miR-101-3p suppresses proliferation of orbital fibroblasts by targeting pentraxin-3 in thyroid eye disease

Abstract

Background 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. Methods 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 via 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. Results 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. Conclusion 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.