Myofibroblast transdifferentiation is associated with changes in cellular and extracellular vesicle miRNA abundance

Abstract

Transforming growth factor-beta 1 (TGF-β1), a pro-fibrotic tumour-derived factor promotes fibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer-associated fibroblasts (CAFs) by promoting myofibroblast differentiation. miRNA dysregulation has been demonstrated in myofibroblast transdifferentiation and CAF activation, however, their expression varies among cell types and with the method of fibroblast induction. Here, the expression profile of miRNA in human primary oral fibroblasts treated with TGF-β1, to derive a myofibroblastic, CAF-like phenotype, was determined compared to untreated fibroblasts. Myofibroblast transdifferentiation was determined by the expression of alpha-smooth muscle actin (α-SMA) and fibronectin-1 extra domain A (FN-EDA1) using quantitative real-time PCR (qRT-PCR) and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility were assessed using collagen contraction assays. Extracellular vesicles (EVs) were purified by using size exclusion chromatography and ultracentrifugation and their size and concentration were determined by nanoparticle tracking analysis. miRNA expression profiling in oral fibroblasts treated with TGF-β1 and their extracellular vesicles was carried out using tiling low-density array cards. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to perform functional and pathway enrichment analysis of target genes. In this study, TGF-β1 induced a myofibroblastic phenotype in normal oral fibroblasts as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TaqMan Low-Density Array (TLDA) analysis demonstrated that miR-503 and miR-708 were significantly upregulated, while miR-1276 was significantly downregulated in TGF-β1-treated oral fibroblasts (henceforth termed experimentally-derived CAF, eCAF). The gene functional enrichment analysis showed that the candidate miRNAs have the potential to modulate various pathways; including the Ras associated protein 1 (Rap1), PI3K-Akt, and tumour necrosis factor (TNF) signalling pathways. In addition, altered levels of several miRNAs were detected in eCAF EV, including miR-142 and miR-222. No differences in size or abundance of EV were detected between eCAF and normal oral fibroblast (NOF). Little overlap was observed between changes in cellular and EV miRNA profiles, suggesting the possibility of selective loading of EV miRNA. The study reveals miRNA expression signature could be involved in myofibroblast transdifferentiation and the miRNA cargo of their EV, providing novel insight into the involvement of miRNA in CAF development and function.

Targeting Galectin 3 illuminates its contributions to the pathology of uterine serous carcinoma

“…In the present study, we found that fibroblasts cultured in CM from Gal3-positive cells expressed more αSMA, a marker of CAFs, than cells cultured in CM from Gal3-KO. Like others we observed that treatment with TGF-β resulted in an increase in αSMA concurrent with a shift towards CAFs [ 48 , 49 ]. We showed this increase in αSMA was inversely associated with a decrease in endogenous Gal3.…”

“…4i, j ). Others have shown TGF-β to induce the transition of normal fibroblasts towards a CAF-like phenotype, as determined by an increase in αSMA [ 48 , 49 ]. Thus, we used this strategy as a positive control.…”

Promoting cell proliferation and collagen production with ascorbic acid 2-phosphate-releasing poly(l-lactide-co-ε-caprolactone) membranes for treating pelvic organ prolapse

“…The amount of myofibroblasts increases during tissue healing as they are the major cell type to produce ECM during tissue regeneration. Therefore, an increased presence of myofibroblasts could provide enhanced support for the pelvic floor [ 64–66 ]. In the present study, more α-SMA appears to be present in hVFs on PLCL A2P and hPLCL A2P membranes compared to PLCL and hPLCL membranes, yet the amount of mRNA was significantly higher only compared to hPLCL.…”