Abstract
Hepatocyte nuclear factor (HNF) 4alpha is a key transcription factor regulating endo/xenobiotic-metabolizing enzymes and transporters. We investigated whether microRNAs are involved in the regulation of human HNF4alpha. Potential recognition elements for miR-24 (MRE24) were identified in the coding region and the 3'-untranslated region (3'-UTR), and those for miR-34a (MRE34a) were identified in the 3'-UTR in HNF4alpha mRNA. The HNF4alpha protein level in HepG2 cells was markedly decreased by the overexpression of miR-24 and miR-34a. The HNF4alpha mRNA level was significantly decreased by the overexpression of miR-24 but not by miR-34a. In luciferase analyses in HEK293 cells, the reporter activity of plasmid containing the 3'-UTR of HNF4alpha was significantly decreased by miR-34a. The reporter activity of plasmid containing the HNF4alpha coding region downstream of the luciferase gene was significantly decreased by miR-24. These results suggest that the MRE24 in the coding region and MRE34a in the 3'-UTR are functional in the negative regulation by mRNA degradation and translational repression, respectively. The down-regulation of HNF4alpha by these microRNAs resulted in the decrease of various target genes such as cytochrome P450 7A1 and 8B1 as well as morphological changes and the decrease of the S phase population in HepG2 cells. We also clarified that the expressions of miR-24 and miR-34a were regulated by protein kinase C/mitogen-activated protein kinase and reactive oxygen species pathways, respectively. In conclusion, we found that human HNF4alpha was down-regulated by miR-24 and miR-34a, the expression of which are regulated by cellular stress, affecting the metabolism and cellular biology.
Highlights
Bile acids are important regulatory molecules mediating cholesterol synthesis and glucose metabolism as well as their own synthesis [3]
Because the HNF4␣ expression and/or activity are changed in response to signals derived from bile acids, we examined whether bile acids affect the expression of miR-24 and miR-34a
To investigate whether the decrease of these mRNAs resulted from the decrease of the HNF4␣ protein level but not the direct effects of miRNAs, we introduced siHNF4␣ into the HepG2 cells
Summary
HNF4␣ CYP7A1 CYP8B1 CYP27A1 PEPCK p16 p21 p27 GAPDH U6 snRNA Pre-miR-24-1 Pre-miR-24-2 Pre-miR-34a miR-24 miR-34a. Two independent studies revealed that the knockdown of all miRNAs in liver by conditional knock-out of Dicer resulted in apoptosis and inflammation [13] or a disruption of hepatic zonation [14]. These findings indicate the physiological and biological significance of miRNAs in liver function. We examined the possibility that miRNAs might regulate the expression of human HNF4␣, resulting in the modulation of liver function
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