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RNA sequencing reveals distinct mechanisms underlying BET inhibitor JQ1-mediated modulation of the LPS-induced activation of BV-2 microglial cells.

Jung KH, Das A, Chai JC, Kim SH, Morya N, Park KS, Lee YS, Chai YG - J Neuroinflammation (2015)

Bottom Line: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively.Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively).Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan, Gyeonggi-do, 426-791, South Korea. khjung2@gmail.com.

ABSTRACT

Background: Microglial cells become rapidly activated through interaction with pathogens, and their persistent activation is associated with the production and secretion of various pro-inflammatory genes, cytokines, and chemokines, which may initiate or amplify neurodegenerative diseases. Bromodomain and extraterminal domain (BET) proteins are a group of epigenetic regulators that associate with acetylated histones and facilitate the transcription of target genes. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities by inhibiting the expression of IL-6 and Tnf-α in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in microglia.

Methods: The present study was aimed at evaluating the anti-inflammatory function and underlying genes targeted by JQ1 in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells using two transcriptomic techniques: global transcriptomic biological duplicate RNA sequencing and quantitative real-time PCR. Associated biological pathways and functional gene ontology were also evaluated.

Results: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively. Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively). Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice. Utilizing functional group analysis, the genes affected by JQ1 were classified into four categories related to biological regulation, immune system processes, and response to stimuli. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1-treated BV-2 microglial cells.

Conclusions: These unprecedented results suggest the BET inhibitor JQ1 as a candidate for the prevention or therapeutic treatment of inflammation-mediated neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus

RNA-Seq analysis reveals LPS-stimulated pro-inflammatory gene expression in BV-2 microglial cells. (A and B) A heat map representing RNA-Seq gene expression of top 100 upregulated (P ≤ 0.01 and fold change ≥1.5 log2) inflammatory genes in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.
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Fig2: RNA-Seq analysis reveals LPS-stimulated pro-inflammatory gene expression in BV-2 microglial cells. (A and B) A heat map representing RNA-Seq gene expression of top 100 upregulated (P ≤ 0.01 and fold change ≥1.5 log2) inflammatory genes in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.

Mentions: To identify the response of BV-2 cells to LPS (10 ng/mL), BV-2 cells were stimulated for two different time periods, that is, 2 and 4 h. RNA-Seq analysis revealed differentially expressed genes in the LPS-stimulated BV-2 cells at both time points: 270 genes for 2 h and 396 genes for 4 h (increased and decreased in expression fold change ≥1.5 log2 and P ≤ 0.01, respectively) were differentially regulated. Among them, 214 and 301 genes were upregulated, whereas 56 and 95 genes were downregulated at 2 and 4 h, respectively, after LPS treatment (Figure 2A,B and Additional file 2: Table S1 and S2). Notably, most of the upregulated genes included the following inflammatory response- and immune response-related genes: iNOS, interleukin and interleukin-related genes (Il1-β, Il1a, Il18, Il1rn); Tnf-α and Tnf-α-related genes (Tnfaip3, Tnip3, Tnip1, Tnfaip2); a prostaglandin-related gene, Ptgs2; NF-κB-related genes (Nfkbiz, Nfkbia, Nfkb2, Relb, Nfkbie, Nfkb1); interferon-related genes (Ifit1, interferon regulatory factors (Irf) Irf1, Irf7, Irf9); and cytokines or chemokines (Cxcl10, Ccl4, Ccl7, Ccl2, Ccl3, Ccl12, Ccl9) (Figure 2A,B,C,D). We selected these genes based on their biological processes and the molecular functions of their gene ontology. As the downregulated genes were not associated with inflammation, only upregulated genes were studied further. We confirmed by a GO analysis (FDR 0.05) using DAVID Bioinformatics Resources that LPS downregulated transcripts were associated with regulation of biological and cellular processes in BV-2 microglial cells (Figure 3C,D).Figure 2


RNA sequencing reveals distinct mechanisms underlying BET inhibitor JQ1-mediated modulation of the LPS-induced activation of BV-2 microglial cells.

