Abstract
Transforming growth factor-β (TGF-β) pathway presents dysregulation in pathological scarring and mediates hypertrophic scar (HS) formation. The study aims to analyze the potential mechanism of long non-coding RNA NORAD (LncRNA NORAD) and microRNA (miR-26a) regulation of the TGF-β pathway in hypertrophic scar fibroblasts (HSFs). Hypertrophic scar tissues were collected and assayed for LncRNA NORAD, miR-26a, transforming growth factor β receptor I (TGF-βR1) and TGF-βR2, with enzyme-linked immunosorbent assay (ELISA) or qualitative polymerase chain reaction (qPCR). LncRNA NORAD interfering plasmids were transfected into HSFs and induced with TGF-β1. Cell Counting Kit-8 (CCK-8) assays were performed to assess HSF proliferation, and flow cytometry to analyze apoptosis and the cell cycle. TGF-βR1, TGF-βR2, Smad2, and p-Smad2 levels were detected using western blot (WB). The related proteins (p21, cyclin D1 and cyclin-dependent kinase 4 (CDK4)) regulating the cell cycle, and apoptosis-related proteins (caspase-3 and Bcl-2) were also detected using WB. The binding sites of miRNA-26a and LncRNA NORAD, TGF-βR2, or UBE3A were predicted using Starbase and confirmed with dual luciferase reporter assay. RNA immunoprecipitation (RIP) was utilized to explore the interplay of miR-26a with its target genes. LncRNA NORAD is decreased, miR-26a is increased and TGF-β receptors show abnormal expression in scar tissue. LncRNA NORAD knockdown inhibits proliferation of HSF cells induced by TGF-β1 treatment. In addition, cell apoptotic levels are markedly increased and cell numbers in G0/G1 phase are increased. Moreover, the TGF-β/Smad pathway is regulated by decreasing endogenous LncRNA NORAD levels, possibly by affecting the relative levels of TGF-βR1. p21 is notably upregulated, while cyclin D1 and CDK4 are downregulated. Apoptosis-related proteins are significantly affected. LncRNA NORAD may act as a sponge, binding miR-26a and changing its expression. Finally, RIP shows that miR-26a targets the 3'UTRs of TGF-βR2 and UBE3A. LncRNA NORAD regulates HSF proliferation via miR-26a mediating the regulation of TGF-βR2/R1. LncRNA NORAD/miR-26a could be a potential target for treating HS.
Highlights
Transforming growth factor-β (TGF-β) pathway presents dysregulation in pathological scarring and mediates hypertrophic scar (HS) formation
LncRNA NORAD knockdown inhibits proliferation of hypertrophic scar fibroblasts (HSFs) cells induced by TGF-β1 treatment
The TGF-β/Smad pathway is regulated by decreasing endogenous LncRNA NORAD levels, possibly by affecting the relative levels of TGF-βR1. p21 is notably upregulated, while cyclin D1 and CDK4 are downregulated
Summary
Transforming growth factor-β (TGF-β) pathway presents dysregulation in pathological scarring and mediates hypertrophic scar (HS) formation. Hypertrophic scarring (HS) is a common skin disorder, mostly following burns or skin trauma, or after an operation.[1] The process of collagen secretion and the metabolism of fibroblasts are strictly modulated during skin wound healing. Hypertrophic scarring occurs under excess proliferation of fibroblasts and massive collagen deposition.[2] TGF-β signaling affects cell proliferation and extracellular matrix (ECM) production in the process of wound healing. Phosphorylation of TGF-βR1 is necessary for this activation,[3] while its ubiquitination contributes to its degradation and inactivates the Smad pathway. TGF-β signaling has been demonstrated to be dysregulated in pathological scarring,[4,5] and TGF-βR2 expression is increased in keloid fibroblasts.[6]
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