Summary of microarray and qRT-PCR analyses comparing differences in gene expression between stable BE(2)M17 clones expressing miR-142 and miR-null.

<p>Genes that were found to be down-regulated in the miR-142 clones with a p-value <0.001 in the microarray analysis are shown here. Only MAOA and AKAP12 showed significant (p < 0.05) down-regulation in the post-validation experiment using RT-PCR.</p

miR-142 downregulates MAOA protein expression in human neurons.

<p>Human neurons grown in vitro for 11 days were transduced with either miR-142 or GFP lentivirus. On day 6 after transduction, cells were harvested and Western blot analysis was performed for MAOA. Representative Western blots from three human neuron donors indicating a decrease in MAOA protein level after miR-142 transduction compared to control GFP transduction are shown. Quantification of Western blots from 5 human neuron donors revealed a significant decrease in MAOA level after miR-142 transduction (fold change -1.9, p<.05). Blots were normalized to β-actin and paired t-test was performed.</p

miR-142 downregulates MAOA protein expression and enzyme activity in BE(2)M17 cells.

<p>(A) Representative Western blot for MAOA in stable BE(2)M17 clones expressing miR-142 and in control miR-null clones. (B) Quantification of three independent Western blots showing that MAOA protein level is lower (-3.5 fold) in stable BE(2)M17 clones expressing miR-142 compared to miR-null clones. Blots were normalized to β-actin and unpaired t-test was performed. *** represent p<0.001, error bars are standard error of mean. (C) Total MAO as well as MAOA and MAOB specific activity was measured in the miR-142 and miR-null BE(2)M17 clones. Both total MAO and MOA specific activity was approximately 40% lower in miR-142 clones compared to miR-null clones. There was no difference in MAOB specific activity. Y-axis represents fold change of enzyme activity, error bars represent standard error of mean, n = 3.</p

Schematic representation of downstream effects of miR-142 upregulation in neurons.

<p>Normally, uninfected neurons have minimal miR-142 expression. Most of the SIRT1 mRNA is therefore available for translation, maintaining the SIRT1 protein pool. SIRT1 deacetylates a lysine residue in the transcription factor NHLH2 and activates it. NHLH2 in turn increases MAOA transcription. In HIVE/SIVE, miR-142 expression is upregulated in neurons. One of the two mature strands of this miR, miR-142-5p, binds to the 3′UTR of SIRT1 mRNA and prevents its translation. This leads to reduction of SIRT1 protein level. Therefore SIRT1 is no longer available for induction if MAOA expression, finally resulting in decrease in MAOA expression and activity.</p

Overexpression of SIRT1 in BE(2)M17 clones leads to increase in MAOA protein levels.

<p>(A) Representative Western blot for SIRT1 and MAOA in BE(2)M17 clones 1A (miR-null) and 6B (miR-142). As predicted, clone 6B has lower levels of SIRT1 compared to clone 1A. Transfection of SIRT1 results in overexpression of the same, as well as increase in MAOA protein levels in both the clones. (B) Quantification of MAOA expression by Western blot analysis after three independent transfection experiments. MAOA protein level was increased by 1.7-fold in clone 1A and 1.8-fold in clone 6B after transfection of SIRT1. Blots were normalized to β-actin and one-way ANOVA was performed followed by Bonferroni's multiple correction test. ** represent p<0.01, * represents p<0.05, error bars are standard error of mean, NT =  non-transfected.</p