Neural Regeneration Research (Jan 2024)

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

  • Yuanyuan Ding,
  • Yu Sun,
  • Hongyan Wang,
  • Hongqin Zhao,
  • Ruihua Yin,
  • Meng Zhang,
  • Xudong Pan,
  • Xiaoyan Zhu

DOI
https://doi.org/10.4103/NRR.NRR-D-23-01355
Journal volume & issue
Vol. 19, no. 11
pp. 2488 – 2498

Abstract

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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.

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