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Long noncoding RNA LINC00305 promotes inflammation by activating the AHRR-NF- κ B pathway in human monocytes

View Article: PubMed Central - PubMed

ABSTRACT

Accumulating data from genome-wide association studies (GWAS) have provided a collection of novel candidate genes associated with complex diseases, such as atherosclerosis. We identified an atherosclerosis-associated single-nucleotide polymorphism (SNP) located in the intron of the long noncoding RNA (lncRNA) LINC00305 by searching the GWAS database. Although the function of LINC00305 is unknown, we found that LINC00305 expression is enriched in atherosclerotic plaques and monocytes. Overexpression of LINC00305 promoted the expression of inflammation-associated genes in THP-1 cells and reduced the expression of contractile markers in co-cultured human aortic smooth muscle cells (HASMCs). We showed that overexpression of LINC00305 activated nuclear factor-kappa beta (NF-κB) and that inhibition of NF-κB abolished LINC00305-mediated activation of cytokine expression. Mechanistically, LINC00305 interacted with lipocalin-1 interacting membrane receptor (LIMR), enhanced the interaction of LIMR and aryl-hydrocarbon receptor repressor (AHRR), and promoted protein expression as well as nuclear localization of AHRR. Moreover, LINC00305 activated NF-κB exclusively in the presence of LIMR and AHRR. In light of these findings, we propose that LINC00305 promotes monocyte inflammation by facilitating LIMR and AHRR cooperation and the AHRR activation, which eventually activates NF-κB, thereby inducing HASMC phenotype switching.

No MeSH data available.


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LINC00305 promotes inflammation in THP-1 cells and phenotype switching in co-cultured HASMCs.(A) QRT-PCR analysis of pro-inflammatory gene expression in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). The upper and lower panels show different levels of LINC00305 overexpression. Gene expression levels in the control groups were assigned a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. (B,C) GO analysis and heat map of the microarray data demonstrated upregulation of inflammation-associated genes in LINC00305-overexpressing THP-1 cells. The gene expression profiles of THP-1 cells stably expressing LINC00305 and the control cells were examined using microarray analysis (Affymetrix, Human Exon 1.0 ST array). Genes upregulated in cells overexpressing LINC00305 were analysed by GO enrichment analysis (B) and the genes involved in inflammation, monocyte activation and chemotaxis (GO:002544, 002548, 0042177, 0090025) were analyzed with MeV software to show the heat map of the inflammation-relevant genes in the microarray assay (C). (D) QRT-PCR analysis of contractile markers in untreated HASMCs (blank) and in HASMCs co-cultured with wild type THP-1 cells, THP-1 cells stably expressing LINC00305 (Linc00305 oe THP-1) or the control vector (Control THP-1). The relative mRNA expression levels were normalized to the GAPDH internal control. The genes expression levels in the Blank group were assigned a value of 1.0. Data are presented as the mean ± sem of 3 replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group.
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f3: LINC00305 promotes inflammation in THP-1 cells and phenotype switching in co-cultured HASMCs.(A) QRT-PCR analysis of pro-inflammatory gene expression in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). The upper and lower panels show different levels of LINC00305 overexpression. Gene expression levels in the control groups were assigned a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. (B,C) GO analysis and heat map of the microarray data demonstrated upregulation of inflammation-associated genes in LINC00305-overexpressing THP-1 cells. The gene expression profiles of THP-1 cells stably expressing LINC00305 and the control cells were examined using microarray analysis (Affymetrix, Human Exon 1.0 ST array). Genes upregulated in cells overexpressing LINC00305 were analysed by GO enrichment analysis (B) and the genes involved in inflammation, monocyte activation and chemotaxis (GO:002544, 002548, 0042177, 0090025) were analyzed with MeV software to show the heat map of the inflammation-relevant genes in the microarray assay (C). (D) QRT-PCR analysis of contractile markers in untreated HASMCs (blank) and in HASMCs co-cultured with wild type THP-1 cells, THP-1 cells stably expressing LINC00305 (Linc00305 oe THP-1) or the control vector (Control THP-1). The relative mRNA expression levels were normalized to the GAPDH internal control. The genes expression levels in the Blank group were assigned a value of 1.0. Data are presented as the mean ± sem of 3 replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group.

