NF-кB-regulated micro RNAs (miRNAs) in primary human brain cells

Abstract

Micro RNAs (miRNAs), small and labile ~22 nucleotide-sized fragments of single stranded RNA, are important regulators of messenger (mRNA) complexity and in shaping the transcriptome of a cell. In this communication, we utilized amyloid beta 42 (Aβ42) peptides and interleukin-1beta (IL-1β) as a combinatorial, physiologically-relevant stress to induce miRNAs in human primary neural (HNG) cells (a co-culture of neurons and astroglia). Specific miRNA up-regulation was monitored using miRNA arrays, Northern micro-dot blots and RT-PCR. Selective NF-кB translocation and DNA binding inhibitors, including the chelator and anti-oxidant pyrollidine dithiocarbamate (PDTC) and the polyphenolic resveratrol analog CAY10512 (trans-3,5,4'-trihydroxystilbene), indicated the NF-кB sensitivity of several brain miRNAs, including miRNA-9, miRNA-125b and miRNA-146a. The inducible miRNA-125b and miRNA-146a, and their verified mRNA targets, including 15-lipoxygenase (15-LOX), synapsin-2 (SYN-2), complement factor H (CFH) and tetraspanin-12 (TSPAN12), suggests complex and highly interactive roles for NF-кB, miRNA-125b and miRNA-146a. These data further indicate that just two NF-кB-mediated miRNAs have tremendous potential to contribute to the regulation of neurotrophic support, synaptogenesis, neuroinflammation, innate immune signaling and amyloidogenesis in stressed primary neural cells of the human brain.

Figure 1

(A) control human neuronal-glial (HNG) cells in primary culture stained with antibody to glial fibrillary acidic protein (GFAP), a glial-specific cytoplasmic marker (green fluorescence; λ_max=556 nm); with antibody to βTUBIII, a neuron-specific cytoplasmic marker (red; λ_max=702 nm), and with Hoescht 33258 to highlight the morphological features of both glial- and neuronal-cell nuclei (blue; λ_max=461 nm; 2 weeks in culture; 20x magnification) (Zhao et al., 2011); note large nuclear area, relative to both glial or neuronal cytoplasmic area, indicative of high levels of transcriptional activity (Cui et al., 2005); (B) relative induction of NF-κB in HNG cells by IL-1β and Aβ42, either alone or in combination, and effects of PDTC and CAY10512, as analyzed by human NF-κB gel-shift assay (Lukiw and Bazan, 1998; Pogue et al., 2009); IL-1β and Aβ42 together show synergistic effects as previously described in older HNG cell cultures (Lukiw et al., 2008); (C) Aβ42+IL-1β-induced HNG cells in primary culture show significant up-regulation of miRNA-9, miRNA-125b and miRNA-146a, and inhibition by the metal ion chelator, anti-oxidant and NF-κB inhibitor PDTC, and CAY10512 (Lukiw et al., 2008; Cui et al., 2010). Aβ42+IL-1β has been previously shown to induce NF-κB and selective miRNA expression in several different human brain cell types (Li et al., 2011); other classes of NF-κB inhibitors, including the polyphenolic free radical scavenger curcumin are also known to significantly quench the up-regulation of brain-enriched miRNAs (Lukiw et al., 2008; Cui et al., 2010).

Figure 2

(A) Cluster diagram of NF-κB-up-regulated miRNAs in Aβ42+IL1β-stressed HNG primary cells (N=3) compared to untreated controls (N=3); hsa miR = homo sapiens micro RNA; (B) dot blot confirmation of up-regulated miRNA-9, miRNA-125b and miRNA-146a abundance in stressed HNG cells compared to control 5SRNA and miRNA-183 (N=2 control; N=2 Aβ42+IL1β-stressed); and (C) RT-PCR confirmation of up-regulated miRNA-146a, miRNA-125b and miRNA-9 (N=5 control; N=5 Aβ42+IL1β-stressed); note in parts (B) and (C) 5SRNA was loaded at 1/20 the amount of all other miRNAs; as miRNAs have been shown to possess relatively short half-lives, down-regulated miRNA abundance values are suspect to degradation interference and were not considered in these experiments (Sethi and Lukiw, 2009). Members of the Let7 miRNA family were also found to be up-regulated by stress in these studies cells but did not reach statistical significance (data not shown). Genbank-based DNA sequence analysis indicates miRNA-9, miRNA-125b and miRNA-146a all contain canonical, and often multiple, NF-κB binding sites in their respective pre-miRNA promoters, a feature absent within the 15-LOX, SYN-2, CFH and TSPAN12 immediate promoters (Lukiw et al., 2008; Pogue et al., 2010, Pogue et al., 2011; see also Fig. 1; unpublished observations). In (C) a dashed horizontal line at 100 indicates levels of the 5SRNA control for ease of comparison; *p<0.05, ANOVA).

