Additional file 1: Figure S1. of Ethanol induces cytostasis of cortical basal progenitors

Ethanol dose dependently decreases cell proliferation: BrdU incorporation as an index of cell proliferation was determined after exposing the cells with 2.5 mg/ml (56 mM) and 4 mg/ml (86 mM) ETOH for 24 h. Absorbance was determined at 450 nm. Results were analyzed by one-way analysis of variance (ANOVA) and Newman-Keul’s posthoc test. *p < 0.05 when compared with untreated control. (PPTX 43 kb

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

<div><p>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 <em>in-silico</em> 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.</p> </div

Additional file 1: Table S1. of Differential regulation of miRNA and mRNA expression in the myocardium of Nrf2 knockout mice

Complete List of Differentially Expressed Genes: All mRNAs determined to be significantly altered in Nrf2 knockout hearts following RNA sequencing analysis. Each gene symbol is presented with its official name, Log2 fold change (Log2FC) and adjusted p-value to account for false discovery rate (q-value). (DOCX 41 kb

Mutating miR144, miR153, miR27a and miR142-5p binding sites in Nrf2 3′ UTR confirms Nrf2 as a direct target of miR144/153/27a/142-5p.

<p>(A) Schematic representation of different Nrf2 3′ UTR luciferase reporters used in the transfection experiments is depicted. Constructs a, and e were generated in Dr. Jen-Tsan Chi’s laboratory and constructs b, c, d were generated in our laboratory and sequence verified for incorporation of mutagenized nucleotides (data not shown). (B) SH-SY5Y cells were co-transfected with either miR144 or scramble miR and 200 ng pGL3 reporter construct containing wild type or miR144 site mutated 3′ UTR of Nrf2. The relative firefly luciferase activity normalized with renilla luciferase was measured 48 h post-transfection and results are plotted as percentage change over respective control. (C) Cells were co-transfected with both the reporter gene (either wild type or miR 153 site mutated Nrf2 3′ UTR) constructs and scramble miR mimic/miR153 mimic. 48 h following transfection, firefly luciferase activity normalized to renilla activity was determined. The results are represented as percentage change over respective controls. (D) 100 nM of pre-miR scramble control (or) pre-miR27a was transfected into SH-SY5Y cells together with a wild-type or miR27a binding site mutated Nrf2 3′ UTR construct. Cells were lysed 48 h post-transfection and luciferase signal was measured. Firefly luciferase signals were normalized to renilla luciferase signal and percentage change over corresponding controls was represented. (E) Luciferase reporters containing wild-type or miR142-5p site mutant of human Nrf2 3′ UTR were co-transfected with scramble control miRNA or miR142-5p precursors into SH-SY5Y cells. 48 h after transfection, dual luciferase activity was measured. After normalization for renilla luciferase activity, the results were plotted as percentage change and compared to the corresponding controls. In (B-E), values are expressed as means±s.e.m from 3 independent samples and * indicate significantly (p<0.05) different when compared with wild-type Nrf2 3′ UTR construct by one way ANOVA/Newman Keuls post-test; ns-not significant when compared with the respective miRNA target site mutated Nrf2 3′ UTR constructs.</p

miRs144/153/27a/142-5p repression of Nrf2 is Keap1 independent.

<p>(A) Indicated miR mimics or scramble miR mimic was transfected into SH-SY5Y cells for 48 h. 30 µg of whole cell proteins from different samples was immunoblotted for Keap1. GAPDH was used as loading control. Lower panel depicts densitometric analysis of Keap1 normalized to GAPDH (n = 5). ns indicates not significant (p<0.05) vs scramble control miR transfected cells as analyzed by one way ANOVA followed by Newman Keul’s post-test.</p

Sequences of mutagenic oligonucleotides.

<p>Sequences of mutagenic oligonucleotides.</p

Nrf2 transactivation and ARE-driven NQO1 gene expression were reduced by overexpression of different miRs, miR144, miR153, miR27a and miR142-5p.

<p>(A) Cytosolic and (B) nuclear extracts from either scramble miR or indicated miR transfected cells were immunoblotted for Nrf2. GAPDH and laminb1 served as controls for purity of cytosolic and nuclear fraction respectively. * and # indicate 100 kDA and 68 kDA Nrf2 bands respectively and ns indicate non-specific bands. Low exposure of Panel B was provided to appreciate the reduction in 100 kDA. Three independent experiments were performed and a representative blot is given in A & B. (C) Endogenous NQO1 mRNA expression from cells transfected with or without individual miRs were estimated by Taqman based real-time qRT-PCR analysis. NQO1 mRNA levels were normalized to GAPDH (n = 5). (D) As indicated, 48 h after co-transfection of luciferase constructs containing NQO1 ARE sequences with individual miRs, Nrf2 transactivation was determined in terms of measuring the reporter activity. The graph represents the luciferase activity normalized to protein values (n = 6). Panel (C & D), * indicates significant differences between miR and scramble control miR transfected cells as analyzed by one way ANOVA followed by Newman Keul’s post-test.</p

Individual overexpression of miR144, miR153, miR27a and miR142-5p directly target Nrf2 3′ UTR and downregulate expression of Nrf2 transcript.

