MicroRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility

Amelia Mazzone, Peter R. Strege, Simon J. Gibbons, Constanza Alcaino, Vikram Joshi, Andrew J. Haak, Daniel J. Tschumperlin, Cheryl E. Bernard, Robert R. Cima, David W. Larson, Heidi K. Chua, Rondell P. Graham, Mona El Refaey, Peter J. Mohler, Yujiro Hayashi, Tamas Ordog, Stefan Calder, Peng Du, Gianrico Farrugia, Arthur Beyder

Research output: Contribution to journalArticle

Abstract

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 NaV1.5. 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 NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

Original languageEnglish (US)
Article number318747
JournalGut
DOIs
StateAccepted/In press - Jan 1 2019

Fingerprint

Constipation
MicroRNAs
Smooth Muscle
Reverse Transcription
Smooth Muscle Myocytes
Atomic Force Microscopy
Electrophysiology
Traction
Colon
Cell Line
Polymerase Chain Reaction
Messenger RNA

Keywords

  • constipation
  • genetics
  • intestinal ion transport
  • intestinal motility
  • motility disorders

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Mazzone, A., Strege, P. R., Gibbons, S. J., Alcaino, C., Joshi, V., Haak, A. J., ... Beyder, A. (Accepted/In press). MicroRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility. Gut, [318747]. https://doi.org/10.1136/gutjnl-2019-318747

MicroRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility. / Mazzone, Amelia; Strege, Peter R.; Gibbons, Simon J.; Alcaino, Constanza; Joshi, Vikram; Haak, Andrew J.; Tschumperlin, Daniel J.; Bernard, Cheryl E.; Cima, Robert R.; Larson, David W.; Chua, Heidi K.; Graham, Rondell P.; El Refaey, Mona; Mohler, Peter J.; Hayashi, Yujiro; Ordog, Tamas; Calder, Stefan; Du, Peng; Farrugia, Gianrico; Beyder, Arthur.

In: Gut, 01.01.2019.

Research output: Contribution to journalArticle

Mazzone, A, Strege, PR, Gibbons, SJ, Alcaino, C, Joshi, V, Haak, AJ, Tschumperlin, DJ, Bernard, CE, Cima, RR, Larson, DW, Chua, HK, Graham, RP, El Refaey, M, Mohler, PJ, Hayashi, Y, Ordog, T, Calder, S, Du, P, Farrugia, G & Beyder, A 2019, 'MicroRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility', Gut. https://doi.org/10.1136/gutjnl-2019-318747
Mazzone, Amelia ; Strege, Peter R. ; Gibbons, Simon J. ; Alcaino, Constanza ; Joshi, Vikram ; Haak, Andrew J. ; Tschumperlin, Daniel J. ; Bernard, Cheryl E. ; Cima, Robert R. ; Larson, David W. ; Chua, Heidi K. ; Graham, Rondell P. ; El Refaey, Mona ; Mohler, Peter J. ; Hayashi, Yujiro ; Ordog, Tamas ; Calder, Stefan ; Du, Peng ; Farrugia, Gianrico ; Beyder, Arthur. / MicroRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility. In: Gut. 2019.
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abstract = "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 NaV1.5. 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 NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.",
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T1 - MicroRNA overexpression in slow transit constipation leads to reduced NaV1.5 current and altered smooth muscle contractility

AU - Mazzone, Amelia

AU - Strege, Peter R.

AU - Gibbons, Simon J.

AU - Alcaino, Constanza

AU - Joshi, Vikram

AU - Haak, Andrew J.

AU - Tschumperlin, Daniel J.

AU - Bernard, Cheryl E.

AU - Cima, Robert R.

AU - Larson, David W.

AU - Chua, Heidi K.

AU - Graham, Rondell P.

AU - El Refaey, Mona

AU - Mohler, Peter J.

AU - Hayashi, Yujiro

AU - Ordog, Tamas

AU - Calder, Stefan

AU - Du, Peng

AU - Farrugia, Gianrico

AU - Beyder, Arthur

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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 NaV1.5. 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 NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

AB - 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 NaV1.5. 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 NaV1.5. Within this set, a novel NaV1.5 regulator, let-7f, resulted in decreased NaV1.5 expression, current density and reduced motility of GI smooth muscle. These results suggest NaV1.5 and miRNAs as novel diagnostic and potential therapeutic targets in STC.

KW - constipation

KW - genetics

KW - intestinal ion transport

KW - intestinal motility

KW - motility disorders

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