Functional predication of differentially expressed circRNAs/lncRNAs in the prefrontal cortex of Nrf2-knockout mice

doi:10.18632/aging.202688

. 2021 Mar 10;13(6):8797-8816.

doi: 10.18632/aging.202688. Epub 2021 Mar 10.

Functional predication of differentially expressed circRNAs/lncRNAs in the prefrontal cortex of Nrf2-knockout mice

Yan-Jing Gao¹, Run-Jiao Zhang¹, Qing Liu¹, Shao-Guang Sun², Mao-Yang Qi³, Yue Wang³, Dan-Dan Geng¹, Lei Wang¹

Affiliations

PMID: 33714958
PMCID: PMC8034947
DOI: 10.18632/aging.202688

Functional predication of differentially expressed circRNAs/lncRNAs in the prefrontal cortex of Nrf2-knockout mice

Yan-Jing Gao et al. Aging (Albany NY). 2021.

. 2021 Mar 10;13(6):8797-8816.

doi: 10.18632/aging.202688. Epub 2021 Mar 10.

Authors

Yan-Jing Gao¹, Run-Jiao Zhang¹, Qing Liu¹, Shao-Guang Sun², Mao-Yang Qi³, Yue Wang³, Dan-Dan Geng¹, Lei Wang¹

Affiliations

¹ Department of Human Anatomy, Institute of Medicine and Health, Hebei Medical University, Shijiazhuang 050017, Hebei, China.
² Department of Biochemistry and Molecular Biology, Hebei Medical University, Shijiazhuang 050017, Hebei, China.
³ School of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, Hebei, China.

PMID: 33714958
PMCID: PMC8034947
DOI: 10.18632/aging.202688

Abstract

In the central nervous system, nuclear factor erythroid-2-related factor 2 (Nrf2) protects neurons from oxidant injury, thereby ameliorating neurodegeneration. We explored the key circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs) involved in Nrf2-induced neuroprotection. We used microarrays to examine the circRNAs (DEcircRNAs), lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) differentially expressed between Nrf2 (+/+) and Nrf2 (-/-) mice and identified DEcircRNA/DElncRNA-miRNA-DEmRNA interaction networks. In total, 197 DEcircRNAs, 685 DElncRNAs and 356 DEmRNAs were identified in prefrontal cortical tissues from Nrf2 (-/-) mice. The expression patterns of selected DEcircRNAs (except for mmu_circ_0003404) and DElncRNAs in qRT-PCR analyses were generally consistent with the microarray analysis results. Functional annotation of the DEmRNAs in the DEcircRNA/DElncRNA-miRNA-DEmRNA networks indicated that five non-coding RNAs (mmu_circ_0000233, ENSMUST00000204847, NONMMUT024778, NONMMUT132160 and NONMMUT132168) may contribute to Nrf2 activity, with the help of mmu_circ_0015035 and NONMMUT127961. The results also revealed that four non-coding RNAs (cicRNA.20127, mmu_circ_0012936, ENSMUST00000194077 and NONMMUT109267) may influence glutathione metabolism. Additionally, 44 DEcircRNAs and 7 DElncRNAs were found to possess coding potential. These findings provide clues to the molecular pathways through which Nrf2 protects neurons.

Keywords: Nrf2; circular RNA; long non-coding RNA.

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Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

