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

Siti Amalina Inche Zainal Abidin,Daniel W Lambert,Ian Charles Paterson,Samuel Higginbotham,Stuart Hunt,Ryan Charles Pink,Klaus Roemer

doi:10.1371/journal.pone.0256812

Siti Amalina Inche Zainal Abidin, Daniel W Lambert + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0256812

Copy DOI

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.

Highlights

Several lines of evidence have demonstrated that the altered stroma microenvironment makes a significant contribution to the malignant progression of cancers, including oral squamous cell carcinomas (OSCC) [1]
In order to establish a model of cancer-associated fibroblasts (CAFs)-like myofibroblast differentiation, we exposed normal oral fibroblast (NOF) to TGF-β1
In keeping with previous reports, TGF-β1 induced expression of α-SMA and FN-EDA1 transcripts in establish a model of CAF-like (eCAF) with maximum α-SMA and FN-EDA1 expressions occurring at 48 h post-treatment at a concentration of 5 ng/ml (Fig 1A and 1B)

Summary

Introduction

Several lines of evidence have demonstrated that the altered stroma microenvironment makes a significant contribution to the malignant progression of cancers, including oral squamous cell carcinomas (OSCC) [1]. The tumour microenvironment is a dynamic milieu consisting of a mixture of cancerous cells together with stromal cell populations. The majority of tumour stromal cells are activated fibroblasts which commonly express α-SMA [2] and FN-EDA1 [3]. Other growth factors and cytokines mediate myofibroblast transdifferentiation including interleukin-6 (IL-6), platelet-derived growth factor (PDGF) and connective tissue growth factor (CTGF) [6]. Various cytokines and growth factors are capable of modulating the myofibroblast transdifferentiation, the precise signals, and underlying molecular mechanisms, promoting the formation of myofibroblastic CAF in vivo remain to be fully elucidated

Methods

Results

Conclusion