Extracellular vesicle microRNAs from human bone marrow MSCs suppress fibrogenesis of hepatic stellate cells by downregulation of RhoA signaling

We have developed a liver regeneration therapy for decompensated liver cirrhosis (DLC) using autologous bone marrow mesenchymal stem cells (MSCs), and it is currently under trial (jRCT2063200014). However, the mechanism underlying the anti-fibrotic action of MSCs remains unclear. Therefore, we aimed to investigate the anti-fibrotic effects of microRNAs (miRNAs) in extracellular vesicles (EVs) derived from human bone marrow MSCs. We performed a comprehensive microarray analysis of miRNA expression profiles of MSC-EVs from healthy individuals, patients with DLC, and hepatic stellate cells (HSCs). We identified 11 miRNAs that showed a normalized intensity > 1,000 in MSC-EVs, which was 50-fold higher than that in HSCs. MiRNA mimics for the 11 miRNAs were transfected into HSCs, and five miRNAs (miR-204-3p, miR-7977, miR-1237-5p, miR-5787, and miR-6089) that suppressed the expression of fibrosis-related genes (α-SMA, collagen I, and elastin) were identified. Transfection of the five-miRNA cocktail into HSCs decreased the expression of all fibrosis-related genes. Subsequently, pathway analysis revealed the downregulation of the RhoA signaling pathway, a key pathway in HSC activation. RhoA activity and migration were suppressed in HSCs transfected with the five-miRNA cocktail. This study identified five anti-fibrotic miRNAs enriched in MSC-EVs and elucidated the underlying mechanism of the anti-fibrotic action of MSCs.