**Figure 1**
**Expression profiles of circRNAs, lncRNAs and mRNAs in prefrontal cortex tissues from Nrf2 (-/-) and Nrf2 (+/+) mice.** (A–C) Scatter plots showing the expression variation of circRNAs (A), lncRNAs (B) and mRNAs (C) between Nrf2 (-/-) and Nrf2 (+/+) prefrontal cortex tissues. The values of the X and Y axes in the scatter plots are the normalized signal values of the samples (log₂-scaled). The red (up-regulated) and blue (down-regulated) points represent the DEcircRNAs (A), DElncRNAs (B) and DEmRNAs (C) with more than FCs > 1.5 between Nrf2 (-/-) and Nrf2 (+/+) prefrontal cortex tissues. (D–F) Volcano plots showing the differential expression of circRNAs (D), lncRNAs (E) and mRNAs (F) between Nrf2 (-/-) and Nrf2 (+/+) prefrontal cortex tissues. The vertical lines correspond to 1.5-fold upregulation and downregulation, and the horizontal line represents a p-value of 0.05. The red (up-regulated) and blue (down-regulated) points represent the DEcircRNAs (D), DElncRNAs (E) and DEmRNAs (F) with statistical significance. g1: group 1, refers to the Nrf2 (+/+) group. g2: group 2, refers to the Nrf2 (-/-) group. (G–I) Hierarchical clustering analyses were performed to depict the DEcircRANs (G), DElncRNAs (H) and DEmRNAs (I) in Nrf2 (-/-) prefrontal cortex compared with Nrf2 (+/+) prefrontal cortex tissues. The clustering analysis was used to group samples based on their expression values so that the relationships among samples could be predicted. ‘Red’ denotes high relative expression, and ‘green’ denotes low relative expression.

**Figure 2**
**Validation of the DEcircRNAs and DElncRNAs.** The levels of six DEcircRNAs and eight DElncRNAs were validated in prefrontal cortex samples from Nrf2 (-/-) and Nrf2 (+/+) mice using qRT-PCR and normalized to the internal reference gene (β-actin). (A) The qRT-PCR results for five of the verified circRNAs (all but mmu_circ_0003404) were consistent with the microarray results. (B) The qRT-PCR results for all eight verified lncRNAs were consistent with the microarray results. All experiments were replicated three times. The deep red column indicates the FCs of the DEcircRNAs/DElncRNAs determined through microarray analyses; the blue column indicates the expression FCs of the DEcircRNAs/DElncRNAs determined through qRT-PCR experiments. The presented values are the means ± standard deviations. Values > 1 indicate up-regulated DEcircRNAs/DElncRNAs, and values < 1 indicate down-regulated DEcircRNAs/DElncRNAs. *p < 0.05, **p < 0.01.

**Figure 3**
**DEcircRNA/DElncRNA-miRNA-DEmRNA crosstalk network.** Through linear regression model analysis and seed sequence matching method based on gene expression, five DEcircRNAs and seven DElncRNAs were established as a regulatory network of miRNA sponge adsorbents. (A–E) The network of five verified circRNAs (mmu_circ_0000233 (A), mmu_circ_0015035 (B), mmu_circ_0012936 (C), mmu_circ_0008393 (D) and cicRNA.20127 (E)). (F–L) The network of seven verified lncRNAs (NONMMUT132168 (F), NONMMUT127961 (G), NONMMUT132160 (H), ENSMUST00000204847 (I), NONMMUT024778 (J), NONMMUT109267 (K) and ENSMUST00000194077 (L)). Red rhombi: up-regulated DEcircRNAs. Green rhombi: down-regulated DEcircRNAs. Red squares: up-regulated DElncRNAs. Green squares: down-regulated DElncRNAs. Gray V: miRNAs. Red circles: up-regulated DEmRNAs. Green circles: down-regulated DEmRNAs.

**Figure 4**
**Predicted functions of the DEcircRNAs/DElncRNAs involved in the DEcircRNA/DElncRNA-miRNA-DEmRNA network.** (A, B) The top 30 most significantly enriched GO (A) and KEGG (B) pathways of the DEmRNAs that were targets of the five DEcircRNAs (mmu_circ_0000233, mmu_circ_0015035, mmu_circ_0012936, mmu_circ_0008393 and cicRNA.20127) and seven DElncRNAs (NONMMUT132168, NONMMUT127961, NONMMUT132160, ENSMUST00000204847, NONMMUT024778, NONMMUT109267 and ENSMUST00000194077). The X-axis displays the enrich factor in the GO and KEGG pathways and the Y-axis displays the GO and KEGG pathways. The color scale depicts the p-values. The sizes of the nodes represent the counts of genes enriched in the GO and KEGG pathways. (C, D) Diagram of the various functions of the DEcircRNAs/DElncRNAs.

