A Translational Approach to Identify Microrna that Regulate the Voltage-Gated Potassium Channel, Kcnh2

Abdullah Assiri, Purdue University

Abstract

The human ether-a-go-go-related gene (hERG, KCNH2) potassium channel has been implicated in diverse physiological and pathological processes. The KCNH2 gene encodes a rectifier voltage-gated potassium channel (Kv 11.1) that governs the chief repolarizing current, IKr, which is essential for normal electrical activity in excitable cells such as cardiomyocytes. It is also involved in cell growth and apoptosis regulation in non-excitable cells, such as tumor cells. Dysfunction of hERG is associated with potentially lethal complications, including diseases and sudden death under certain circumstances. While the mechanisms regulating KCNH2 expression remain unclear, recent data suggested that microRNAs (miRNAs) are involved, particularly in the context of several pathologic effects. miRNA is a class of RNA defined by its conserved, short, non-coding nature. miRNAs are important regulators of gene expression at the post-transcriptional level that bind through complimentary annealing to the 3’ untranslated regions (3’ UTRs) of target mRNAs, resulting in mRNA destabilization and translational repression. The primary objectives of this research were to 1) identify miRNAs regulating KCNH2 expression in cancer, 2) investigate the potential association between miR-362-3p expression and risk of drug-induced QT interval lengthening, and 3) identify miRNAs potentially regulating KCNH2 expression and function in cardiac cells. Through bioinformatics approaches, five miRNAs were identified to potentially regulate KCNH2 expression and function in breast cancer cells. The five identified miRNAs were validated through a Dual-Luciferase Assay using the KCNH2 3′ UTR. Only miR-362-3p was validated to bind to the KCNH2 3’ UTR, decreasing luciferase activity by 10% ± 2.3 (P < 0.001, n = 3) when compared to cells transfected with luciferase plasmid alone. miR-362-3p was also the only miRNA that its expression positively correlated with overall survival of patients with breast cancer from The Cancer Genome Atlas-Cancer Genome (TCGA) database by log-rank test (HR: 0.39, 95% CI: 0.18 to 0.82, P = 0.012). Cell proliferation was assessed by MTS assay (3-(4,5-dimethylthiazol-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) 48 hours following transfection in breast cancer cell lines, including SK-BR-3 and MCF-7. miR-362-3p significantly decreased proliferation of SK-BR-3 and MCF-7 cells by 23% ± 8.7 (P = 0.014, n = 3) and 11.7% ± 1.0 (P < 0.001, n = 3), respectively. Cell cycle phases in SK-BR-3 and MCF-7 cells were differentiated by flow cytometry 48 hours following transfection. miR-362-3p and hERG siRNA (positive control) significantly increased the accumulation of cells in G0/G1 phase in MCF-7 by 11.7% (from 51.1% ± 0.64 to 57.1 ± 0.96, P = 0.002, n = 3) and 10% (from 51.1% ± 0.64 to 56.8 ± 0.96, P < 0.001, n = 3), respectively. The demonstrated ability of miR-362-3p to regulate hERG in breast cancer cells coupled with previously published data that indicated an alteration of miR-362-3p expression during HF and a potential association between its expression and QT interval prolongation suggesting an important role for this miRNA in regulation of hERG function during HF. Therefore, the contribution of miR-362-3p to hERG function was investigated in patients administered the QT prolonging drug ibutilide, known to inhibit hERG. A total of 22 patients completed a prospective, parallel-group comparative study during which they received subtherapeutic doses (0.003 mg/kg) of ibutilide. The study was originally designed to investigate the influence of heart failure with preserved ejection fraction (HFpEF) on response to drug-induced QT prolongation.

Degree

Ph.D.

Advisors

Overholser, Purdue University.

Subject Area

Physiology|Biochemistry|Cellular biology|Genetics|Medicine|Oncology|Pharmaceutical sciences

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