β2-induced proliferation of HTFs and led to G1 phase cell cycle arrest and the significant downregulation of cyclin D1 expression. The migration ability of the HTFs decreased following the silencing of ILK, while the downregulation of α-smooth muscle actin expression and the upregulation of E-cadherin expression were observed. The findings of our study indicate that the silencing of ILK attenuates the abnormal proliferation and migration of HTFs induced by TGF-β2, which reveals the therapeutic potential of ILK inhibition in the prevention of scarring following glaucoma filtration surgery.
细胞因子转化生长因子-β（TGF-β）在伤口愈合和组织修复中起关键作用。整合素连接激酶（ILK）是TGF-β信号通路的下游因子。对ILK的研究主要集中在其在癌细胞侵袭和转移中的作用。然而，关于ILK在人眼球筋膜成纤维细胞（HTF）中的作用报道较少。在本研究中，我们调查了ILK在暴露于TGF-β2的HTF增殖和迁移中的作用。从三个候选物中筛选出靶向ILK的慢病毒载体，实验结果表明RNA干扰可用于在mRNA和蛋白质水平抑制ILK表达。采用逆转录定量聚合酶链反应（RT-qPCR）评估ILK mRNA表达。通过MTT法对细胞增殖进行定量，并通过流式细胞术分析检测细胞周期进程。通过伤口愈合试验测量迁移。观察到ILK沉默抑制了TGF-β2诱导的HTF增殖，并导致G1期细胞周期停滞以及细胞周期蛋白D1表达的显著下调。ILK沉默后HTF的迁移能力下降，同时观察到α-平滑肌肌动蛋白表达下调和E-钙黏蛋白表达上调。我们的研究结果表明，ILK沉默减弱了TGF-β2诱导的HTF异常增殖和迁移，这揭示了抑制ILK在预防青光眼滤过术后瘢痕形成中的治疗潜力。
