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Role of lncRNA-SPANXA2-OT1 in macrophage chemotaxis

ABSTRACT: Coronary artery disease (CAD) remains the leading cause of mortality worldwide. Macrophages play a crucial role in recruiting immune cells and regulating the inflammatory milieu through the release of a diverse array of cytokines, chemokines, and other immune mediators in CAD. Long noncoding RNAs (lncRNAs) interact with DNA, RNA, miRNA, and proteins, making them attractive therapeutic targets for regulating gene expression. Methods and Results: Gene-expression meta-analysis and weighted gene co-expression network analysis (WGCNA) of human CAD datasets identified 26 lncRNA-mRNA co-expression modules. Network prioritization of top co-expression modules identified SPANXA2-OT1 as a potential key candidate. Conservation analysis revealed that SPANXA2-OT1 is human specific and conserved only in primates. We validated the candidate coding-noncoding RNA regulatory triad in human primary macrophages derived from healthy human peripheral blood mononuclear cells (PBMCs). IL-1β induced the expression of SPANXA2-OT1. RNA in situ hybridization localized SPANXA2-OT1 mRNA in cytoplasm of macrophages. Loss-of-function experiments using antisense oligonucleotide (ASO) against SPANXA2-OT1 demonstrated decreased monocyte/macrophage chemotaxis signature after SPANXA2-OT1 silencing, as demonstrated by unbiased global proteomics and RNAseq data. Luciferase assay established that SPANXA2-OT1 binds to miR-338 through its miRNA response elements. Gain-of-function (miR-338 mimic) and loss-of-function (SPANXA2-OT1 ASO) experiments revealed that SPANXA2-OT1-miR-338 axis regulates the expression of monocyte chemotactic genes (e.g., IL-8) that may contribute to the pathophysiology of CAD. CRISPR/Cas9 mediated deletion of the SPANXA2-OT1 functional domain (exon 3, which harbors the miR-338 binding site) in human primary macrophages resulted in decreased IL-8 expression, alteration of the chemokine profile, and decreased macrophage chemotaxis. Conclusion: Our results indicate that the lncRNA SPANXA2-OT1 regulates chemokine signatures and macrophage chemotaxis. One such mechanism involves SPANXA2-OT1 binding to miR-338, making it unavailable to regulate IL-8 expression. Our findings may provide a molecular basis for the future identification of novel biomarkers and therapeutic targets for CAD.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Primary Cell, Cell Culture, Macrophage

SUBMITTER: Prabhash Jha

LAB HEAD: Masanori Aikawa

PROVIDER: PXD059474 | Pride | 2025-09-10

REPOSITORIES: Pride

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			Action	DRS
	12_PKJ_SPANXA2-OT1_project_hmac.mzML	Mzml
	12_PKJ_SPANXA2-OT1_project_hmac.mzid.gz	Mzid
	12_PKJ_SPANXA2-OT1_project_hmac.pdResult	Other
	12_PKJ_SPANXA2-OT1_project_hmac_Control_1_donor_1.raw	Raw
	12_PKJ_SPANXA2-OT1_project_hmac_Control_2_donor_2.raw	Raw

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Project description:A favourable immunotherapeutic strategy in the treatment of chronic inflammatory disease, is to dampen the pro-inflammatory macrophage response and upregulate the anti-inflammatory macrophage response. Previous studies have shown that Arginase-2 (Arg2), a mitochondrial enzyme, is a key regulator of the macrophage anti-inflammatory response with an essential role in IL-10 signalling. Here we show that IL-10 in the presence of LPS increased Arg2 expression in human macrophages and peripheral blood mononuclear cells. Target site blockers (TSBs) (locked nucleic acid antisense oligonucleotides) specifically designed to target the 3’ UTR of Arg2 messenger RNA were used to inhibit binding of specific microRNAs thus enhancing Arg2 expression. Eleven Arg2-TSBs were screened and the TSB targeting miR-155 (TSB-155) and the TSB targeting miR-3202 (TSB-3202) were found to increase Arg2 expression in human macrophages resulting in decreased gene expression and cytokine production of TNF-α and CCL2. In addition, following TSB-3202 stimulation, there were statistically significant increases in the ‘M2-like’ macrophage marker, CD206; and CD16, associated with an IL-10 response, and a decrease in the ‘M1-like’ macrophage marker, HLA-DR, indicating a shift in the macrophage phenotype. Proteomic analysis demonstrated that multiple pro-inflammatory responsive proteins including Signal Transducer and Activator of Transcription 1 (STAT-1), Toll-like receptors, Signalling Lymphocytic Activation Molecule F7 (SLAMF7) and Interferon Induced Protein with Tetratricopeptide Repeats 3 (IFIT3), were downregulated by TSB-155 and TSB-3202 while Sequestomsome-1 (SQSTM1) was increased after treatment. In silico analysis of significantly dysregulated proteins predicted that TSB-3202 supressed several upstream pro-inflammatory regulators including STAT-1 and Interferon α2 (IFNA2) while enhancing anti-inflammatory associated interleukin 1 receptor antagonist (IL1RN) and STAT-3. Proteomic data was validated by confirming increased levels of SQSTM1, decreased levels of phosphoylated-STAT-1 and STAT-1, and downregulation of Ifit3 and Slamf7 by TSB treatment. In conclusion, upregulation of Arg2 protein by TSBs inhibits several pro-inflammatory signalling proteins resulting in reduced pro-inflammatory cytokine secretion, reduced ‘M1-like’ marker expression and enhanced ‘M2-like’ macrophage marker expression. Arg2 is a promising novel therapeutic target to modulate inflammatory signalling in macrophages.

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Role of lncRNA-SPANXA2-OT1 in macrophage chemotaxis

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