Circ_0047835 Combines with miR-144-3p to Promote the Proliferation, Invasion, Migration, and Fibrosis of TGF-β1-Treated Human Tenon’s Capsule Fibroblasts by Upregulating SP1

Glaucoma is the leading cause of blindness worldwide with complex pathogenesis. Circular RNAs (circRNAs) play critical roles in various diseases, including glaucoma. The purpose of this study was to investigate the role of circ_0047835 and underlying mechanisms in the development of fibrosis after glaucoma filtration surgery. Human Tenon’s capsule fibroblasts (HTFs) were stimulated using transforming growth factor-β1 (TGF-β1) to mimic a cellular model of glaucoma <i>in vitro</i>. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2’-deoxyuridine (EdU) assay. Cell invasion and migration were detected by transwell assay and wound healing assay, respectively. Western blot assay was used to measure protein levels. The expression levels of circ_0047835, microRNA-144-3p (miR-144-3p) and specific protein 1 (SP1) mRNA were determined by real-time quantitative polymerase chain reaction (RT-qPCR). The interaction between miR-144-3p and circ_0047835 or SP1 was confirmed by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Circ_0047835 expression was elevated in glaucoma tissues and TGF-β1-treated HTFs. Circ_0047835 or SP1 knockdown suppressed the proliferation, migration, invasion, and fibrosis of TGF-β1-treated HTFs. MiR-144-3p was a target of circ_0047835, and miR-144-3p inhibition reversed the effects of circ_0047835 knockdown in TGF-β1-treated HTFs. Moreover, SP1 was identified as a target of miR-144-3p, and miR-144-3p overexpression weakened TGF-β1-induced proliferation, migration, invasion, and fibrosis by targeting SP1 in HTFs. Furthermore, circ_0047835 combined with miR-144-3p to regulate SP1 expression. Circ_0047835 might contribute to fibrosis progression after glaucoma surgery by regulating the miR-144-3p/SP1 axis.

青光眼（Glaucoma）是全球范围内首要的致盲性眼病，其发病机制复杂。环状RNA（Circular RNAs, circRNAs）在包括青光眼在内的多种疾病中发挥关键作用。本研究旨在探讨circ_0047835在青光眼滤过手术后纤维化发生发展中的作用及其潜在机制。使用转化生长因子-β1（transforming growth factor-β1, TGF-β1）刺激人Tenon囊成纤维细胞（Human Tenon’s capsule fibroblasts, HTFs）以构建青光眼的体外细胞模型。采用细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）实验与5-乙炔基-2’-脱氧尿苷（5-ethynyl-2’-deoxyuridine, EdU）实验评估细胞增殖能力；分别通过Transwell实验与划痕愈合实验检测细胞侵袭与迁移能力。采用蛋白质印迹（Western blot）实验检测蛋白水平；通过实时定量聚合酶链反应（real-time quantitative polymerase chain reaction, RT-qPCR）检测circ_0047835、微小RNA-144-3p（microRNA-144-3p, miR-144-3p）及特异性蛋白1（specific protein 1, SP1）的mRNA表达水平。采用双荧光素酶报告基因实验与RNA免疫沉淀（RNA Immunoprecipitation, RIP）实验验证miR-144-3p与circ_0047835或SP1之间的相互作用。研究结果显示，circ_0047835在青光眼组织及TGF-β1处理的HTFs中表达上调。敲低circ_0047835或SP1可抑制TGF-β1处理的HTFs的增殖、迁移、侵袭及纤维化进程。miR-144-3p是circ_0047835的靶基因，抑制miR-144-3p可逆转敲低circ_0047835对TGF-β1处理的HTFs的上述影响。此外，SP1被证实为miR-144-3p的靶基因，过表达miR-144-3p可通过靶向SP1削弱TGF-β1诱导的HTFs增殖、迁移、侵袭及纤维化。进一步研究发现，circ_0047835可通过miR-144-3p调控SP1的表达。综上，circ_0047835可能通过调控miR-144-3p/SP1轴参与青光眼术后纤维化的进展。