**Figure 5**
**Expression correlations between DEcircRNAs/DElncRNAs and their host mRNAs.** (A) The expression correlations between DEcircRNAs and their host mRNAs. (B) The expression correlations between DElncRNAs and their host mRNAs. The blue area represents the DEcircRNAs/DElncRNAs that correlated with their host mRNAs as a percentage of all the DEcircRNAs/DElncRNAs. The gray area represents the remaining DEcircRNAs/DElncRNAs. The orange area represents the DEcircRNAs/DElncRNAs that correlated positively with their host mRNAs, and the green area represents the DEcircRNAs/DElncRNAs that correlated negatively with their host mRNAs, as percentages of the DEcircRNAs/DElncRNAs that correlated with their host mRNAs.

**Figure 6**
**The intersections of DEcircRNAs, DElncRNAs and DEmRNAs in the prefrontal cortex (cor), substantia nigra (SN), corpus striatum (CPU) and hippocampus (hip).** (A–D) Venn diagrams of up-regulated DEcircRNAs/DElncRNAs (A), down-regulated DEcircRNAs/DElncRNAs (B), up-regulated DEmRNAs (C) and down-regulated DEmRNAs (D) were showed respectively.

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References

1. Chan K, Lu R, Chang JC, Kan YW. NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci U S A. 1996; 93:13943–48. 10.1073/pnas.93.24.13943 - DOI - PMC - PubMed
1. Venugopal R, Jaiswal AK. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci U S A. 1996; 93:14960–65. 10.1073/pnas.93.25.14960 - DOI - PMC - PubMed
1. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997; 236:313–22. 10.1006/bbrc.1997.6943 - DOI - PubMed
1. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007; 47:89–116. 10.1146/annurev.pharmtox.46.120604.141046 - DOI - PubMed
1. Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, Johnson JA, Murphy TH. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci. 2003; 23:3394–406. 10.1523/JNEUROSCI.23-08-03394.2003 - DOI - PMC - PubMed

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[1] Chan K, Lu R, Chang JC, Kan YW. NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci U S A. 1996; 93:13943–48. 10.1073/pnas.93.24.13943 - DOI - PMC - PubMed

[2] Chan K, Lu R, Chang JC, Kan YW. NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci U S A. 1996; 93:13943–48. 10.1073/pnas.93.24.13943 - DOI - PMC - PubMed

[3] Venugopal R, Jaiswal AK. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci U S A. 1996; 93:14960–65. 10.1073/pnas.93.25.14960 - DOI - PMC - PubMed

[4] Venugopal R, Jaiswal AK. Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene. Proc Natl Acad Sci U S A. 1996; 93:14960–65. 10.1073/pnas.93.25.14960 - DOI - PMC - PubMed

[5] Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997; 236:313–22. 10.1006/bbrc.1997.6943 - DOI - PubMed

[6] Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I, Yamamoto M, Nabeshima Y. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997; 236:313–22. 10.1006/bbrc.1997.6943 - DOI - PubMed

[7] Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007; 47:89–116. 10.1146/annurev.pharmtox.46.120604.141046 - DOI - PubMed

[8] Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu Rev Pharmacol Toxicol. 2007; 47:89–116. 10.1146/annurev.pharmtox.46.120604.141046 - DOI - PubMed

[9] Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, Johnson JA, Murphy TH. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci. 2003; 23:3394–406. 10.1523/JNEUROSCI.23-08-03394.2003 - DOI - PMC - PubMed

[10] Shih AY, Johnson DA, Wong G, Kraft AD, Jiang L, Erb H, Johnson JA, Murphy TH. Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. J Neurosci. 2003; 23:3394–406. 10.1523/JNEUROSCI.23-08-03394.2003 - DOI - PMC - PubMed

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Functional predication of differentially expressed circRNAs/lncRNAs in the prefrontal cortex of Nrf2-knockout mice

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Functional predication of differentially expressed circRNAs/lncRNAs in the prefrontal cortex of Nrf2-knockout mice

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Abstract

Conflict of interest statement

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