关键词: 刚地弓形虫, 瞬时受体电位M8型通道蛋白, 巨噬细胞, N⁶-甲基腺嘌呤

Abstract:

Objective To investigate the role of N⁶-methyladenosine (m⁶A) modification in regulation of transient receptor potential cation channel subfamily M member 8 (TRPM8) protein in macrophages following Toxoplasma gondii infection and unravel its underlying mechanisms. Methdos Transcriptome sequencing (RNA-seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) data were sourced from the NCBI Gene Expression Omnibus (GEO) dataset GSE288205. Transient receptor potential (TRP) channel protein family-coding genes were subjected to bioinformatics analyses with the OmicStudio platform, and volcano plots for m⁶A modifications of TRP channel-associated factor 1 (TCAF1) and TRPM8 mRNA were generated based on MeRIP-seq data using ggplot2 in the RStudio package. THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs) and mouse RAW264.7 cells were seeded onto 6-well plates at a density of 1 × 10⁶ cells per well. Cells infected with T. gondii RH strain tachyzoites at a cell-to-parasite ratio of 1∶1 served as the infection group, while cells in the control group received an equal volume of parasite-free medium. Total RNA was extracted from cells using the TRIzol reagent, reversely transcribed into cDNA. The relative mRNA expression of TRPM8 (human) and Trpm8 (mouse) was quantified in cells using quantitative fluorescent real-time PCR (qPCR) assay, and the relative TCAF1 and TCAF2 mRNA levels were detected in THP-1-derived macrophages with and without infected with T. gondii tachyzoites infections using qPCR assay. Intracellular Ca²⁺ influx was detected in cells using flow cytometry. THP-1-derived macrophages, fat mass and obesity-associated protein (FTO)-knockdown THP-1-derived macrophages (shFTO-THP-1), and THP-1-derived macrophages carrying non-targeted control shRNA (shNC-THP-1) were seeded onto 6-well plates at a density of 1 × 10⁶ cells per well. Cells in the infection group were infected with T. gondii RH strain tachyzoites at a cell-to-parasite ratio of 1∶1, while cells in the control group received an equal volume of parasite-free medium. Changes in m⁶A modification on TCAF1 mRNA in THP-1-derived macrophages were validated using m⁶A methylated RNA immunoprecipitation-real time quantitative PCR (m⁶A-IP-qPCR) assay. The expression of TCAF1 mRNA binding the m⁶A recognition protein YTH domain family protein 2 (YTHDF2) was detected in THP-1-derived macrophages infected with T. gondii tachyzoites using RNA immunoprecipitation (RIP) assay. The rate of TCAF1 mRNA degradation was compared between the shFTO-THP-1 and shNC-THP-1 groups using qPCR assay 0, 2, 4, and 6 hours post-treatment with actinomycin D. Differences of means between groups were tested for statistical significance with independent-sample Student’s t-test. Results RNA-seq revealed significant downregulation of TRP channel protein-coding genes except transient receptor potential cation channel subfamily C member 4-related protein, which was upregulated. Notably, TRPM8 exhibited the most pronounced downregulation, with its transcription level at 0.16-fold of the control group. qPCR assay quantified lower TRPM8 (human) and Trpm8 (mouse) mRNA expression in THP-1-derived macrophages [(0.445 ± 0.118) vs. (1.000 ± 0.036); t = 7.828, P < 0.05], PBMCs [(0.302 ± 0.040) vs. (1.042 ± 0.381); t = 3.345, P < 0.05], and RAW264.7 cells [(0.365 ± 0.234) vs. (1.004 ± 0.105); t = 4.312, P < 0.05] infected with T. gondii tachyzoites than in controls, and flow cytometry detected lower intracellular fluorescence intensities of Ca²⁺ in THP-1-derived macrophages [(19 500.0 ± 2 427.0) vs. (23 500.0 ± 2 64.6); t = 2.838, P < 0.05], PBMCs [(3 569.0 ± 313.9) vs. (13 050.0 ± 1 072.0); t = 16.970, P < 0.05], and RAW264.7 cells [(6 513.0 ± 348.0) vs. (7 683.0 ± 245.2); t = 4.762, P < 0.05] infected with T. gondii tachyzoites than in controls. qPCR assay quantified lower TCAF1 mRNA expression in THP-1-derived macrophages [(0.617 ± 0.132) vs. (1.005 ± 0.115); t = 3.832, P < 0.05] infected with T. gondii tachyzoites than in controls, and no significant differences in TCAF2 mRNA expression between the infection and control groups [THP-1-derived macrophages: (0.973 ± 0.030) vs. (1.015 ± 0.209), t = 0.343, P > 0.05]. Volcano plots of MeRIP-seq data displayed that m⁶A modification in the 3′ untranslated region (3′UTR) of TCAF1 mRNA was upregulated by 552 folds in THP-1-derived macrophages infected with T. gondii tachyzoites than in controls, and m⁶A-IP-qPCR assay detected a higher m⁶A modification level in the 3′ UTR region of TCAF1 mRNA in THP-1-derived macrophages infected with T. gondii tachyzoites than in controls [(4.794 ± 0.854) vs. (1.000 ± 0); t = 7.696, P < 0.05]. YTHDF2-RIP analysis showed a higher transcriptional level of TCAF1 mRNA binding to YTHDF2 in THP-1-derived macrophages infected with T. gondii tachyzoites than in controls [(2.423 ± 0.782) vs. (1.010 ± 0.180); t = 3.048, P < 0.05]. In addition, a higher rate of TCAF1 mRNA degradation was seen in shFTO-THP-1 cells infected with T. gondii tachyzoites than in shNC-THP-1 cells infected with T. gondii tachyzoites. Conclusion m⁶A modification contributes to downregulation of the cold receptor TRPM8 in both human and mouse macrophages following T. gondii infection.

Key words: Toxoplasma gondii, Transient receptor potential cation channel subfamily M member 8, Macrophage, N ⁶-methyladenosine