Abstract
Unveiling the regulatory pathways maintaining hepatic stellate cells (HSC) in a quiescent (q) phenotype is essential to develop new therapeutic strategies to treat fibrogenic diseases. To uncover the miRNA-mRNA regulatory interactions in qHSCs, HSCs were FACS-sorted from healthy livers and activated HSCs (aHSCs) were generated in vitro. MiRNA Taqman array analysis showed HSCs expressed a low number of miRNAs (n = 259), from which 47 were down-regulated and 212 up-regulated upon activation. Computational integration of miRNA and gene expression profiles revealed that 66% of qHSC-associated miRNAs correlated with more than 6 altered target mRNAs (17,28 ± 10,7 targets/miRNA) whereas aHSC-associated miRNAs had an average of 1,49 targeted genes. Interestingly, interaction networks generated by miRNA-targeted genes in qHSCs were associated with key HSC activation processes. Next, selected miRNAs were validated in healthy and cirrhotic human livers and miR-192 was chosen for functional analysis. Down-regulation of miR-192 in HSCs was found to be an early event during fibrosis progression in mouse models of liver injury. Moreover, mimic assays for miR-192 in HSCs revealed its role in HSC activation, proliferation and migration. Together, these results uncover the importance of miRNAs in the maintenance of the qHSC phenotype and form the basis for understanding the regulatory networks in HSCs.
Highlights
Pathways involved in the maintenance of hepatic stellate cells (HSC) quiescence have barely been addressed
QHSCs isolated by density gradient from the same patients were cultured until passage four to obtain pure populations of activated HSCs (aHSCs)
To assess the purity of the different cell populations we measured the expression of specific markers for each liver cell type. qHSC and liver sinusoidal endothelial cells (LSECs) sorted fractions were highly enriched in PDGFRB and CD32b respectively and no significant expression of albumin was detected (Fig. 1A)
Summary
Pathways involved in the maintenance of HSC quiescence have barely been addressed. most studies have been conducted in rodent models of liver disease and little information is available on human HSCs. Several miRNAs involved in HSC activation have been reported and their potential anti-fibrotic effects have been shown by inhibiting miRNAs function in vivo and in vitro[5,6,7]. One of the best studied miRNAs in HSCs is miR-21, which has been shown to be up-regulated in human and murine fibrotic liver samples. A limited number of miRNAs have been described in both quiescent and activated HSCs (aHSCs)[8,9,10,11,12], no global miRNA analysis has been performed on qHSCs. far, little is known about miRNAs expressed in human qHSCs and their potential functional role in promoting the maintenance of quiescence. We have identified a panel of miRNAs with predicted target genes associated with HSC activation, and with a potential role in the repression of activation
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