Cholangiocyte-derived exosomal miR-381-3p promotes hepatic stellate cell activation and cholestatic liver fibrosis via targeting Klf6

Introduction

The activation of hepatic stellate cells (HSCs) is a central mechanism driving the progression of chronic cholestatic liver diseases (CCLD). MicroRNAs (miRNAs) have been increasingly recognized for their regulatory roles in various liver pathologies. This study aimed to identify specific miRNAs involved in the progression of CCLD and elucidate their underlying molecular mechanisms.

Methods

A bile duct ligation (BDL) mouse model was established to mimic cholestatic liver injury. Exosomes were isolated from mouse large cholangiocytes (MLE) and co-cultured with HSCs to investigate their functional effects. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to assess the expression levels of fibrotic marker genes. Exosomal miRNA microarray assay was used to screen differentially expressed miRNAs. Furthermore, dual-luciferase reporter assays and RNA pull-down experiments were conducted to validate the interaction between miR-381-3p and its putative target gene, kinesin family member 6 (Klf6).

Results

The results confirmed the successful isolation of exosomes from MLE. Furthermore, exosomes derived from BDL-MLE were shown to promote the activation of HSCs and exacerbate hepatic fibrosis. Dicer knockout reduced the mRNA expression of key fibrotic markers. In addition, miR-381-3p was found to be upregulated both in BDL-MLE–derived exosomes and in HSCs treated with transforming growth factor (TGF)-β and BDL-MLE–exosomes. Mechanistic investigations identified Klf6 as a direct target gene of miR-381-3p. Rescue experiments further revealed that overexpression of Klf6 alleviated the pro-fibrotic effects caused by miR-381-3p overexpression, thereby attenuating HSC activation and mitigating cholestatic liver fibrosis.

Conclusion

This study highlighted the role of MLE-derived exosomal miR-381-3p in promoting HSC activation and cholestatic liver fibrosis through the regulation of Klf6. These findings provide novel insights into the molecular mechanisms underlying CCLD.