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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

Confirmation of differentially expressed genes by quantitative reverse transcription-polymerase chain reaction. (A and B) The Irf9, Irf1, Irak3, Ccl2, Ccl7, Ccl4, Ccl12, Cxcl10, Ptgs2, Irg1, and Il1a genes were significantly downregulated in JQ1-treated BV-2 microglial cells. Gene expression was normalized to GAPDH transcript levels. *P < 0.05 and **P < 0.001 compared with the control. The data represent three independent experiments. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Fig7: Confirmation of differentially expressed genes by quantitative reverse transcription-polymerase chain reaction. (A and B) The Irf9, Irf1, Irak3, Ccl2, Ccl7, Ccl4, Ccl12, Cxcl10, Ptgs2, Irg1, and Il1a genes were significantly downregulated in JQ1-treated BV-2 microglial cells. Gene expression was normalized to GAPDH transcript levels. *P < 0.05 and **P < 0.001 compared with the control. The data represent three independent experiments. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Mentions: A large number of genes that were identified as differentially regulated by the RNA-Seq analysis were subjected to validation by qRT-PCR using GAPDH as the reference gene. Most were selected to be validated according to the distinct effects of JQ1 on the LPS-affected genes. To measure gene expression, mRNA was reverse transcribed into cDNA using Prime Script TM Reverse Transcriptase (Takara Bio Inc., Shiga, Japan); the qRT-PCR assays were repeated several times using at least three mRNA preparations from independent experiments. The results are expressed as the fold change relative to control levels. Thirteen genes were selected for verification; the RNA-Seq expression pattern confirmed for eleven (Irf9, Irf1, Irak3, Ccl2, Ccl7, Ccl4, Ccl12, Cxcl10, Ptgs2, Irg1, Il1a; Figure 7A,B), and two were non-significant (data not shown) in the qRT-PCR analysis compared to the RNA-Seq experiments. Overall, the qRT-PCR data correlated with the RNA-Seq data (Tables 2 and 3). To confirm the distinct effects of JQ1 in primary microglial, we incubated primary microglial cells under inflammatory conditions (LPS 10 ng/mL), which induced inflammatory genes. More importantly, JQ1 suppressed the expression of key LPS-inducible inflammation- and immunity-related genes, including Ccl7, Cxcl10, Irf7, Irg1, Ccl12, Ccl2, Irf1, Il1a and Il1b, in primary microglial cells (Figure 8A,B). However, it should be noted that Ptgs2 gene was not affected by the treatment of LPS. In addition, we analyzed cytokines/chemokines in the supernatants of treated primary microglial cells with ELISAs. Compared to untreated cells Ccl2, Ccl7, and Cxcl10 in the supernatants were increased in primary microglial cells following 2 and 4 h LPS (10 ng/mL) treatment. Co-treatment with JQ1 (500 nM) led to significant reduction of Ccl2, Ccl7, and Cxcl10 in primary microglial cells (Figure 9).Figure 7


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)

Confirmation of differentially expressed genes by quantitative reverse transcription-polymerase chain reaction. (A and B) The Irf9, Irf1, Irak3, Ccl2, Ccl7, Ccl4, Ccl12, Cxcl10, Ptgs2, Irg1, and Il1a genes were significantly downregulated in JQ1-treated BV-2 microglial cells. Gene expression was normalized to GAPDH transcript levels. *P < 0.05 and **P < 0.001 compared with the control. The data represent three independent experiments. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: Confirmation of differentially expressed genes by quantitative reverse transcription-polymerase chain reaction. (A and B) The Irf9, Irf1, Irak3, Ccl2, Ccl7, Ccl4, Ccl12, Cxcl10, Ptgs2, Irg1, and Il1a genes were significantly downregulated in JQ1-treated BV-2 microglial cells. Gene expression was normalized to GAPDH transcript levels. *P < 0.05 and **P < 0.001 compared with the control. The data represent three independent experiments. LPS, lipopolysaccharide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Mentions: A large number of genes that were identified as differentially regulated by the RNA-Seq analysis were subjected to validation by qRT-PCR using GAPDH as the reference gene. Most were selected to be validated according to the distinct effects of JQ1 on the LPS-affected genes. To measure gene expression, mRNA was reverse transcribed into cDNA using Prime Script TM Reverse Transcriptase (Takara Bio Inc., Shiga, Japan); the qRT-PCR assays were repeated several times using at least three mRNA preparations from independent experiments. The results are expressed as the fold change relative to control levels. Thirteen genes were selected for verification; the RNA-Seq expression pattern confirmed for eleven (Irf9, Irf1, Irak3, Ccl2, Ccl7, Ccl4, Ccl12, Cxcl10, Ptgs2, Irg1, Il1a; Figure 7A,B), and two were non-significant (data not shown) in the qRT-PCR analysis compared to the RNA-Seq experiments. Overall, the qRT-PCR data correlated with the RNA-Seq data (Tables 2 and 3). To confirm the distinct effects of JQ1 in primary microglial, we incubated primary microglial cells under inflammatory conditions (LPS 10 ng/mL), which induced inflammatory genes. More importantly, JQ1 suppressed the expression of key LPS-inducible inflammation- and immunity-related genes, including Ccl7, Cxcl10, Irf7, Irg1, Ccl12, Ccl2, Irf1, Il1a and Il1b, in primary microglial cells (Figure 8A,B). However, it should be noted that Ptgs2 gene was not affected by the treatment of LPS. In addition, we analyzed cytokines/chemokines in the supernatants of treated primary microglial cells with ELISAs. Compared to untreated cells Ccl2, Ccl7, and Cxcl10 in the supernatants were increased in primary microglial cells following 2 and 4 h LPS (10 ng/mL) treatment. Co-treatment with JQ1 (500 nM) led to significant reduction of Ccl2, Ccl7, and Cxcl10 in primary microglial cells (Figure 9).Figure 7

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