Identification of Novel microRNAs in Post-Transcriptional Control of Nrf2 Expression and Redox Homeostasis in Neuronal, SH-SY5Y Cells

Abstract

Nuclear factor-erythroid 2-related factor 2 (Nrf2/NFE2L2), a redox-sensitive transcription factor plays a critical role in adaptation to cellular stress and affords cellular defense by initiating transcription of antioxidative and detoxification genes. While a protein can be regulated at multiple levels, control of Nrf2 has been largely studied at post-translational regulation points by Keap1. Importantly, post-transcriptional/translational based regulation of Nrf2 is less understood and to date there are no reports on such mechanisms in neuronal systems. In this context, studies involving the role of microRNAs (miRs) which are normally considered as fine tuning regulators of protein production through translation repression and/or post-transcriptional alterations, are in place. In the current study, based on in-silico analysis followed by immunoblotting and real time analysis, we have identified and validated for the first time that human NFE2L2 could be targeted by miR153/miR27a/miR142-5p/miR144 in neuronal, SH-SY5Y cells. Co-transfection studies with individual miR mimics along with either WT 3′ UTR of human Nrf2 or mutated miRNA targeting seed sequence within Nrf2 3′ UTR, demonstrated that Nrf2 is a direct regulatory target of these miRs. In addition, ectopic expression of miR153/miR27a/miR142-5p/miR144 affected Nrf2 mRNA abundance and nucleo-cytoplasmic concentration of Nrf2 in a Keap1 independent manner resulting in inefficient transactivating ability of Nrf2. Furthermore, forced expression of miRs diminished GCLC and GSR expression resulting in alteration of Nrf2 dependent redox homeostasis. Finally, bioinformatics based miRNA-disease network analysis (MDN) along with extended computational network analysis of Nrf2 associated pathologic processes suggests that if in a particular cellular scenario where any of these miR153/miR27a/miR142-5p/miR144 either individually or as a group is altered, it could affect Nrf2 thus triggering and/or determining the fate of wide range of disease outcomes.

Caloric restriction confers persistent anti-oxidative, pro-angiogenic, and anti-inflammatory effects and promotes anti-aging miRNA expression profile in cerebromicrovascular endothelial cells of aged rats

“…2B). miR-144 is a known regulator of Nrf2 expression (58,68). Because miR-144 was not among the targets present on the TaqMan Array Rodent MicroRNA A Cards Set v3.0, its expression was analyzed by QRT-PCR separately.…”

Nuclear Factor–Erythroid-2–Related Factor 2 in Aging and Lung Fibrosis

“…Post-translational modifications mediated by AMPK, 91 sumoylation, 92 and miRNAs 93 are also known to regulate Nrf2 signaling. In endothelial cells, activation of AMPK stimulates the expression of the antioxidant enzyme HO-1 through Nrf2 signaling.…”

“…96 The miRNAs, miR-144, miR-27a, and miR-153, have been reported to regulate Nrf2 gene expression. 93,97 In addition, miRNAs are also known to target other Nrf2 regulators and alter Nrf2 activation. 98 Although age-related changes in the expression of miRNAs have been reported, 97 investigating their role in regulating Nrf2 activation and downstream transcriptional responses in cellular/animal models of aging requires further investigation.…”

MicroRNA Pharmacoepigenetics: Posttranscriptional Regulation Mechanisms behind Variable Drug Disposition and Strategy to Develop More Effective Therapy

“…BCRP/ABCG2, another important efflux transporter in ADME and MDR, is also regulated by a number of miRNAs via direct or indirect mechanisms ( Table 2). The MDR of S1 colon carcinoma cell line is Zhao et al, 2008;Guttilla et al, 2012;Wei et al, 2014a Let-7 Reporter assay, mRNA/protein expression, and function study Zhao et al, 2011 miR-18a Reporter assay, protein expression, association and function study Liu et al, 2009 miR-18a/b, -19b, -20b, -193b, -206 Reporter assay, protein expression Castellano et al, 2009;Leivonen et al, 2009b miR-145 Reporter assay, mRNA and protein expression, and function study Spizzo et al, 2010 miR-22 Reporter assay, protein expression, and function study Pandey and Picard, 2009;Xiong et al, 2010 miR-206 Reporter assay, protein expression, and function study Adams et al, 2007;Kondo et al, 2008 miR-27a via zinc finger and BTB domain containing 10 (ZBTB10) Li et al, 2010a miR-219-5p Reporter assay, mRNA/protein expression, association and function Huang et al, 2015 miR-135b Reporter assay, mRNA and protein expression Aakula et al, 2015 NFE2L2/Nrf2 miR-144 Reporter assay, mRNA and protein expression, and function study Sangokoya et al, 2010 miR-28 Reporter assay, mRNA and protein expression Yang et al, 2011 miR-200a (Activation) via Kelch-like ECH-associated protein 1 (Keap1) Eades et al, 2011;Yang et al, 2014 miR-153, -27a, -142-5p, -144 Reporter assay, mRNA and protein expression, and function study Narasimhan et al, 2012Narasimhan et al, , 2014 Protein expression, and function study Singh et al, 2013 miR-507, -634, -450, -129-5p Reporter assay, mRNA and protein expression, and function study Yamamoto et al, 2014 miR-34a mRNA and protein expression Huang et al, 2014 miR-340 Reporter assay, mRNA and protein expression, and function study Shi et al, 2014a miR-141 via Kelch-like ECH-associated protein 1 (Keap1) …”

“…In addition to direct targeting of CYP3A4 (Pan et al, 2009a), CYP1B1 (Tsuchiya et al, 2006), and ABCB1 (Zhu et al, 2008), miR27b/a was able to act on a number of xenobiotic receptors and transcription factors including NR1I1/ (Pan et al, 2009a), NR1C1/ PPARa (Kida et al, 2011), ERa/NR3A1 (Li et al, 2010a), NFE2L2/ Nrf2 (Narasimhan et al, 2012), and RXRa/NR2B1 (Ji et al, 2009). Therefore, alteration of enzyme/transporter protein outcome by miR27b/a could be an accumulative effect from direct targeting of the enzyme/transporter and "indirect" targeting of regulatory factors including xenobiotic receptors and transcription factors (Yu, 2009;Yu and Pan, 2012).…”