Mesenchymal stem cells improve liver fibrosis and protect hepatocytes by promoting microRNA-148a-5p-mediated inhibition of Notch signaling pathway.

BACKGROUND

Mesenchymal stem cells (MSCs) are considered to be a potential therapeutic tool for liver fibrosis. Inhibiting the activation of hepatic stellate cells (HSCs) and protecting hepatocytes are important mechanisms for the anti-fibrotic effect of MSCs. However, how MSCs inhibit liver fibrosis by regulating the expression of microRNAs (miRNAs) has not been fully clarified.

METHODS

Transforming growth factor-β1 (TGF-β1)-activated HSCs LX-2 were single cultured or co-cultured with human umbilical cord mesenchymal stem cells (HUC-MSCs). High-throughput sequencing was used to evaluate the differentially expressed microRNAs (DEMs) between the two groups. Quantitative real-time PCR (qRT-PCR), Western blot, and transfection experiments were used to investigate and screen the most significantly up-regulated DEM. Bioinformatics analysis was used to predict the target mRNAs and the potential functions of the DEM. The possible mechanism of HUC-MSCs against liver fibrosis was analyzed by co-culture experiment of HUC-MSCs with LX-2 cells, and HUC-MSCs treatment of Bile duct ligation (BDL)-induced liver fibrosis in mice. Finally, the mechanism of the DEM regulating liver fibrosis was confirmed in human liver fibrosis specimens.

RESULTS

MicroRNA-148a-5p (miR-148a-5p) was the most significantly up-regulated DEM in activated LX-2 cells co-cultured with HUC-MSCs compared with LX-2 cells single cultured. Up-regulation of the expression of miR-148a-5p in activated LX-2 cells could significantly inhibit the expression of hepatic fibrosis markers α-SMA and Col1α1. Notch2 was one target gene of miR-148a-5p. Co-cultured with HUC-MSCs could inhibit the activation of LX-2 cells by inhibiting the expression of the Notch2 and the Notch signaling pathway. In addition, HUC-MSCs treatment could up-regulate the expression of miR-148a-5p in liver tissue and hepatocytes, promote the proliferation and avoid the apoptosis of hepatocytes, and reduce the degree of fibrosis by inhibiting expression of the Notch2 and the Notch signaling pathway in BDL-induced liver fibrosis mice. Moreover, miR-148a-5p was down-regulated and Notch2 was up-regulated in fibrotic human liver tissues compared with the normal livers.

CONCLUSIONS

HUC-MSCs treatment could inhibit HSCs activation, protect hepatocytes, and alleviate BDL-induced liver fibrosis in mice by up-regulating the expression of miR-148-5p and inhibiting the Notch signaling pathway. The down-regulation of miR-148-5p and up-regulation of Notch2 could be used as biomarkers to monitor the progression of liver fibrosis.