Elevated hepatic MDR3/ABCB4 is directly mediated by MiR-378a-5p in human obstructive cholestasis

The function of MDR3 is important in bile acid transport. The miRNAs can suppress the expression of gene through combining mRNA of target gene. The regulation about MDR3 mediated by FXR or PPARα in cholestasis is clear, but the mechanism through miRNA is hardly reported. We aimed to find out the miRNA, which could suppress MDR3 expression and the significance of this connection in cholestasis.

We measured hsa-miR-378a-5p expression level in liver tissues from 20 patients with cholestasis and 15 patients without cholestasis by quantitative PCR. We also tested the level of clinical features of the same group. HepG2 cell lines were performed experiments to discover the connection between hsa-miR-378a-5p and MDR3, including transient transfection, RNA and protein extraction, qPCR, Western blotting and luciferase reporter assay.

A significant decrease of miR-378a-5p was observed in obstructive cholestasis patient liver tissues compared to control group. We also find that the miR-378a-5p expression is correlated to several clinical features, which are important biomarkers in cholestatic liver injury. Then we predicted that MDR3 may be the target gene of miR-378a-5p through miRanda v3.3a. We programed the transient transfection of mimics and inhibitor on HepG2 cell lines, and detected the mRNA and protein expression of MRP2, MRP3 and MDR3. The results suggested that miR-378a-5p could negatively regulate MDR3 expression in both mRNA and protein expression level, and this regulation is specific. We didn't find same regulation in MRP2 and MRP3. Dual luciferase assays proved this regulation is mediated by a direct binding between miR-378a-5p and CDS of MDR3.

We found that hsa-miR-378a-5p expression was down-regulated in cholestatic liver tissues, compared to control liver tissues. Transient transfection and luciferase reporter assay in HepG2 cell lines results suggest that hsa-miR-378a-5p can directly combine MDR3 mRNA and suppress MDR3 protein expression. The down-regulated hsa-miR-378a-5p may cause a protective alteration through up-regulating MDR3 expression in cholestasis.