Mentions: Monocyte-mediated inflammation plays an important role in atherogenesis8. To investigate the function of LINC00305, THP-1 cells were infected with the LINC00305-expressing or the empty control lentiviruses at two different levels. Both levels of LINC00305 overexpression significantly enhanced the expression of inflammatory genes in THP-1 cells, and the stimulative effect is concentration-dependent (Fig. 3A). However, the expression of SERPINBs, genes located adjacent to the LINC00305 locus were unaffected, indicating that LINC00305 functions in a trans-regulatory fashion (Fig. S4). To further study the downstream effects of LINC00305, the transcriptional profile of LINC00305- and empty vector-transfected THP-1 cells were analysed using chip assays (Affymetrix, Human Exon 1.0 ST array). Gene Ontology (GO) analysis demonstrated that LINC00305-upregulated genes are enriched for inflammation-associated genes, and similar result was observed in the heat map of inflammation-associated genes in the microarray data, indicating that LINC00305 promotes inflammation in THP-1 cells (Fig. 3B,C). In atherosclerotic plaques, the cytokines secreted by inflammatory cells promote the shift of VSMCs from a contractile to a synthetic phenotype, an event that contributes to the development of plaques3233. To investigate the functional significance of LINC00305 in atherosclerosis, HASMCs were co-cultured with wild type THP-1, THP-1 cells stably expressing LINC00305 or the control empty vector. QRT-PCR analysis revealed that co-culture with both wild type and control transfected THP-1 cells decreased the expression of genes associated with the contractile phenotype in HASMCs, and THP-1 cells stably expressing LINC00305 further down-regulated the expression of these genes (Fig. 3D), suggesting that LINC00305 overexpression promotes the switch of co-cultured HASMCs from a contractile phenotype to a synthetic phenotype. Together, these results support the hypothesis that LINC00305 expression plays an important role in the progression of atherosclerosis.