<p>(A) SH-SY5Y cells were co-transfected with 100 nM of indicated miRs along with wildtype Nrf2 3′UTR construct as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051111#s2" target="_blank">Materials and Methods</a>”. After 48 h, luciferase reporter assay was performed and the relative luciferase normalized to protein was graphed (n = 4). (B) Cells were transfected with different miRs for 48 h and Taqman based real time qRT-PCR analysis was performed using primers specific against Nrf2 3′ UTR and GAPDH. Endogenous expression of Nrf2 3′ UTR was quantified relative to GAPDH levels from 4 independent samples. (C) TaqMan based quantitative real time RT-PCR analysis of Nrf2 mRNA level in SH-SY5Y cells transiently transfected with indicated miRs. GAPDH mRNA levels were used to normalize those of Nrf2 mRNA (n = 5). In panel (A–C), one way ANOVA was used to establish the statistical significance and * - p<0.05 compared with scramble miR control.</p

<i>In-silico</i> based identification of Nrf2 dependent molecular pathway and complex network of disease processes that could be regulated at the intersection of miR144, miR153, miR27a and miR142-5p.

<p>(A) “mirDIP” based computational analysis of tested miRs (miR144/153/27a/142-5p) and identification of number of genes that could be potentially targeted at the intersection of the 4 miRs. mirDIP generated list was used as an input file and a network was generated using another bioinformatic program, “Cytoscape_v2.8.2″. NFE2L2, which is at the intersection of 4 miRs is represented by a bold colored lines. While all the other target genes which are at the intersection of 4 miRs are represented by independent colored lines (miR144-brown; miR153-red; miR27a-black; miR142-5p-purple). (B) KEGG pathway mapping identified Prion disease as the predominant pathway at the intersection of miR144, miR153, miR27a, miR142-5p involving NFE2L2, one of the genes that is predicted to be regulated with a high –ln(p-value) of 20.79 by “DIANA-mirPath”. (C) Using the concept of network biology, we constructed miRNA-associated disease network (MDN) to visualize the relationship of miRs of our interest with multiple pathologic/pathogenic conditions. The network was generated by compiling the data manually from Pubmed (till 05/07/2012) along with the information from two other databases, Human miRNA Disease Database (HMDD - <a href="http://202.38.126.151/hmdd/mirna/md/" target="_blank">http://202.38.126.151/hmdd/mirna/md/</a>) and miR2Disease (<a href="http://www.mir2disease.org/" target="_blank">http://www.mir2disease.org/</a>). This compiled data was used in “Cytoscape_v2.8.2″ to establish connections between miRs and associated diseases. Different diseases associated with indicated miRs are represented as light green colored nodes. Diseases associated with individual miRs are connected by specific colored edges (lines) and any two different miRs that share one common disease are connected by red colored edge. Neuro specific pathologies are represented in red font.</p

Overexpression of miR144, miR153, miR27a and miR142-5p downregulates Nrf2 protein expression in SH-SY5Y cells.

<p>(A) <i>In-silico</i> bioinformatic algorithm based prediction using “miRecords” to identify different miRs that target human Nrf2 3′ UTR and the top 4 miRs generated by atleast 6 individual databases are listed. (B) Sequence alignment and comparison of evolutionary conservation between different miRNA (miR27a, miR142-5p, miR153, miR144(site1), miR144(site2)) seed sequences and its canonical targeting sequences on human Nrf2 3′-UTRs in cross species. Hsa, human; Ptr, chimpanzee; Mml, rhesus; Oga, bushbaby; Tbe, treeshrew; Mmu, mouse; Rno, rat. The sequences recognized by individual miRNA on human Nrf2 3′ UTR are marked above with a black, bold line. (C) SH-SY5Y cells were transiently transfected with 100 nM each of indicated miR mimics individually for 48 h. Whole cell protein lysates were immunoblotted for Nrf2. GAPDH and tubulin were used for normalization and a representative immunoblot is shown. (D) * and # indicate 100 kDA and 68 kDA Nrf2 bands respectively and ns indicate non-specific bands. Using ImageJ based densitometric analysis, the intensity of 100 kDA and 68 kDA Nrf2 levels were normalized to GAPDH and plotted. Comparison between scramble control miR and indicated miRs were performed using one way ANOVA followed by Newman-Keul’s post-test analysis. Values are means±s.e.m from 4 independent replicates and * indicates significantly different from scramble control miR (p<0.05).</p