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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

Induction of inflammatory response-related genes in LPS-stimulated BV-2 microglial cells. Quantitative real-time reverse transcriptase PCR analysis of the expression of inflammatory genes in BV-2 microglial cells stimulated with LPS (10 ng/mL). Inflammatory genes were significantly upregulated in cells treated with LPS compared to untreated cells (*P < 0.05 and **P < 0.001) at the indicated times. Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are the mean ± SD of triplicate wells. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tnf-α, tumor necrosis factor alpha.
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Fig1: Induction of inflammatory response-related genes in LPS-stimulated BV-2 microglial cells. Quantitative real-time reverse transcriptase PCR analysis of the expression of inflammatory genes in BV-2 microglial cells stimulated with LPS (10 ng/mL). Inflammatory genes were significantly upregulated in cells treated with LPS compared to untreated cells (*P < 0.05 and **P < 0.001) at the indicated times. Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are the mean ± SD of triplicate wells. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tnf-α, tumor necrosis factor alpha.

Mentions: We began our study by examining the timing of gene activation induced by LPS by performing an expression analysis of BV-2 microglial cells treated with LPS (10 ng/mL) for 10 min to 24 h and compared the results with the expression in untreated cells under normal culture conditions. We found a significant, time-dependent upregulation of inflammatory response-related genes with up to 4 h of LPS treatment (Figure 1). Because we found that most of the inflammatory response-related genes were upregulated at the 2- and 4-h time points, we chose of these time points for transcriptional profiling; these time points were also used in other studies [7,25,26] that investigated the general induction pattern of microglial activation by LPS.Figure 1


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)

Induction of inflammatory response-related genes in LPS-stimulated BV-2 microglial cells. Quantitative real-time reverse transcriptase PCR analysis of the expression of inflammatory genes in BV-2 microglial cells stimulated with LPS (10 ng/mL). Inflammatory genes were significantly upregulated in cells treated with LPS compared to untreated cells (*P < 0.05 and **P < 0.001) at the indicated times. Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are the mean ± SD of triplicate wells. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tnf-α, tumor necrosis factor alpha.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Induction of inflammatory response-related genes in LPS-stimulated BV-2 microglial cells. Quantitative real-time reverse transcriptase PCR analysis of the expression of inflammatory genes in BV-2 microglial cells stimulated with LPS (10 ng/mL). Inflammatory genes were significantly upregulated in cells treated with LPS compared to untreated cells (*P < 0.05 and **P < 0.001) at the indicated times. Gene expression was normalized to GAPDH transcript levels. The data represent three independent experiments. The values are the mean ± SD of triplicate wells. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Tnf-α, tumor necrosis factor alpha.
Mentions: We began our study by examining the timing of gene activation induced by LPS by performing an expression analysis of BV-2 microglial cells treated with LPS (10 ng/mL) for 10 min to 24 h and compared the results with the expression in untreated cells under normal culture conditions. We found a significant, time-dependent upregulation of inflammatory response-related genes with up to 4 h of LPS treatment (Figure 1). Because we found that most of the inflammatory response-related genes were upregulated at the 2- and 4-h time points, we chose of these time points for transcriptional profiling; these time points were also used in other studies [7,25,26] that investigated the general induction pattern of microglial activation by LPS.Figure 1

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