Long noncoding RNA LINC00305 promotes inflammation by activating the AHRR-NF- κ B pathway in human monocytes
LINC00305 promotes inflammation in THP-1 cells and phenotype switching in co-cultured HASMCs.(A) QRT-PCR analysis of pro-inflammatory gene expression in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). The upper and lower panels show different levels of LINC00305 overexpression. Gene expression levels in the control groups were assigned a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. (B,C) GO analysis and heat map of the microarray data demonstrated upregulation of inflammation-associated genes in LINC00305-overexpressing THP-1 cells. The gene expression profiles of THP-1 cells stably expressing LINC00305 and the control cells were examined using microarray analysis (Affymetrix, Human Exon 1.0 ST array). Genes upregulated in cells overexpressing LINC00305 were analysed by GO enrichment analysis (B) and the genes involved in inflammation, monocyte activation and chemotaxis (GO:002544, 002548, 0042177, 0090025) were analyzed with MeV software to show the heat map of the inflammation-relevant genes in the microarray assay (C). (D) QRT-PCR analysis of contractile markers in untreated HASMCs (blank) and in HASMCs co-cultured with wild type THP-1 cells, THP-1 cells stably expressing LINC00305 (Linc00305 oe THP-1) or the control vector (Control THP-1). The relative mRNA expression levels were normalized to the GAPDH internal control. The genes expression levels in the Blank group were assigned a value of 1.0. Data are presented as the mean ± sem of 3 replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group.
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f3: LINC00305 promotes inflammation in THP-1 cells and phenotype switching in co-cultured HASMCs.(A) QRT-PCR analysis of pro-inflammatory gene expression in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). The upper and lower panels show different levels of LINC00305 overexpression. Gene expression levels in the control groups were assigned a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. (B,C) GO analysis and heat map of the microarray data demonstrated upregulation of inflammation-associated genes in LINC00305-overexpressing THP-1 cells. The gene expression profiles of THP-1 cells stably expressing LINC00305 and the control cells were examined using microarray analysis (Affymetrix, Human Exon 1.0 ST array). Genes upregulated in cells overexpressing LINC00305 were analysed by GO enrichment analysis (B) and the genes involved in inflammation, monocyte activation and chemotaxis (GO:002544, 002548, 0042177, 0090025) were analyzed with MeV software to show the heat map of the inflammation-relevant genes in the microarray assay (C). (D) QRT-PCR analysis of contractile markers in untreated HASMCs (blank) and in HASMCs co-cultured with wild type THP-1 cells, THP-1 cells stably expressing LINC00305 (Linc00305 oe THP-1) or the control vector (Control THP-1). The relative mRNA expression levels were normalized to the GAPDH internal control. The genes expression levels in the Blank group were assigned a value of 1.0. Data are presented as the mean ± sem of 3 replicate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the indicated group.
Mentions: Monocyte-mediated inflammation plays an important role in atherogenesis8. To investigate the function of LINC00305, THP-1 cells were infected with the LINC00305-expressing or the empty control lentiviruses at two different levels. Both levels of LINC00305 overexpression significantly enhanced the expression of inflammatory genes in THP-1 cells, and the stimulative effect is concentration-dependent (Fig. 3A). However, the expression of SERPINBs, genes located adjacent to the LINC00305 locus were unaffected, indicating that LINC00305 functions in a trans-regulatory fashion (Fig. S4). To further study the downstream effects of LINC00305, the transcriptional profile of LINC00305- and empty vector-transfected THP-1 cells were analysed using chip assays (Affymetrix, Human Exon 1.0 ST array). Gene Ontology (GO) analysis demonstrated that LINC00305-upregulated genes are enriched for inflammation-associated genes, and similar result was observed in the heat map of inflammation-associated genes in the microarray data, indicating that LINC00305 promotes inflammation in THP-1 cells (Fig. 3B,C). In atherosclerotic plaques, the cytokines secreted by inflammatory cells promote the shift of VSMCs from a contractile to a synthetic phenotype, an event that contributes to the development of plaques3233. To investigate the functional significance of LINC00305 in atherosclerosis, HASMCs were co-cultured with wild type THP-1, THP-1 cells stably expressing LINC00305 or the control empty vector. QRT-PCR analysis revealed that co-culture with both wild type and control transfected THP-1 cells decreased the expression of genes associated with the contractile phenotype in HASMCs, and THP-1 cells stably expressing LINC00305 further down-regulated the expression of these genes (Fig. 3D), suggesting that LINC00305 overexpression promotes the switch of co-cultured HASMCs from a contractile phenotype to a synthetic phenotype. Together, these results support the hypothesis that LINC00305 expression plays an important role in the progression of atherosclerosis.

View Article: PubMed Central - PubMed

ABSTRACT

Accumulating data from genome-wide association studies (GWAS) have provided a collection of novel candidate genes associated with complex diseases, such as atherosclerosis. We identified an atherosclerosis-associated single-nucleotide polymorphism (SNP) located in the intron of the long noncoding RNA (lncRNA) LINC00305 by searching the GWAS database. Although the function of LINC00305 is unknown, we found that LINC00305 expression is enriched in atherosclerotic plaques and monocytes. Overexpression of LINC00305 promoted the expression of inflammation-associated genes in THP-1 cells and reduced the expression of contractile markers in co-cultured human aortic smooth muscle cells (HASMCs). We showed that overexpression of LINC00305 activated nuclear factor-kappa beta (NF-&kappa;B) and that inhibition of NF-&kappa;B abolished LINC00305-mediated activation of cytokine expression. Mechanistically, LINC00305 interacted with lipocalin-1 interacting membrane receptor (LIMR), enhanced the interaction of LIMR and aryl-hydrocarbon receptor repressor (AHRR), and promoted protein expression as well as nuclear localization of AHRR. Moreover, LINC00305 activated NF-&kappa;B exclusively in the presence of LIMR and AHRR. In light of these findings, we propose that LINC00305 promotes monocyte inflammation by facilitating LIMR and AHRR cooperation and the AHRR activation, which eventually activates NF-&kappa;B, thereby inducing HASMC phenotype switching.

No MeSH data available.


Related in: MedlinePlus