Downexpression of miR-200c-3p Contributes to Achalasia Disease by Targeting the Gene.

Achalasia is an esophageal smooth muscle motility disorder with unknown pathogenesis. Taking into account our previous results on the downexpression of miR-200c-3p in tissues of patients with achalasia correlated with an increased expression of , , and genes, our aim was to explore the unknown biological interaction between these genes and human miR-200c-3p and if this relation could unravel their functional role in the etiology of achalasia. To search for putative miR-200c-3p binding sites in the 3'-UTR of , and , a bioinformatics tool was used. To test whether , , and are targeted by miR-200c-3p, a dual-luciferase reporter assay and quantitative PCR on HEK293 and fibroblast cell lines were performed. To explore the biological correlation between and miR-200c-3p, an immunoblot analysis was carried out. The overexpression of miR-200c-3p reduced the luciferase activity in cells transfected with a luciferase reporter containing a fragment of the 3'-UTR regions of , , and which included the miR-200c-3p seed sequence. The deletion of the miR-200c-3p seed sequence from the 3'-UTR fragments abrogated this reduction. A negative correlation between miR-200c-3p and , , and expression levels was observed. Finally, a reduction of the endogenous level of in cells overexpressing miR-200c-3p was detected. Our study provides, for the first time, functional evidence about the gene as a direct target and and as potential indirect substrates of miR-200c-3p and suggests the involvement of NO/cGMP/PKG signaling in the pathogenesis of achalasia.