microRNA overexpression in slow transit constipation leads to reduced Na(V)1.5 current and altered smooth muscle contractility

OBJECTIVE: This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). DESIGN: All human tissue samples were from the muscularis externa of the colon. Expression of 372 miRNAs was examined in a discovery cohort of four patients with STC versus three age/sex-matched controls by a quantitative PCR array. Upregulated miRNAs were examined by quantitative reverse transcription qPCR (RT-qPCR) in a validation cohort of seven patients with STC and age/sex-matched controls. The effect of a highly differentially expressed miRNA on a custom human smooth muscle cell line was examined in vitro by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat muscularis externa organotypic cultures. RESULTS: The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target SCN5A, the gene that encodes the Na(+) channel Na(V)1.5. The expression of SCN5A mRNA was decreased in STC samples. Let-7f significantly decreased Na(+) current density in vitro in human smooth muscle cells. In rat muscularis externa organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. CONCLUSIONS: A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na(+) channel Na(V)1.5. Within this set, a novel Na(V)1.5 regulator, let-7f, resulted in decreased Na(V)1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na(V)1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.