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. 2022 Jan-Dec:14:17590914221101704.
doi: 10.1177/17590914221101704.

Identification of microRNA-mRNA regulatory network associated with oxidative DNA damage in human astrocytes

Chukwumaobim Daniel Nwokwu  1 , Adam Y Xiao  2 , Lynn Harrison  2 , Gergana G Nestorova  3
Affiliations

Identification of microRNA-mRNA regulatory network associated with oxidative DNA damage in human astrocytes

Chukwumaobim Daniel Nwokwu et al. ASN Neuro. 2022 Jan-Dec.

Abstract

The high lipid content of the brain, coupled with its heavy oxygen dependence and relatively weak antioxidant system, makes it highly susceptible to oxidative DNA damage that contributes to neurodegeneration. This study is aimed at identifying specific ROS-responsive miRNAs that modulate the expression and activity of the DNA repair proteins in human astrocytes, which could serve as potential biomarkers and lead to the development of targeted therapeutic strategies for neurological diseases. Oxidative DNA damage was established after treatment of human astrocytes with 10μM sodium dichromate for 16 h. Comet assay analysis indicated a significant increase in oxidized guanine lesions. RT-qPCR and ELISA assays confirmed that sodium dichromate reduced the mRNA and protein expression levels of the human base-excision repair enzyme, 8-deoxyguanosine DNA glycosylase 1 (hOGG1). Small RNAseq data were generated on an Ion Torrent™ system and the differentially expressed miRNAs were identified using Partek Flow® software. The biologically significant miRNAs were selected using miRNet 2.0. Oxidative-stress-induced DNA damage was associated with a significant decrease in miRNA expression: 231 downregulated miRNAs and 2 upregulated miRNAs (p < 0.05; >2-fold). In addition to identifying multiple miRNA-mRNA pairs involved in DNA repair processes, this study uncovered a novel miRNA-mRNA pair interaction: miR-1248:OGG1. Inhibition of miR-1248 via the transfection of its inhibitor restored the expression levels of hOGG1. Therefore, targeting the identified microRNA candidates could ameliorate the nuclear DNA damage caused by the brain's exposure to mutagens, reduce the incidence and improve the treatment of cancer and neurodegenerative disorders.

Keywords: DNA repair; astrocytes; microRNA; oxidative stress.

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Conflict of interest statement

Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Schematics of experimental workflow that includes induction of oxidative DNA damage using 10µM sodium dichromate treatment followed by small RNA sequencing, computational analysis of differentially expressed targets, and miR inhibition functional analysis.
Figure 2.
Figure 2.
Bright-field image of human astrocytes, 10× magnification (A) control, and (B) treated with 10µM sodium dichromate.
Figure 3.
Figure 3.
Sodium dichromate increases oxidative DNA base damage. The alkaline comet assay with FPG treatment was used to detect oxidative base damage following 10 μM Na2Cr2O7 treatment for 16 h (A) and the tail moment was measured using OpenComet (B). Analysis was performed on one experiment with at least 70 cells in each experimental group. Error bars represent SD and **** represents P < 0.0001 using a Student's t-test. (C) ROS levels in untreated and treated cells, n  =  10.
Figure 4.
Figure 4.
A) Pre-alignment QA/QC showing average base quality score per reading. The Phred quality scores of the analyzed samples ranged from 28% to 31%. B) Principal Component Analysis (PCA) plot showing clustering of the treated and control samples.
Figure 5.
Figure 5.
A) Volcano plot depicting the distribution of upregulated and downregulated miRNA genes in treated samples relative to controls. (B) Unsupervised hierarchical clustering using the differentially expressed miRNAs between treated and control samples represented as a heat map. The heat map colors correspond to microRNA expression as indicated in the color key: Red (up-regulated) and Green (down-regulated).
Figure 6.
Figure 6.
A coherent group of miRNAs target DNA repair proteins. Network constructed on miRNet 2.0.
Figure 7.
Figure 7.
RT-qPCR confirmed (A) downregulation of miR-335, and (B) upregulation of its target mRNA, PARP-1 (p < <0.001), n  =  3.
Figure 8.
Figure 8.
Effect of sodium dichromate treatment (10µM, 16 h) on (A) miR-1248, P < 0.05, (B) OGG1 mRNA, and (C) OGG1 protein expression levels (n  =  3).
Figure 9.
Figure 9.
OGG1 and miR-1248 expression analysis after inhibition experiments. (A) OGG1 mRNA upregulation (p < 0.01), (B) increased OGG1 protein expression, and (C) miR-1248 downregulation (p < 0.05), after inhibition of miR-1248 in human astrocytes (n  =  3).
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