Jung KH, Das A, Chai JC, Kim SH, Morya N, Park KS, Lee YS, Chai YG - J Neuroinflammation (2015)

RNA-Seq analysis reveals LPS-stimulated pro-inflammatory gene expression in BV-2 microglial cells. (A and B) A heat map representing RNA-Seq gene expression of top 100 upregulated (P ≤ 0.01 and fold change ≥1.5 log2) inflammatory genes in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4359438&req=5

Fig2: RNA-Seq analysis reveals LPS-stimulated pro-inflammatory gene expression in BV-2 microglial cells. (A and B) A heat map representing RNA-Seq gene expression of top 100 upregulated (P ≤ 0.01 and fold change ≥1.5 log2) inflammatory genes in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. Biological replicates (n = 2) for each condition were combined separately, and the heat map were generated with the Multi Experiment Viewer (version 4.8) software. (C and D) UCSC Browser images representing the normalized RNA-Seq read density in 2- and 4-h LPS-stimulated BV-2 microglial cells compared to the control, respectively. LPS, lipopolysaccharide.
Mentions: To identify the response of BV-2 cells to LPS (10 ng/mL), BV-2 cells were stimulated for two different time periods, that is, 2 and 4 h. RNA-Seq analysis revealed differentially expressed genes in the LPS-stimulated BV-2 cells at both time points: 270 genes for 2 h and 396 genes for 4 h (increased and decreased in expression fold change ≥1.5 log2 and P ≤ 0.01, respectively) were differentially regulated. Among them, 214 and 301 genes were upregulated, whereas 56 and 95 genes were downregulated at 2 and 4 h, respectively, after LPS treatment (Figure 2A,B and Additional file 2: Table S1 and S2). Notably, most of the upregulated genes included the following inflammatory response- and immune response-related genes: iNOS, interleukin and interleukin-related genes (Il1-β, Il1a, Il18, Il1rn); Tnf-α and Tnf-α-related genes (Tnfaip3, Tnip3, Tnip1, Tnfaip2); a prostaglandin-related gene, Ptgs2; NF-κB-related genes (Nfkbiz, Nfkbia, Nfkb2, Relb, Nfkbie, Nfkb1); interferon-related genes (Ifit1, interferon regulatory factors (Irf) Irf1, Irf7, Irf9); and cytokines or chemokines (Cxcl10, Ccl4, Ccl7, Ccl2, Ccl3, Ccl12, Ccl9) (Figure 2A,B,C,D). We selected these genes based on their biological processes and the molecular functions of their gene ontology. As the downregulated genes were not associated with inflammation, only upregulated genes were studied further. We confirmed by a GO analysis (FDR 0.05) using DAVID Bioinformatics Resources that LPS downregulated transcripts were associated with regulation of biological and cellular processes in BV-2 microglial cells (Figure 3C,D).Figure 2

Bottom Line: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively.Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively).Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Life Science, Hanyang University, 1271 Sa 3-dong, Ansan, Gyeonggi-do, 426-791, South Korea. khjung2@gmail.com.

ABSTRACT

Background: Microglial cells become rapidly activated through interaction with pathogens, and their persistent activation is associated with the production and secretion of various pro-inflammatory genes, cytokines, and chemokines, which may initiate or amplify neurodegenerative diseases. Bromodomain and extraterminal domain (BET) proteins are a group of epigenetic regulators that associate with acetylated histones and facilitate the transcription of target genes. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities by inhibiting the expression of IL-6 and Tnf-α in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in microglia.

Methods: The present study was aimed at evaluating the anti-inflammatory function and underlying genes targeted by JQ1 in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells using two transcriptomic techniques: global transcriptomic biological duplicate RNA sequencing and quantitative real-time PCR. Associated biological pathways and functional gene ontology were also evaluated.

Results: With a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively. Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively). Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice. Utilizing functional group analysis, the genes affected by JQ1 were classified into four categories related to biological regulation, immune system processes, and response to stimuli. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1-treated BV-2 microglial cells.

Conclusions: These unprecedented results suggest the BET inhibitor JQ1 as a candidate for the prevention or therapeutic treatment of inflammation-mediated neurodegenerative diseases.

No MeSH data available.


Related in: MedlinePlus