MicroRNA‐23b cluster microRNAs regulate transforming growth factor‐beta/bone morphogenetic protein signaling and liver stem cell differentiation by targeting Smads†

Abstract

Transforming growth factor-beta / bone morphogenetic protein (TGFbeta/BMP) signaling has a gradient of effects on cell fate choice in the fetal mouse liver. The molecular mechanism to understand why adjacent cells develop into bile ducts or grow actively as hepatocytes in the ubiquitous presence of both TGFbeta ligands and receptors has been unknown. We hypothesized that microRNAs (miRNAs) might play a role in cell fate decisions in the liver. miRNA profiling during late fetal development in the mouse identified miR-23b cluster miRNAs comprising miR-23b, miR-27b, and miR-24-1 and miR-10a, miR-26a, and miR-30a as up-regulated. In situ hybridization of fetal liver at embryonic day 17.5 of gestation revealed miR-23b cluster expression only in fetal hepatocytes. A complementary (c)DNA microarray approach was used to identify genes with a reciprocal expression pattern to that of miR-23b cluster miRNAs. This approach identified Smads (mothers against decapentaplegic homolog), the key TGFbeta signaling molecules, as putative miR-23b cluster targets. Bioinformatic analysis identified multiple candidate target sites in the 3' UTRs (untranslated regions) of Smads 3, 4, and 5. Dual luciferase reporter assays confirmed down-regulation of constructs containing Smad 3, 4, or 5, 3' UTRs by a mixture of miR-23b cluster mimics. Knockdown of miR-23b miRNAs during hepatocytic differentiation of a fetal liver stem cell line, HBC-3, promoted expression of bile duct genes, in addition to Smads, in these cells. In contrast, ectopic expression of miR-23b mimics during bile duct differentiation of HBC-3 cells blocked the process. Our data provide a model in which miR-23b miRNAs repress bile duct gene expression in fetal hepatocytes while promoting their growth by down-regulating Smads and consequently TGFbeta signaling. Concomitantly, low levels of the miR-23b miRNAs are needed in cholangiocytes to allow TGFbeta signaling and bile duct formation.