Figure 3

In silico computation and bioinformatics analysis using www.mirbase.org and/or www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/b algorithms; predicted miRNA-mRNA complementarity maps for miRNA-125b (A–C) and miRNA-146a (D–F) of ≤ −21.71 kcal/mol (Cui et al., 2010). Specific mRNA targets for miRNA-9 are currently being investigated. miRNA-mRNA oligonucleotide complementarity yielding free energies of at least ≤ −21kcal/mol may favor selective miRNA-mRNA targeting and down-regulation of specific gene expression (unpublished observations). As indicated, miRNA-125b and miRNA-146a sequences are highlighted in yellow, and complementary sequence in the 3′ un-translated region (3′ UTR) of target mRNAs are highlighted in red; an “|” between the miRNA and mRNA indicates a hydrogen bond; an “:” between the miRNA and mRNA indicates a partial hydrogen bond. Energies of association (E_A), chromosomal location and Genbank accession numbers of target mRNAs are indicated. miRNA-125b-mediated down-regulation of CDKN2A, 15-LOX and SYN II has implications for, respectively, glial cell proliferation (Pogue et al., 2010), neurotrophism (Lukiw et al., 2005; Zhao et al., 2011) and synaptic signaling (Yao et al., 2003; Lukiw 2004). Evidence for miRNA-146a targeting of CFH, IRAK-1 and TSPAN12 (also known as NET-2 or TM4SF12) mRNA, and down-regulation of CFH, IRAK-1 and TSPAN12 expression in human brain cells is further supported by recent studies (Lukiw et al., 2008; Cui et al., 2009; Li et al., 2011a; Li et al., 2011b).

Figure 4

(A) Western analysis indicating down-regulation of 2 miRNA-125b targets (15-LOX and SYN-2) and 2 miRNA-146a targets (CFH and TSPAN12) in control and Aβ42+IL1β-stressed HNG primary cells, compared to a β-actin internal control within the same sample; 15-LOX, SYN-2, CFH and TSPAN12 protein levels (molecular weights ~63, 74, 150, and 35 kDa, respectively) were found to be down-regulated between 0.21 and 0.29 of controls; note that in the ‘stressed’ panel (part A, bottom) total proteins were loaded at 1.5 times the amount as in the ‘control’ panel so that the faint bands for ‘stressed’ 15-LOX, SYN-2, CFH and TSPAN12 became more clearly visible for a publication quality photo; (B) Comparison of RNA and protein signal strengths for 15-LOX, SYN-2, CFH and TSPAN12; mRNA signal strengths (gel data not shown) were found to be down-regulated between 0.54 and 0.63 of controls; relative RNA and protein control levels were both set to 1.0 (dashed horizontal line) for ease of comparison; N=3; *p<0.05, **p<0.01 (ANOVA).

Figure 5

Several key pathological features of AD - glial cell proliferation, synaptic failure, neurotrophic failure, neuroinflammation and amyloidogenesis - can be explained in part by the actions of just two up-regulated miRNAs - miRNA-125b and miRNA-146a. Initially, in this pathogenic cascade, the combination of Aβ42 peptide+IL-1β up-regulates the pro-inflammatory transcription factor NF-κB which drives the transcription of miRNA-125b and miRNA-146a (Lukiw et al., 2007; Pogue et al., 2009). Up-regulated miRNA-125b results in down-regulation in the expression of CDKN2A, a negative regulator of glial cell proliferation (Pogue et al., 2010), down-regulation in the abundance of SYN-2, an essential neuronal phosphoprotein implicated in synaptogenesis and the modulation of neurotransmitter release (Figure 4), and down-regulation in the abundance of 15-LOX, a key enzyme in the biosynthesis of neuroprotectin D1 (NPD1) from the essential omega-3 fatty acid docosahexaneoic acid (DHA; Lukiw et al., 2005; Lukiw and Bazan 2008; Zhao et al., 2011). Similarly, an up-regulated miRNA-146a targets the mRNAs for CFH, IRAK-1 and TSPAN12, down-regulates CFH, IRAK-1 and TSPAN12 gene expression, and this has implications for up-regulated neuroinflammation and amyloidogenesis (notably a down-regulated IRAK-1 is associated with a compensatory surge in the abundance of IRAK-2; Cui et al., 2010). At this time we cannot exclude the participation of other misegulated miRNAs that may contribute epigenetically to the initiation or advancement of the AD process.