NET silencing by let-7i in postural tachycardia syndrome
Abdul Waheed Khan, … , Murray D. Esler, Assam El-Osta
Abdul Waheed Khan, … , Murray D. Esler, Assam El-Osta
Published March 23, 2017
Citation Information: JCI Insight. 2017;2(6):e90183. https://doi.org/10.1172/jci.insight.90183.
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Research Article Cardiology

NET silencing by let-7i in postural tachycardia syndrome

Abstract

While strongly implicated in postural tachycardia syndrome (POTS), considerable controversy exists regarding norepinephrine transporter (NET) loss of function. POTS is characterized by the clinical symptoms of orthostatic intolerance, lightheadedness, tachycardia, and syncope or near syncope with upright posture. Abnormal sympathetic nervous system activity is typical, of a type which suggests dysfunction of the NET, with evidence that the gene responsible is under tight epigenetic control. Using RNA of isolated chromatin combined with massive parallel sequencing (RICh-seq) we show that let-7i miRNA suppresses NET by methyl-CpG-binding protein 2 (MeCP2). Vorinostat restores epigenetic control and NET expression in leukocytes derived from POTS participants.

Authors

Abdul Waheed Khan, Mark Ziemann, Susan J. Corcoran, Harikrishnan K.N, Jun Okabe, Haloom Rafehi, Scott S. Maxwell, Murray D. Esler, Assam El-Osta

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Figure 1

Optimization of RNA of isolated chromatin (RICh) assay.

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Optimization of RNA of isolated chromatin (RICh) assay. (A) RICh protoco...
(A) RICh protocol. Briefly, cells (HeLa or leukocytes) were crosslinked with 1% formaldehyde and soluble chromatin prepared by sonication. Chromatin was denatured and hybridized with locked nucleic acid (LNA) probes. Chromatin-LNA probe hybrids were captured with streptavidin beads. For RICh optimization, DNA was isolated and assessed for norepinephrine transporter (NET) promoter amplification by quantitative PCR (qPCR). (B) Location of sequence-specific LNA probes targeting the NET promoter. (C) NET promoter regions are represented and correspond to those captured using sequence-specific LNA probes. Primers in schematic are abbreviated as P. (D–F) NET promoter DNA amplification by qPCR using RICh-captured DNA. (G) Amplification of exon 5 of the NET gene from RICh-captured DNA shows no difference between the NET-specific and scrambled (scr) probes. This is a measure of assay specificity; n = 3, mean ± SD. *P < 0.05 by Student’s t test.