Journal of Molecular Biology
Post-Transcriptional Regulation of Renalase Gene by miR-29 and miR-146 MicroRNAs: Implications for Cardiometabolic Disorders
Introduction
Renalase, a recently discovered flavoprotein with oxidoreductase activity, is expressed in several tissues including kidney, heart and nervous system [1], [2]. Apart from its enzymatic function, renalase may also act as a signaling molecule to protect against acute kidney injury via interaction with a cell surface receptor, such as the plasma membrane calcium ATPase isoform PMCA4b [3], [4]. The renalase gene is positioned on chromosome 19 and 10 in mouse and human, respectively [5], [6]. Human renalase (RNLS) occurs as 7 isoforms (hRenalase1–hRenalase7 yielding proteins of 342, 315, 232, 200, 181, 163 and 139 amino acid residues, respectively) [3]; among these isoforms, RNLS isoform 1 and RNLS isoform 2 are prominently expressed [2], [7]. On the other hand, mouse renalase (Rnls) has been reported to have 2 isoforms (Rnls isoform 1 and Rnls isoform 2 yielding proteins of 342 and 249 amino acids, respectively) [5], [8].
Accumulating evidence in the literature shows that renalase is a major regulator for hypertension, diabetes, stroke and coronary heart disease [9], [10], [11]. For example, knockout mouse for renalase exhibited increased plasma catecholamines and high blood pressure; consistently, injection of purified renalase into rats significantly decreased the mean arterial blood pressure [1], [12]. The amount of this protein is profoundly diminished in sub-nephrectomized rats and end-stage renal disease and chronic kidney disease patients [6], [11], [13]. In addition, three common single nucleotide polymorphisms (SNPs) in human RNLS (viz. rs2576178 located in the promoter region, rs2296545 located in exon 2 and rs10887800 located in intron 6) have been associated with cardiometabolic disease states including hypertension, stroke and diabetes in different human populations [9], [10], [14], [15], [16], [17]. Therefore, it is important to understand the regulation of this gene under basal and pathophysiological conditions.
Recent studies by us and others have revealed that several transcription factors (viz. Sp1, STAT3 and ZBP89) play crucial roles to regulate renalase transcription and that nicotine, dopamine and epinephrine activate renalase gene expression in various cell types [11], [18], [19]. The mouse Rnls promoter has also been shown to harbor multiple hypoxia response elements that bind to hypoxia-inducible factor 1α and enhance the renalase expression providing protection against myocardial ischemic reperfusion injury [20]. However, the mechanism of regulation of renalase at the post-transcriptional level remains completely unknown.
Here, we probed for the possible regulatory role of microRNAs (miRNAs) in governing the expression of renalase. Systematic functional analysis by mouse Rnls 3′-UTR (untranslated region)/luciferase reporter assays and measurement of endogenous renalase protein levels after ectopic expression/inhibition of specific miRNAs revealed interactions of miR-29 and miR-146 miRNAs with mouse/human renalase 3′-UTR. In corroboration, genetically hypertensive blood pressure high (BPH) mice showed diminished protein levels of renalase and elevated levels of miR-29b as compared to genetically hypotensive blood pressure low (BPL) mice. Moreover, a common genetic variation in the 3′-UTR of human RNLS gene (C → T; rs10749571) altered interaction with miR-146a and the SNP displayed association with several physiological/biochemical cardiometabolic traits, suggesting potential role of miR-146a in regulating human RNLS gene in vivo. These observations provide new insights into the pathogenetic mechanisms of cardiometabolic disease states.
Section snippets
Prediction of putative miRNA binding sites in the mouse renalase 3′-UTR
To identify miRNAs that may interact with the mouse Rnls 3′-UTR, we used 11 computational analysis programs (viz. miRWalk, Microt4, mirBridge, miRmap, miRNAMap, PICTAR2, RNA22, RNAhybrid, TargetScan, miRanda and PITA), resulting in the prediction of 1012 miRNAs. Among those potential miRNAs, four were predicted by at least seven programs (viz. mmu-miR-29b-3p, mmu-miR-146a-5p, mmu-miR-146b-5p and mmu-miR-758-3p; Table 1). Interestingly, the putative binding sites for these miRNAs are closely
Discussion
Post-transcriptional regulation of genes by miRNAs is gaining significance in recent years since these small non-coding RNAs may be dysregulated under pathological conditions including cardiovascular diseases [23], [24], [25]. We undertook a systematic computational and experimental approach to identify potential miRNAs that may regulate renalase expression.
As miRNA:mRNA interactions follow incomplete base pairing in animals, the predictions of miRNA binding sites for animal genes are more
Computational analysis for prediction of potential miRNA binding sites in mouse and human renalase 3′-UTR
The following computational programs were used for prediction of putative miRNA binding sites in the 3′-UTR of the mouse and human renalase genes: miRWalk [42], Microt4 [43], mirBridge [44], miRmap [45], miRNAMap [46], PICTAR2 [47], RNA22 [48], RNAhybrid [49], TargetScan [36], miRanda [50] and PITA [51]. The Renalase gene ID or the 3′-UTR sequence was given as input. The seed sequence length of 6 nucleotide complementarity with no mismatch and the G:U base pairing were defined for predictions.
Acknowledgements
Authors thank Prof. D. Karunagaran and Dr. M. Subramanian, Department of Biotechnology, Indian Institute of Technology Madras, Chennai, for kindly providing them with miR-29 expression plasmids; Mark Boldin, Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, for the miR-146b expression plasmid; and Anindya Dutta, Department of Biochemistry and Molecular Genetics Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville,
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