MicroRNA-7a overexpression in VMH restores the sympathoadrenal response to hypoglycemia
Rahul Agrawal, … , Sankar Swaminathan, Simon J. Fisher
Rahul Agrawal, … , Sankar Swaminathan, Simon J. Fisher
Published October 17, 2019
Citation Information: JCI Insight. 2019;4(20):e130521. https://doi.org/10.1172/jci.insight.130521.
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Research Article Endocrinology

MicroRNA-7a overexpression in VMH restores the sympathoadrenal response to hypoglycemia

Abstract

It is proposed that the impaired sympathoadrenal response to hypoglycemia induced by recurrent insulin-induced hypoglycemia (RH) is an adaptive phenomenon induced by specific changes in microRNA expression in the ventromedial hypothalamus (VMH). To test this hypothesis, genome-wide microRNAomic profiling of the VMH by RNA-sequencing was performed in control rats and rats treated for RH. Differential expression analysis identified microRNA-7a-5p and microRNA-665 as potential mediators of this phenomenon. To further test this hypothesis, experiments were conducted consisting of targeted lentiviral-mediated overexpression of microRNA-7a-5p and downregulation of microRNA-665 in the VMH. Hyperinsulinemic hypoglycemic clamp experiments demonstrated that targeted overexpression of microRNA-7a-5p (but not downregulation of microRNA-665) in the VMH of RH rats restored the epinephrine response to hypoglycemia. This restored response to hypoglycemia was associated with a restoration of GABAA receptor gene expression. Finally, a direct interaction of microRNA-7a-5p with the 3′-UTR of GABAA receptor α1-subunit (Gabra1) gene was demonstrated in a luciferase assay. These findings indicate that (a) the impaired sympathoadrenal response RH induces is associated with changes in VMH microRNA expression and (b) microRNA-7a-5p, possibly via direct downregulation of GABA receptor gene expression, may serve as a mediator of the altered sympathoadrenal response to hypoglycemia.

Authors

Rahul Agrawal, Griffin Durupt, Dinesh Verma, Michael Montgomery, Adriana Vieira-de Abreu, Casey Taylor, Sankar Swaminathan, Simon J. Fisher

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Figure 8

Restoration of the microRNA-7a-5p negatively regulates the expression of Gabra1 gene by binding to its 3′-UTR region.

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Restoration of the microRNA-7a-5p negatively regulates the expression of...
(A) Schematic diagram of rat Gabra1 mRNA containing the predicted conserved target site of microRNA-7a-5p at microRNA regulatory element (MRE) on 3′-UTR. The seed region is indicated in the box. (B) Schematic diagram of SV-40–driven firefly luciferase reporter construct containing a DNA insert at XbaI site (nt 1934), ~100-bp oligonucleotide sequence from the 3′-UTR region of rat Gabra1 gene, containing predicted rno-miR-7a-5p target site. (C) Relative luciferase activity and (D) microRNA-7a-5p expression in HEK293T cells, cotransfected with firefly luciferase reporter construct (shown in B), renilla luciferase plasmid (transfection control), and with either microRNA mimic for rat microRNA-7a-5p (miR-7a-5p mimic) or microRNA mimic negative control (miR-NC). Relative luciferase activity (firefly normalized to renilla) values of miR-7a-5p were normalized to miR-NC–transfected controls. MicroRNA-7a-5p expression was measured by qPCR and represented as relative fold change expression after normalization with reference to the expression of U6 snRNA endogenous control. Data are expressed as mean ± SEM of 3 independent experiments. **P < 0.01 for difference between miR-NC– and miR-7a-5p mimic–treated samples; unpaired 2-tailed Student’s t test.