Neural Regeneration Research ›› 2024, Vol. 19 ›› Issue (11): 2488-2498.doi: 10.4103/NRR.NRR-D-23-01355

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Atherosis-associated lnc_000048 activates PKR to enhance STAT1-mediated polarization of THP-1 macrophages to M1 phenotype

Yuanyuan Ding1, #, Yu Sun1, #, Hongyan Wang2, Hongqin Zhao1, Ruihua Yin1, Meng Zhang1, *, Xudong Pan1, *, Xiaoyan Zhu3, *   

  1. 1Department of Neurology, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China; 2Qingdao Cadre Health Care Service Center, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China; 3Department of Critical Care Medicine, The Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, China
  • Online:2024-11-15 Published:2024-03-29
  • Contact: Xiaoyan Zhu, PhD, zxysdjm@qdu.edu.cn; Xudong Pan, PhD, drpan022@qdu.edu.cn; Meng Zhang, MD, 2021010088@qdu.edu.cn.
  • Supported by:
    This work was supported by the Natural Science Foundation of Shandong Province, No. ZR2020MH138 (to XZ).

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Abstract: 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.

Key words: atherosclerosis, inflammation, lnc_000048, lncRNA, macrophage, polarization, protein kinase RNA-activated (PKR), STAT1