目的:探讨microRNA146a(miR-146a)在大鼠青光眼滤过手术(GFS)及转化生长因子(TGF)-β1刺激大鼠Tenon囊成纤维细胞后纤维化改变中的作用及其机制。
方法:用TGF-β1处理培养的大鼠Tenon的囊膜成纤维细胞,并用微阵列分析mRNA谱分析以验证miR-146a为靶标。然后在体外TGF-β1刺激后,分别用慢病毒介导的miR-146a模拟物或抑制剂转染处理Tenon's荚膜成纤维细胞,而GFS是在大鼠眼中进行的,术中分别给予miR-146a,丝裂霉素C(MMC),或体内5-氟尿嘧啶(5-FU)。促纤维化基因表达水平(纤连蛋白,胶原蛋白Iα,NF-KB,IL-1β,TNF-α,SMAD4和α-平滑肌肌动蛋白)通过qPCR测定,西方印迹,免疫荧光染色和/或组织化学分析在体外和体内。靶向SMAD4的siRNA进一步与miR-146a干预组合用于治疗成纤维细胞以证实其在潜在机制中的作用。
结果:miR-146a上调可降低TGF-β1体外诱导的大鼠Tenon’s囊成纤维细胞的增殖率和促纤维化变化,并减轻结膜下纤维化,以延长体内GFS后的滤过泡存活率,其中miR-146a降低NF-KB-SMAD4相关基因的表达水平,如纤连蛋白,胶原蛋白Iα,NF-KB,IL-1β,TNF-α,SMAD4和α-平滑肌肌动蛋白(α-SMA)。此外,SMAD4是miR-146a抑制纤维化过程中的关键靶基因。
结论:MiR-146a在体外和体内均能有效减轻TGF-β1诱导的大鼠Tenon囊成纤维细胞纤维化,可能通过NF-KB-SMAD4信号通路。MiR-146a有望成为预防纤维化和提高GFS成功率的新型治疗靶标。
OBJECTIVE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-β1 in rat Tenon\'s capsule fibroblasts.
METHODS: Cultured rat Tenon\'s capsule fibroblasts were treated with TGF-β1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon\'s capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-β1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1β, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms.
RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon\'s capsule fibroblasts induced by TGF-β1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1β, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis.
CONCLUSIONS: MiR-146a effectively reduced TGF-β1-induced fibrosis in rat Tenon\'s capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.