piRNA-Annotated hsa_piR_016984 Regulates Extracellular Matrix Homeostasis in Human Scleral Fibroblasts by Modulating TGF-β1 mRNA Stability.

Scleral extracellular matrix (ECM) remodeling is a pathological feature of axial myopia. The relationship between small-RNA-associated regulation and cellular responses to mechanical loading remains incompletely understood. This study examines hsa_piR_016984-associated regulation in human scleral fibroblasts under a defined cyclic-stretch regimen. Text mining and bioinformatics identified myopia-associated hub genes. Fibroblasts underwent cyclic stretch (10% elongation, 1 Hz, 48 h) in vitro. Gene expression was evaluated by quantitative polymerase chain reaction (qPCR), and matrix-associated staining was assessed using toluidine blue. Sequencing dataset analysis and aqueous humor qPCR screened differentially expressed piRNA-annotated small RNAs. RNA pull-down, dual-luciferase reporter assays, and transcript time-course measurements examined their relationship with target messenger RNAs (mRNAs). Eight hub genes, including transforming growth factor beta 1 (TGF-β1) and various matrix metalloproteinases (MMPs), were identified via bioinformatic mining. In vitro, mechanical stretch reduced toluidine blue staining intensity, alongside increased MMP expression and decreased TGF-β1 expression, accompanied by a more pronounced decline in TGF-β1 qPCR signals relative to 0 h after actinomycin D addition. The piRNA-annotated small RNA hsa_piR_016984 was significantly downregulated in myopic aqueous humor. RNA pull-down showed TGFB1 mRNA co-enrichment with biotin-labeled hsa_piR_016984 relative to the negative-control probe. Separately, hsa_piR_016984 manipulation was associated with changes in TGFB1 qPCR signals over time and attenuation of the stretch-induced reduction in matrix staining intensity. Co-enrichment does not itself demonstrate direct RNA-RNA base pairing. Exogenous TGF-β1 increased hsa_piR_016984 expression, whereas BI-4659 decreased its expression under the tested in vitro conditions. These changes do not establish direct SMAD-mediated transcriptional regulation or a positive-feedback loop. Conclusions: The findings describe hsa_piR_016984-associated changes in TGF-β1 transcript signals and matrix staining in cultured human scleral fibroblasts under the tested conditions. They do not establish intrinsic mechanosensitivity or effects on myopia progression in vivo. The descriptive time courses, including post-washout measurements, do not establish mRNA half-life differences.