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

Abstract 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 h WNK4 expression. The 3′-untranslated region (3′-UTR) is an important area for gene regulation. Here, we focused on the 3′-UTR of h WNK4 to investigate how it regulates h WNK4 expression. A luciferase assay showed that the h WNK4 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 h WNK4 promoter in a cell-specific manner. Following the deletion of the promoter, the enhancer action of the h WNK4 3′-UTR fell away, indicating that the motifs within the promoter could be essential for the function of the h WNK4 3′-UTR. The h WNK4 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 h WNK4 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 h WNK4 3′-UTR plays an enhancer role by crosstalking with the promoter, and miR-296 suppresses h WNK4 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.