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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

Functional annotation and biological pathways of the JQ1-downregulated genes. (A) Analysis of GO term enrichment for the ‘biological process’ category of JQ1 downregulated genes. The top GO terms are ranked by the number of counts. (B) The most highly represented biological pathways of JQ1 downregulated genes in BV-2 microglial cells. GO, gene ontology.
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Fig6: Functional annotation and biological pathways of the JQ1-downregulated genes. (A) Analysis of GO term enrichment for the ‘biological process’ category of JQ1 downregulated genes. The top GO terms are ranked by the number of counts. (B) The most highly represented biological pathways of JQ1 downregulated genes in BV-2 microglial cells. GO, gene ontology.

Mentions: The groups of LPS upregulated genes that showed a change in expression of (P ≤ 0.01 and fold change ≥1.5 log2) were subjected to a GO analysis (FDR 0.05), with functional annotation using DAVID Bioinformatics Resources and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. DAVID revealed that all major biological processes and molecular functions within GO for the LPS upregulated transcripts were, for the most part, genes associated with the immune system process, response to stimulus, and biological regulation (Figure 3A,B). To further functionally classify the JQ1 downregulated genes (P ≤ 0.01 and fold change ≥1.5) with LPS stimulation, we again used DAVID Bioinformatics Resources. Interestingly, we observed that the largest groups of genes are involved in the same biological processes, that is, the immune system process, response to stimulus, and biological regulation (Figure 6A). To determine the possible biological pathways of the JQ1 downregulated genes (P ≤ 0.01 and fold change ≥1.5) in LPS-treated BV-2 cells, we utilized PANTHER classification system version 9.0. The major categories of the biological pathways were inflammation mediated by chemokine and cytokine, Toll receptor, and interleukin signaling pathways (Figure 6B).Figure 6


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)

Functional annotation and biological pathways of the JQ1-downregulated genes. (A) Analysis of GO term enrichment for the ‘biological process’ category of JQ1 downregulated genes. The top GO terms are ranked by the number of counts. (B) The most highly represented biological pathways of JQ1 downregulated genes in BV-2 microglial cells. GO, gene ontology.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Functional annotation and biological pathways of the JQ1-downregulated genes. (A) Analysis of GO term enrichment for the ‘biological process’ category of JQ1 downregulated genes. The top GO terms are ranked by the number of counts. (B) The most highly represented biological pathways of JQ1 downregulated genes in BV-2 microglial cells. GO, gene ontology.
Mentions: The groups of LPS upregulated genes that showed a change in expression of (P ≤ 0.01 and fold change ≥1.5 log2) were subjected to a GO analysis (FDR 0.05), with functional annotation using DAVID Bioinformatics Resources and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. DAVID revealed that all major biological processes and molecular functions within GO for the LPS upregulated transcripts were, for the most part, genes associated with the immune system process, response to stimulus, and biological regulation (Figure 3A,B). To further functionally classify the JQ1 downregulated genes (P ≤ 0.01 and fold change ≥1.5) with LPS stimulation, we again used DAVID Bioinformatics Resources. Interestingly, we observed that the largest groups of genes are involved in the same biological processes, that is, the immune system process, response to stimulus, and biological regulation (Figure 6A). To determine the possible biological pathways of the JQ1 downregulated genes (P ≤ 0.01 and fold change ≥1.5) in LPS-treated BV-2 cells, we utilized PANTHER classification system version 9.0. The major categories of the biological pathways were inflammation mediated by chemokine and cytokine, Toll receptor, and interleukin signaling pathways (Figure 6B).Figure 6

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