Human with-no-lysine kinase-4 3'-UTR acting as the enhancer and being targeted by miR-296

Human with-no-lysine kinase-4 (hWNK4) is a member of the serine-threonine protein kinase family and may be involved in pathophysiological processes of hypertension through regulating diverse ion transporters. We are interested in the molecular mechanism of regulating hWNK4 expression. The 3'-untranslated region (3'-UTR) is an important area for gene regulation. Here, we focused on the 3'-UTR of hWNK4 to investigate how it regulates hWNK4 expression. A luciferase assay showed that the hWNK4 3'-UTR obviously enhanced the transcriptional activity of the promoter, whether homologous or heterologous. Chromosome conformation capture assay further demonstrated that the 3'-UTR enhancer functioned through distant crosstalking with the hWNK4 promoter in a cell-specific manner. Following the deletion of the promoter, the enhancer action of the hWNK4 3'-UTR fell away, indicating that the motifs within the promoter could be essential for the function of the hWNK4 3'-UTR. The hWNK4 3'-UTR, meanwhile, contained a miR-296 binding site, predicted by bioinformatics algorithms and verified as a true target using a reporter assay. Expression of hWNK4 can be downregulated by miR-296 at the posttranscriptional level in cell-specific pattern, shown by real-time quantitative PCR and Western blot assays. These data demonstrate the hWNK4 3'-UTR plays an enhancer role by crosstalking with the promoter, and miR-296 suppresses hWNK4 expression through targeting on its 3'-UTR in a cell-specific fashion. The coordinated modulation of miR-296 and cofactors on the 3'-UTR may be attributed to hWNK4 physiopathological function.