Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype
Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE–/– mice. However, little is known about the role of lnc_000048 in classically activated macrophage (M1) polarization. In this study, we established THP-1-der...
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| Format: | Article |
| Language: | English |
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Wolters Kluwer Medknow Publications
2024-01-01
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| Series: | Neural Regeneration Research |
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| Online Access: | http://www.nrronline.org/article.asp?issn=1673-5374;year=2024;volume=19;issue=11;spage=2488;epage=2498;aulast= |
| _version_ | 1797246011820736512 |
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| author | Yuanyuan Ding Yu Sun Hongyan Wang Hongqin Zhao Ruihua Yin Meng Zhang Xudong Pan Xiaoyan Zhu |
| author_facet | Yuanyuan Ding Yu Sun Hongyan Wang Hongqin Zhao Ruihua Yin Meng Zhang Xudong Pan Xiaoyan Zhu |
| author_sort | Yuanyuan Ding |
| collection | DOAJ |
| description | Our previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE–/– mice. However, little is known about the role of lnc_000048 in classically activated macrophage (M1) polarization. In this study, we established THP-1-derived testing state macrophages (M0), M1 macrophages, and alternately activated macrophages (M2). Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages. Flow cytometry was used to detect phenotypic proteins (CD11b, CD38, CD80). We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048. Flow cytometry, western blot, and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response, while over-expression of lnc_000048 led to the opposite effect. Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization. Moreover, catRAPID prediction, RNA-pull down, and mass spectrometry were used to identify and screen the protein kinase RNA-activated (PKR), then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR. Immunofluorescence (IF)-RNA fluorescence in situ hybridization (FISH) double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage. We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation, leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression. Taken together, these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke. |
| first_indexed | 2024-04-24T19:36:01Z |
| format | Article |
| id | doaj.art-1235e0beeb4b494f93c3527919f5edac |
| institution | Directory Open Access Journal |
| issn | 1673-5374 |
| language | English |
| last_indexed | 2024-04-24T19:36:01Z |
| publishDate | 2024-01-01 |
| publisher | Wolters Kluwer Medknow Publications |
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| series | Neural Regeneration Research |
| spelling | doaj.art-1235e0beeb4b494f93c3527919f5edac2024-03-25T13:02:29ZengWolters Kluwer Medknow PublicationsNeural Regeneration Research1673-53742024-01-0119112488249810.4103/NRR.NRR-D-23-01355Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotypeYuanyuan DingYu SunHongyan WangHongqin ZhaoRuihua YinMeng ZhangXudong PanXiaoyan ZhuOur previous study has demonstrated that lnc_000048 is upregulated in large-artery atherosclerotic stroke and promotes atherosclerosis in ApoE–/– mice. However, little is known about the role of lnc_000048 in classically activated macrophage (M1) polarization. In this study, we established THP-1-derived testing state macrophages (M0), M1 macrophages, and alternately activated macrophages (M2). Real-time fluorescence quantitative PCR was used to verify the expression of marker genes and the expression of lnc_000048 in macrophages. Flow cytometry was used to detect phenotypic proteins (CD11b, CD38, CD80). We generated cell lines with lentivirus-mediated upregulation or downregulation of lnc_000048. Flow cytometry, western blot, and real-time fluorescence quantitative PCR results showed that down-regulation of lnc_000048 reduced M1 macrophage polarization and the inflammation response, while over-expression of lnc_000048 led to the opposite effect. Western blot results indicated that lnc_000048 enhanced the activation of the STAT1 pathway and mediated the M1 macrophage polarization. Moreover, catRAPID prediction, RNA-pull down, and mass spectrometry were used to identify and screen the protein kinase RNA-activated (PKR), then catRAPID and RPIseq were used to predict the binding ability of lnc_000048 to PKR. Immunofluorescence (IF)-RNA fluorescence in situ hybridization (FISH) double labeling was performed to verify the subcellular colocalization of lnc_000048 and PKR in the cytoplasm of M1 macrophage. We speculate that lnc_000048 may form stem-loop structure-specific binding and activate PKR by inducing its phosphorylation, leading to activation of STAT1 phosphorylation and thereby enhancing STAT1 pathway-mediated polarization of THP-1 macrophages to M1 and inflammatory factor expression. Taken together, these results reveal that the lnc_000048/PKR/STAT1 axis plays a crucial role in the polarization of M1 macrophages and may be a novel therapeutic target for atherosclerosis alleviation in stroke.http://www.nrronline.org/article.asp?issn=1673-5374;year=2024;volume=19;issue=11;spage=2488;epage=2498;aulast=atherosclerosis; inflammation; lnc_000048; lncrna; macrophage; polarization; protein kinase rna-activated (pkr); stat1 |
| spellingShingle | Yuanyuan Ding Yu Sun Hongyan Wang Hongqin Zhao Ruihua Yin Meng Zhang Xudong Pan Xiaoyan Zhu Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype Neural Regeneration Research atherosclerosis; inflammation; lnc_000048; lncrna; macrophage; polarization; protein kinase rna-activated (pkr); stat1 |
| title | Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype |
| title_full | Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype |
| title_fullStr | Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype |
| title_full_unstemmed | Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype |
| title_short | Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype |
| title_sort | atherosis associated lnc 000048 activates pkr to enhance stat1 mediated polarization of thp 1 macrophages to m1 phenotype |
| topic | atherosclerosis; inflammation; lnc_000048; lncrna; macrophage; polarization; protein kinase rna-activated (pkr); stat1 |
| url | http://www.nrronline.org/article.asp?issn=1673-5374;year=2024;volume=19;issue=11;spage=2488;epage=2498;aulast= |
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