microRNA overexpression in slow transit constipation leads to reduced Na_V1.5 current and altered smooth muscle contractility.

Abstract

This study was designed to evaluate the roles of microRNAs (miRNAs) in slow transit constipation (STC). All human tissue samples were from the <i>muscularis externa</i> 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 <i>in vitro</i> by RT-qPCR, electrophysiology, traction force microscopy, and ex vivo by lentiviral transduction in rat <i>muscularis externa</i> organotypic cultures. The expression of 13 miRNAs was increased in STC samples. Of those miRNAs, four were predicted to target <i>SCN5A</i>, the gene that encodes the Na⁺ channel Na_V1.5. The expression of <i>SCN5A</i> mRNA was decreased in STC samples. Let-7f significantly decreased Na⁺ current density in vitro in human smooth muscle cells. In rat <i>muscularis externa</i> organotypic cultures, overexpression of let-7f resulted in reduced frequency and amplitude of contraction. A small group of miRNAs is upregulated in STC, and many of these miRNAs target the SCN5A-encoded Na⁺ channel Na_V1.5. Within this set, a novel Na_V1.5 regulator, let-7f, resulted in decreased Na_V1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest Na_V1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.