NSUN6 and HTR7 disturbed the stability of carotid atherosclerotic plaques by regulating the immune responses of macrophages

Tingyu Jin; Han Gao; Danyang Meng; Man Luo; Jin Hu

doi:10.1515/med-2024-1072

NSUN6 and HTR7 disturbed the stability of carotid atherosclerotic plaques by regulating the immune responses of macrophages

Open Med (Wars). 2024 Oct 24;19(1):20241072. doi: 10.1515/med-2024-1072. eCollection 2024.

Authors

Tingyu Jin¹, Han Gao¹, Danyang Meng¹, Man Luo¹, Jin Hu²

Affiliations

¹ Department of Neurology, Affiliated Hospital of Jiaxing University, The First Hospital of Jiaxing, Jiaxing, Zhejiang, China.
² Department of Neurology, Affiliated Hospital of Jiaxing University, The First Hospital of Jiaxing, 1882 Zhonghuan South Road, Chengnan Street, Jiaxing, Zhejiang, China.

Abstract

Background: Ischemic stroke associated with atherosclerosis is globally named atherothrombotic stroke. Presently, the underlying pathogenic genes promoting carotid atherosclerotic plaques transfer from a stable to unstable state remains elusive. This study aims to find the hub genes disturbing the stability of plaques and explore the primary cells affected by these hub genes.

Methods: The optimal hub genes from five datasets for unstable plaques were identified by overlapping genes derived from Boruta and LASSO algorithms. The hub genes' expression levels in stroke patients were confirmed through RT-qPCR. Visualization of the hub genes' expression across various cell clusters was achieved with the aid of the Seurat R package. Then, hub genes were overexpressed or knock-down by lentivirus and siRNA, respectively. The inflammatory factors in the culture medium were detected using an ELISA assay.

Results: Eight genes (APOD, ASXL1, COL4A5, HTR7, INF2, NSUN6, PDSS2, and RBBP7) were identified and confirmed by RT-qPCR. The prognostic model was built upon this eight-gene composite foundation, and the area under the curve was 0.98. Based on CIBERSORT findings, unstable plaques displayed a higher macrophage proportion compared to stable ones (P < 0.05). These eight genes also correlated with infiltrated immune cells, especially macrophages. Then, according to single-cell RNA-seq analysis, we found that the eight hub genes mainly expressed in macrophages. The cellular localization of two hub genes (NSUN6 and HTR7) with high distinguishability was confirmed, and gene set enrichment analysis also clarified the possible biological pathways regulated by them. The findings from the in vitro investigation revealed that TNF-α and IL-6 were reduced in macrophages with NSUN6 overexpression or HTR7 knockdown.

Conclusion: Eight hub genes, especially NSUN6 and HTR7, were found to promote the progression of plaques by regulating the immune responses of macrophages.

Keywords: bioinformatics analysis; carotid atherosclerotic plaques; cellular localization; immune infiltration; macrophages.