Genetic and Epigenetic Modification of Rat Liver Progenitor Cells via HNF4α Transduction and 5' Azacytidine Treatment: An Integrated miRNA and mRNA Expression Profile Analysis.

Neonatal liver-derived rat epithelial cells (rLEC) from biliary origin are liver progenitor cells that acquire a hepatocyte-like phenotype upon sequential exposure to hepatogenic growth factors and cytokines. Undifferentiated rLEC express several liver-enriched transcription factors, including the hepatocyte nuclear factors (HNF) 3 beta and HNF6, but not the hepatic master regulator HNF4 alpha. In this study, we first investigated the impact of the ectopic expression of HNF4 alpha in rLEC on both mRNA and microRNA (miR) level by means of microarray technology. We found that HNF4 alpha transduction did not induce major changes to the rLEC phenotype. However, we next investigated the influence of DNA methyl transferase (DNMT) inhibition on the phenotype of undifferentiated naive rLEC by exposure to 5 ' azacytidine (AZA), which was found to have a significant impact on rLEC gene expression. The transduction of HNF4 alpha or AZA treatment resulted both in significantly downregulated C/EBP alpha expression levels, while the exposure of the cells to AZA had a significant effect on the expression of HNF3 beta. Computationally, dysregulated miRNAs were linked to target mRNAs using the microRNA Target Filter function of Ingenuity Pathway Analysis. We found that differentially regulated miRNA-mRNA target associations predict ectopic HNF4 alpha expression in naive rLEC to interfere with cell viability and cellular maturation (miR-19b-3p/NR4A2, miR30C-5p/P4HA2, miR328-3p/CD44) while it predicts AZA exposure to modulate epithelial/hepatic cell proliferation, apoptosis, cell cycle progression and the differentiation of stem cells (miR-18a-5p/ESR1, miR-503-5p/CCND1). Finally, our computational analysis predicts that the combination of HNF4 alpha transduction with subsequent AZA treatment might cause changes in hepatic cell proliferation and maturation (miR-18a-5p/ESR1, miR-503-5p/CCND1, miR-328-3p/CD44) as well as the apoptosis (miR-16-5p/BCL2, miR-17-5p/BCL2, miR-34a-5p/BCL2and miR-494-3p/HMOX1) of naive rLEC.