Mechanism of strong inhibition of HNF4α1 protein expression by its 5' UTR (749.7)

Abstract

HNF4α is a liver‐enriched master regulator down‐regulated in liver cancer and cirrhosis. Restoration of HNF4α expression can treat both liver cancer and liver fibrosis. However, lack of known activating ligand makes HNF4α “undruggable”. The purpose of this study was to elucidate the mechanism of translational regulation of HNF4α1, the adult‐predominant HNF4α isoform, so as to develop a novel approach to restore HNF4α1 expression for cancer therapy. The 5' UTR of both human and mouse HNF4α1 have potential G‐quadruplex (G4), a stable secondary structure known to inhibit transcription and translation. FRET assay of dual‐labeled oligo from human HNF4α1 5' UTR indicated the formation of G4. Luciferase reporter assay showed the wild‐type, but not G4‐mutated, 5' UTR of HNF4α1 strongly inhibited protein translation without affecting mRNA expression, which is further confirmed by Western blot analysis of protein expression of HNF4α1 in the presence/absence of its 5' UTR. In HEK 293 cells, protein expression of mouse Hnf4α1 was completely inhibited by its 5' UTR. Luciferase assays of the deletion/mutation reporter constructs of Hnf4α1 5' UTR suggest G4 motif and its adjacent stem loop may play the key role in suppressing Hnf4α1 protein expression. In summary, the 5' UTR of human and mouse HNF4α1 markedly inhibit HNF4α1 expression at translational levels, most likely due to interaction of G4 motif with stem loop within 5' UTR. Grant Funding Source: NIH Grant CA143656