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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.


LINC00305 promotes inflammation by activating NF-κB in THP-1 cells.(A) Western blot analysis of the key proteins in the NF-κB pathway (IKKβ, phosphorylated IKKβ, P65, phosphorylated P65 and P50) in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). GAPDH was used as an internal control. (B) QRT-PCR analysis of the indicated cytokine genes in THP-1 cells stably expressing the control vector (THP-1 lenti-eGFP) or LINC00305 (THP-1 lenti-linc00305 oe) treated with DMSO or 10 μM BAY 11-7082 for 30 min. The gene expression levels in the DMSO-treated control group (THP-1 lenti-eGFP) were designated a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, **p < 0.01 vs. the indicated group.
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f4: LINC00305 promotes inflammation by activating NF-κB in THP-1 cells.(A) Western blot analysis of the key proteins in the NF-κB pathway (IKKβ, phosphorylated IKKβ, P65, phosphorylated P65 and P50) in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). GAPDH was used as an internal control. (B) QRT-PCR analysis of the indicated cytokine genes in THP-1 cells stably expressing the control vector (THP-1 lenti-eGFP) or LINC00305 (THP-1 lenti-linc00305 oe) treated with DMSO or 10 μM BAY 11-7082 for 30 min. The gene expression levels in the DMSO-treated control group (THP-1 lenti-eGFP) were designated a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, **p < 0.01 vs. the indicated group.

Mentions: The NF-κB pathway is essential to the regulation of inflammation910. To investigate the mechanism by which LINC00305 promotes inflammation in THP-1 cells, we examined the effect of LINC00305 on the NF-κB pathway. Western blot analysis revealed an increase in IKKβ phosphorylation levels and a significant enhancement of protein levels and phosphorylation of the downstream protein P65 in THP-1 cells stably expressing LINC00305 (Fig. 4A). Furthermore, immunofluorescence assays demonstrated that P65 translocated to the nucleus (Fig. S5A) and P65 binding to the promoters of the upregulated cytokine genes was shown markedly enhanced in LINC00305-overexpressing THP-1 cells (Fig. S5B). Treatment with BAY 11-7082, an inhibitor of NF-κB, abolished the upregulation of cytokine genes observed in THP-1 cells stably expressing LINC00305 (Fig. 4B). These results demonstrate that LINC00305 promotes inflammation by activating NF-κB.


Long noncoding RNA LINC00305 promotes inflammation by activating the AHRR-NF- κ B pathway in human monocytes
LINC00305 promotes inflammation by activating NF-κB in THP-1 cells.(A) Western blot analysis of the key proteins in the NF-κB pathway (IKKβ, phosphorylated IKKβ, P65, phosphorylated P65 and P50) in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). GAPDH was used as an internal control. (B) QRT-PCR analysis of the indicated cytokine genes in THP-1 cells stably expressing the control vector (THP-1 lenti-eGFP) or LINC00305 (THP-1 lenti-linc00305 oe) treated with DMSO or 10 μM BAY 11-7082 for 30 min. The gene expression levels in the DMSO-treated control group (THP-1 lenti-eGFP) were designated a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, **p < 0.01 vs. the indicated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5385552&req=5

f4: LINC00305 promotes inflammation by activating NF-κB in THP-1 cells.(A) Western blot analysis of the key proteins in the NF-κB pathway (IKKβ, phosphorylated IKKβ, P65, phosphorylated P65 and P50) in THP-1 cells stably expressing LINC00305 (OE) or the control vector (Control). GAPDH was used as an internal control. (B) QRT-PCR analysis of the indicated cytokine genes in THP-1 cells stably expressing the control vector (THP-1 lenti-eGFP) or LINC00305 (THP-1 lenti-linc00305 oe) treated with DMSO or 10 μM BAY 11-7082 for 30 min. The gene expression levels in the DMSO-treated control group (THP-1 lenti-eGFP) were designated a value of 1.0, and GAPDH was used as an internal control. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, **p < 0.01 vs. the indicated group.
Mentions: The NF-κB pathway is essential to the regulation of inflammation910. To investigate the mechanism by which LINC00305 promotes inflammation in THP-1 cells, we examined the effect of LINC00305 on the NF-κB pathway. Western blot analysis revealed an increase in IKKβ phosphorylation levels and a significant enhancement of protein levels and phosphorylation of the downstream protein P65 in THP-1 cells stably expressing LINC00305 (Fig. 4A). Furthermore, immunofluorescence assays demonstrated that P65 translocated to the nucleus (Fig. S5A) and P65 binding to the promoters of the upregulated cytokine genes was shown markedly enhanced in LINC00305-overexpressing THP-1 cells (Fig. S5B). Treatment with BAY 11-7082, an inhibitor of NF-κB, abolished the upregulation of cytokine genes observed in THP-1 cells stably expressing LINC00305 (Fig. 4B). These results demonstrate that LINC00305 promotes inflammation by activating NF-κB.

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.