Department of Ophthalmology, Changzheng Hospital of Naval Medical University, Shanghai, China.
PeerJ. 2024 Nov 15;12:e18535. doi: 10.7717/peerj.18535. eCollection 2024.
Excessive proliferation of orbital fibroblasts (OFs) is an essential factor in the pathogenesis of thyroid eye disease (TED). While existing evidence indicates that various microRNAs (miRNAs) significantly contribute to TED development, the precise function and targets of miR-101-3p in TED pathogenesis remain unknown. This research aims to elucidate the effects of miR-101-3p on TED-OFs and identify its potential targets.
Orbital adipose tissues were harvested from both TED patients and healthy controls to culture their fibroblasts. MiR-101-3p mimic or mimic negative control (mimic NC) was transfected into OFs from TED patients, with untreated OFs serving as an additional blank control group. Cell proliferation was assessed using cell counting kit-8 (CCK-8) assay, Ki-67 immunofluorescence staining, and the EdU assay, while apoptosis was evaluated flow cytometry. Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure the expression levels of miR-101-3p and pentraxin-3 (PTX3), and PTX3 protein levels were quantified using western blot. A dual-luciferase assay was conducted to ascertain how miR-101-3p and PTX3 interacted.
The results demonstrated a significant downregulation of miR-101-3p in fibroblasts and TED orbital adipose tissues. Transfection with the miR-101-3p mimic upregulated miR-101-3p levels, significantly reducing OFs proliferation without affecting apoptosis. Overexpression of miR-101-3p led to the downregulation of PTX3 in OFs. The dual-luciferase assay validated miR-101-3p binding to PTX3's 3'UTR, thereby repressing its expression. Moreover, overexpression of PTX3 partially rescued the miR-101-3p mimic's inhibitory effect on TED-OFs proliferation.
Our findings illustrate miR-101-3p's role in targeting PTX3 to regulate TED-OFs proliferation, providing novel insights into the pathological mechanisms underlying TED development.
眼眶成纤维细胞（OFs）的过度增殖是甲状腺眼病（TED）发病机制的一个重要因素。虽然现有证据表明，各种 microRNAs（miRNAs）对 TED 的发展有重要贡献，但 miR-101-3p 在 TED 发病机制中的确切功能和靶标仍不清楚。本研究旨在阐明 miR-101-3p 对 TED-OFs 的影响，并鉴定其潜在的靶标。
从 TED 患者和健康对照者的眼眶脂肪组织中提取眼眶脂肪组织，培养其成纤维细胞。将 miR-101-3p 模拟物或模拟物阴性对照（mimic NC）转染入 TED 患者的 OFs 中，未处理的 OFs 作为空白对照组。通过细胞计数试剂盒-8（CCK-8）检测、Ki-67 免疫荧光染色和 EdU 检测评估细胞增殖，通过流式细胞术评估细胞凋亡。采用定量实时聚合酶链反应（qRT-PCR）检测 miR-101-3p 和 pentraxin-3（PTX3）的表达水平，并用 Western blot 检测 PTX3 蛋白水平。通过双荧光素酶报告基因实验确定 miR-101-3p 和 PTX3 之间的相互作用方式。
结果表明，成纤维细胞和 TED 眼眶脂肪组织中 miR-101-3p 的表达显著下调。转染 miR-101-3p 模拟物上调 miR-101-3p 水平，显著降低 OFs 增殖而不影响细胞凋亡。miR-101-3p 的过表达导致 OFs 中 PTX3 的下调。双荧光素酶报告基因实验验证了 miR-101-3p 与 PTX3 3'UTR 的结合，从而抑制其表达。此外，PTX3 的过表达部分挽救了 miR-101-3p 模拟物对 TED-OFs 增殖的抑制作用。
我们的研究结果表明，miR-101-3p 通过靶向 PTX3 来调节 TED-OFs 的增殖，为 TED 发病机制提供了新的见解。
