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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 associates with LIMR and AHRR.(A) RNA-pull-down assay to identify LINC00305 binding proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and subjected to silver staining (multiple exposures are presented in Supplementary Figure 6). Antisense RNA to LINC00305 (AS) was used as a negative control. The black arrow indicates the band representing the LINC00305-specific binding protein identified by mass spectrometry as LIMR. (B) RIP assay in HeLa cells transfected with LINC00305 and HA-tagged LIMR. RNA was immunoprecipitated using normal rabbit IgG or the anti-HA antibody. GAPDH was used as the negative control. **p < 0.01, ns: no significance vs. the indicated group. (C) GST pull-down assay to identify LIMR-interacting proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and visualized using Coomassie Brilliant Blue Staining. The black arrow indicates the band representing the LIMR-specific interacting protein identified by mass spectrometry as AHRR. (D) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR. Proteins were immunoprecipitated using normal rabbit IgG or anti-His antibody. Input samples and the precipitated proteins were then analysed using anti-His and anti-LIMR antibodies (full length blots are presented in Supplementary Figure 9). (E) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR with or without LINC00305 (full length blots are presented in Supplementary Figure 10).
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f5: LINC00305 associates with LIMR and AHRR.(A) RNA-pull-down assay to identify LINC00305 binding proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and subjected to silver staining (multiple exposures are presented in Supplementary Figure 6). Antisense RNA to LINC00305 (AS) was used as a negative control. The black arrow indicates the band representing the LINC00305-specific binding protein identified by mass spectrometry as LIMR. (B) RIP assay in HeLa cells transfected with LINC00305 and HA-tagged LIMR. RNA was immunoprecipitated using normal rabbit IgG or the anti-HA antibody. GAPDH was used as the negative control. **p < 0.01, ns: no significance vs. the indicated group. (C) GST pull-down assay to identify LIMR-interacting proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and visualized using Coomassie Brilliant Blue Staining. The black arrow indicates the band representing the LIMR-specific interacting protein identified by mass spectrometry as AHRR. (D) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR. Proteins were immunoprecipitated using normal rabbit IgG or anti-His antibody. Input samples and the precipitated proteins were then analysed using anti-His and anti-LIMR antibodies (full length blots are presented in Supplementary Figure 9). (E) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR with or without LINC00305 (full length blots are presented in Supplementary Figure 10).

Mentions: LncRNAs generally coordinate with protein partners to exert their specific function. To investigate the mechanism by which LINC00305 activates NF-κB, we sought to identify proteins that directly bind to LINC00305 using an RNA pull-down assay. The LINC00305 antisense RNA was used as the negative control. Mass spectrometry analysis of the pull-down results revealed that a band that specifically associated with the sense LINC00305 RNA represented lipocalin-interacting membrane receptor (LIMR) (Fig. 5A, Fig. S6, Fig. S7A), a 9-pass transmembrane protein that mediates the endocytosis of lipocalin-1 (LCN1)343536. A RIP assay was subsequently performed in Hela cells expressing HA-tagged LIMR using the anti-HA antibody, and the results confirmed that LIMR binds to LINC00305 in vivo (Fig. 5B). Moreover, the results of RNA-FISH and immunofluorescence assays demonstrated that LINC00305 and LIMR co-localize in THP-1 cells (Fig. S8).


Long noncoding RNA LINC00305 promotes inflammation by activating the AHRR-NF- κ B pathway in human monocytes
LINC00305 associates with LIMR and AHRR.(A) RNA-pull-down assay to identify LINC00305 binding proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and subjected to silver staining (multiple exposures are presented in Supplementary Figure 6). Antisense RNA to LINC00305 (AS) was used as a negative control. The black arrow indicates the band representing the LINC00305-specific binding protein identified by mass spectrometry as LIMR. (B) RIP assay in HeLa cells transfected with LINC00305 and HA-tagged LIMR. RNA was immunoprecipitated using normal rabbit IgG or the anti-HA antibody. GAPDH was used as the negative control. **p < 0.01, ns: no significance vs. the indicated group. (C) GST pull-down assay to identify LIMR-interacting proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and visualized using Coomassie Brilliant Blue Staining. The black arrow indicates the band representing the LIMR-specific interacting protein identified by mass spectrometry as AHRR. (D) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR. Proteins were immunoprecipitated using normal rabbit IgG or anti-His antibody. Input samples and the precipitated proteins were then analysed using anti-His and anti-LIMR antibodies (full length blots are presented in Supplementary Figure 9). (E) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR with or without LINC00305 (full length blots are presented in Supplementary Figure 10).
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Related In: Results  -  Collection

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f5: LINC00305 associates with LIMR and AHRR.(A) RNA-pull-down assay to identify LINC00305 binding proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and subjected to silver staining (multiple exposures are presented in Supplementary Figure 6). Antisense RNA to LINC00305 (AS) was used as a negative control. The black arrow indicates the band representing the LINC00305-specific binding protein identified by mass spectrometry as LIMR. (B) RIP assay in HeLa cells transfected with LINC00305 and HA-tagged LIMR. RNA was immunoprecipitated using normal rabbit IgG or the anti-HA antibody. GAPDH was used as the negative control. **p < 0.01, ns: no significance vs. the indicated group. (C) GST pull-down assay to identify LIMR-interacting proteins in THP-1 cells. The eluted proteins were separated by SDS-PAGE and visualized using Coomassie Brilliant Blue Staining. The black arrow indicates the band representing the LIMR-specific interacting protein identified by mass spectrometry as AHRR. (D) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR. Proteins were immunoprecipitated using normal rabbit IgG or anti-His antibody. Input samples and the precipitated proteins were then analysed using anti-His and anti-LIMR antibodies (full length blots are presented in Supplementary Figure 9). (E) Co-IP assay in 293 T cells transfected with LIMR and His-tagged AHRR with or without LINC00305 (full length blots are presented in Supplementary Figure 10).
Mentions: LncRNAs generally coordinate with protein partners to exert their specific function. To investigate the mechanism by which LINC00305 activates NF-κB, we sought to identify proteins that directly bind to LINC00305 using an RNA pull-down assay. The LINC00305 antisense RNA was used as the negative control. Mass spectrometry analysis of the pull-down results revealed that a band that specifically associated with the sense LINC00305 RNA represented lipocalin-interacting membrane receptor (LIMR) (Fig. 5A, Fig. S6, Fig. S7A), a 9-pass transmembrane protein that mediates the endocytosis of lipocalin-1 (LCN1)343536. A RIP assay was subsequently performed in Hela cells expressing HA-tagged LIMR using the anti-HA antibody, and the results confirmed that LIMR binds to LINC00305 in vivo (Fig. 5B). Moreover, the results of RNA-FISH and immunofluorescence assays demonstrated that LINC00305 and LIMR co-localize in THP-1 cells (Fig. S8).

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