Induction of specific micro RNA (miRNA) species by ROS-generating metal sulfates in primary human brain cells

Abstract

Iron- and aluminum-sulfate together, at nanomolar concentrations, trigger the production of reactive oxygen species (ROS) in cultures of human brain cells. Previous studies have shown that following ROS induction, a family of pathogenic brain genes that promote inflammatory signalling, cellular apoptosis and brain cell death is significantly over-expressed. Notably, iron- and aluminum-sulfate induce genes in cultured human brain cells that exhibit expression patterns similar to those observed to be up-regulated in moderate- to late-stage Alzheimer's disease (AD). In this study we have extended our investigations to analyze the expression of micro RNA (miRNA) populations in iron- and aluminum-sulfate treated human neural cells in primary culture. The main finding was that these ROS-generating neurotoxic metal sulfates also up-regulate a specific set of miRNAs that includes miR-9, miR-125b and miR-128. Notably, these same miRNAs are up-regulated in AD brain. These findings further support the idea that iron- and aluminum-sulfates induce genotoxicity via a ROS-mediated up-regulation of specific regulatory elements and pathogenic genes that redirect brain cell fate towards progressive dysfunction and apoptotic cell death.

Fig 1. Generation of ROS by metal sulfates in HN cell culture.

The evolution of reactive oxygen species (ROS) using 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) assay was quantified in 2 week old HN cells 3.5 days after initial treatment with 100 nM magnesium-, iron-, aluminum-, magnesium plus aluminum- or iron- plus aluminum-sulfate [1]. These were compared to age-matched untreated controls containing no metal sulfate (value arbitrarily set to 1.0; dashed horizontal line) [1]. Because aluminum-sulfate alone and iron- plus aluminum-sulfate together were found to generate the most ROS using the H₂DCFDA assay (Fig.1), these metal sulfates were next used to study their effects on miRNA expression in HN cells. Data are expressed as relative fluorescence yield, defined as fold-changes over 1.0 (dashed horizontal line). N=5; *p<0.05; **p<0.01 (ANOVA).

Fig. 2. miRNA array analysis.

Nylon membrane-bound DNA-equivalents (15 μM) of 13 miRNAs (known to be enriched in brain) and 5S rRNA were probed with total ³²P-radiolabelled miRNA fractions (<25 nucleotides) isolated from magnesium-sulfate, aluminum-sulfate or iron- plus aluminum-sulfate treated HN cells (Table 1) [1,7]. For each panel row A1-A7; miR-219, miR-183, miR-9, miR-125b, miR-124b, miR-125a, miR-128. Row B1−7; 5S rRNA, miR-132, miR-124a, miR-137, miR-139, miR-153, miR-135. Each individual miRNA signal was quantified against the 5S rRNA signal within the same sample. miR-9, miR-125b and miR-128, but not 5S rRNA gave consistently the highest hybridization signals in iron-plus aluminum-sulfate treated cells when compared to magnesium-sulfate treated controls (N=5). We hypothesize that these induced changes in miRNA expression may be ROS-specific in nature since (a) aluminum-sulfate alone gave qualitatively similar but quantitatively smaller signals for miR-9, miR-125b and miR-128 patterns than did iron- plus aluminum-sulfate together (Fig. 1), and (b) incubation of HN cells with 1 uM of hydrogen peroxide (a potent ROS-generating reagent) also emulated these specific miRNA expression profiles (unpublished observations).

Fig 3. Northern analysis.

From miRNA relative abundance data derived from an initial DNA array screening and electronic autoradiography (Fig. 2), specific miRNA signals in magnesium-sulfate, aluminum-sulfate or iron- plus aluminum-sulfate treated HN cells were assayed using Northern gel analysis. Again, signals were quantified against 5S rRNA levels within the same sample. N=5; *p<0.05; **p<0.01 (ANOVA).