Limits...
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 AHRR protein expression and nuclear localization to activate NF-κB.(A) Reporter assay of NF-κB activity in 293 T cells transfected with different combinations of LINC00305, LIMR and AHRR. 293 T cells were transfected with LINC00305, with or without LIMR- and AHRR-expressing vectors, and the pNF-κB-TA-luc and pRL-TK reporters. The luciferase activity (firefly/Renilla) was measured using a Modulus Microplate Multimode Reader. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, ns: no significance vs. the indicated group. (B) Real-time RT-PCR analysis of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. 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. *p < 0.05, ns: no significance vs. the indicated group. (C) Western blotting assay of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. β-Actin and GAPDH were used as the internal controls. (D,E) Immunofluorescence assays of AHRR (D) and AHR (E) localization in THP-1 cells stably expressing LINC00305 or the control vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385552&req=5

f6: LINC00305 promotes AHRR protein expression and nuclear localization to activate NF-κB.(A) Reporter assay of NF-κB activity in 293 T cells transfected with different combinations of LINC00305, LIMR and AHRR. 293 T cells were transfected with LINC00305, with or without LIMR- and AHRR-expressing vectors, and the pNF-κB-TA-luc and pRL-TK reporters. The luciferase activity (firefly/Renilla) was measured using a Modulus Microplate Multimode Reader. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, ns: no significance vs. the indicated group. (B) Real-time RT-PCR analysis of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. 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. *p < 0.05, ns: no significance vs. the indicated group. (C) Western blotting assay of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. β-Actin and GAPDH were used as the internal controls. (D,E) Immunofluorescence assays of AHRR (D) and AHR (E) localization in THP-1 cells stably expressing LINC00305 or the control vector.

Mentions: AHRR represses aryl hydrocarbon receptor (Ahr) and Ahr signalling by competitively binding the AHR nuclear translocator (ARNT)37. Ahr interacts and cooperates with NF-κB in inflammation regulation, and mainly exhibits an inflammation-suppressive role383940. To evaluate the potential pro-inflammatory role of AHRR, reporter assays were carried out in 293 T cells, and demonstrated that co-transfection of LIMR and AHRR markedly activated NF-κB. Although LINC00305 alone does not significantly influence NF-κB activity, it significantly activates NF-κB in the presence of LIMR and AHRR (Fig. 6A).


Long noncoding RNA LINC00305 promotes inflammation by activating the AHRR-NF- κ B pathway in human monocytes
LINC00305 promotes AHRR protein expression and nuclear localization to activate NF-κB.(A) Reporter assay of NF-κB activity in 293 T cells transfected with different combinations of LINC00305, LIMR and AHRR. 293 T cells were transfected with LINC00305, with or without LIMR- and AHRR-expressing vectors, and the pNF-κB-TA-luc and pRL-TK reporters. The luciferase activity (firefly/Renilla) was measured using a Modulus Microplate Multimode Reader. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, ns: no significance vs. the indicated group. (B) Real-time RT-PCR analysis of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. 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. *p < 0.05, ns: no significance vs. the indicated group. (C) Western blotting assay of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. β-Actin and GAPDH were used as the internal controls. (D,E) Immunofluorescence assays of AHRR (D) and AHR (E) localization in THP-1 cells stably expressing LINC00305 or the control vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: LINC00305 promotes AHRR protein expression and nuclear localization to activate NF-κB.(A) Reporter assay of NF-κB activity in 293 T cells transfected with different combinations of LINC00305, LIMR and AHRR. 293 T cells were transfected with LINC00305, with or without LIMR- and AHRR-expressing vectors, and the pNF-κB-TA-luc and pRL-TK reporters. The luciferase activity (firefly/Renilla) was measured using a Modulus Microplate Multimode Reader. Data are presented as the mean ± sem of 3 independent experiments. *p < 0.05, ns: no significance vs. the indicated group. (B) Real-time RT-PCR analysis of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. 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. *p < 0.05, ns: no significance vs. the indicated group. (C) Western blotting assay of AHR and AHRR expression in THP-1 cells stably expressing LINC00305 or the control vector. β-Actin and GAPDH were used as the internal controls. (D,E) Immunofluorescence assays of AHRR (D) and AHR (E) localization in THP-1 cells stably expressing LINC00305 or the control vector.
Mentions: AHRR represses aryl hydrocarbon receptor (Ahr) and Ahr signalling by competitively binding the AHR nuclear translocator (ARNT)37. Ahr interacts and cooperates with NF-κB in inflammation regulation, and mainly exhibits an inflammation-suppressive role383940. To evaluate the potential pro-inflammatory role of AHRR, reporter assays were carried out in 293 T cells, and demonstrated that co-transfection of LIMR and AHRR markedly activated NF-κB. Although LINC00305 alone does not significantly influence NF-κB activity, it significantly activates NF-κB in the presence of LIMR and AHRR (Fig. 6A).

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.