Integrated analysis of bulk transcriptome and single-cell RNA sequencing data reveal RNA modification-related biomarkers in intracerebral hemorrhage

Abstract

Background

RNA modifications play a pivotal role in regulating intracerebral hemorrhage (ICH). However, RNA modification-related genes (RMRGs) in ICH remain largely unexplored. This study aims to identify such biomarkers by integrating bulk and single-cell transcriptomic data.

Methods

Single-cell and transcriptomic data related to ICH were obtained from public databases, and RMRGs were sourced from existing literature. Differentially expressed genes were identified from the GSE216607 dataset and cross-referenced with RMRGs to generate candidate genes. Protein-protein interaction networks were then employed to identify core genes. Gene expression analysis of these core genes was conducted to pinpoint biomarkers in ICH. Functional enrichment analysis followed, and the expression of biomarkers in immune cells was examined. Additionally, drug predictions were made, and single-cell analysis was performed to characterize cell types and identify key cells based on biomarker expression.

Results

Ybx1 and Igf2bp2 were identified as biomarkers, with their expression levels upregulated in ICH samples from both the GSE216607 and GSE206971 datasets. Enrichment analysis indicated that these biomarkers are associated with neuronal systems and other related pathways. Further, these biomarkers were mapped to their human homologs, YBX1 and IGF2BP2. YBX1 exhibited the highest expression in non-classical monocytes, while IGF2BP2 was predominantly expressed in myeloid dendritic cells. Additionally, 3-butylidenephthalide, lithium chloride, and cantharidin were predicted as potential therapeutic agents for ICH. Single-cell analysis revealed monocytes and microglia 1 as key cell types.

Conclusion

Ybx1 and Igf2bp2 were identified as RM-related biomarkers in ICH, offering novel insights for ICH prevention and therapeutic strategies.

Keywords

Intracerebral hemorrhage / RNA modification / Single-cell RNA sequencing / Ybx1 / Igf2bp2

Highlight

This study represents the first systematic investigation of RNA modification-related biomarkers in ICH through integrated transcriptomic and single-cell RNA sequencing analyses.

Ybx1 and Igf2bp2 were identified as key RM-related biomarkers in ICH.

Both Ybx1 and Igf2bp2 were enriched in synaptic signaling and translational pathways.

Ybx1 exhibited dynamic, stage-specific fluctuations in microglia 1 and monocytes, while Igf2bp2 expression remained relatively stable.

Predominant expression of YBX1 was observed in non-classical monocytes, whereas IGF2BP2 was primarily expressed in myeloid dendritic cells.

References

[1] BhuiyanP, ZhangW, LiangG, JiangB, VeraR, ChaeR, KimK, LouisLS, WangY, LiuJ, ChuangDM, WeiH. Intranasal delivery of lithium salt suppresses inflammatory pyroptosis in the brain and ameliorates memory loss and Depression-like behavior in 5XFAD mice. Journal of Neuroimmune Pharmacology: the official Journal of the Society on Neuroimmune Pharmacology, 2025, 2026.

[2] CaoJ, SpielmannM, QiuX, HuangX, IbrahimDM, HillAJ, ZhangF, MundlosS, ChristiansenL, SteemersFJ, TrapnellC, ShendureJ. The single-cell transcriptional landscape of mammalian organogenesis. Nature, 2019, 566: 496-502.

[3] ChenL, ZhangX, ZhangQ, ZhangT, XieJ, WeiW, WangY, YuH, ZhouH. A necroptosis related prognostic model of pancreatic cancer based on single cell sequencing analysis and transcriptome analysis. Frontiers in Immunology, 2022, 131022420.

[4] ChenAY, OwensMC, LiuKF. Coordination of RNA modifications in the brain and beyond. Molecular Psychiatry, 2023, 28: 2737-2749.

[5] ChokkallaAK, MehtaSL, VemugantiR. Epitranscriptomic modifications modulate normal and pathological functions in CNS. Transl Stroke Res, 2022, 13: 1-11.

[6] Coler-Reilly, A., Pincus, Z., Scheller, E. L., Civitelli, R., (2024). Six drivers of aging identified among genes differentially expressed with age. bioRxiv. https://doi.org/10.1101/2024.08.02.606402

[7] DengM, ChenB, LiuZ, WanY, LiD, YangY, WangF. YBX1 mediates alternative splicing and maternal mRNA decay during pre-implantation development. Cell Biosci, 2022, 1212.

[8] EvansMK, MatsuiY, XuB, WillisC, LoomeJ, MilburnL, FanY, PagalaV, PengJC. Ybx1 fine-tunes PRC2 activities to control embryonic brain development. Nature Communications, 2020, 114060.

[9] GeP, DuanH, TaoC, NiuS, HuY, DuanR, ShenA, SunY, SunW. TMAO promotes NLRP3 inflammasome activation of microglia aggravating neurological injury in ischemic stroke through FTO/IGF2BP2. J Inflamm Res, 2023, 16: 3699-3714.

[10] GuZ, HübschmannD. Make interactive complex heatmaps in R. Bioinformatics, 2022, 38: 1460-1462.

[11] GuL, SunM, LiR, ZhangX, TaoY, YuanY, LuoX, XieZ. Didymin suppresses microglia pyroptosis and neuroinflammation through the Asc/Caspase-1/GSDMD pathway following experimental intracerebral hemorrhage. Frontiers in Immunology, 2022, 13810582.

[12] GustavssonEK, ZhangD, ReynoldsRH, Garcia-RuizS, RytenM. Ggtranscript: An R package for the visualization And interpretation of transcript isoforms using ggplot2. Bioinformatics, 2022, 38: 3844-3846.

[13] HongL, ZhuoT, JingS. Silencing of METTL3 inhibits m6A methylation of NEK7 to suppress pyrolysis in an HT-22 cell-based model of intracerebral hemorrhage. Brain Research, 2024, 1831148828.

[14] HuZ, ZuS, LiuJS. SIMPLEs: A single-cell RNA sequencing imputation strategy preserving gene modules and cell clusters variation. NAR Genom Bioinform, 2020, 2lqaa077.

[15] HuangH, WengH, SunW, QinX, ShiH, WuH, ZhaoBS, MesquitaA, LiuC, YuanCL, HuYC, HüttelmaierS, SkibbeJR, SuR, DengX, DongL, SunM, LiC, NachtergaeleS, WangY, HuC, FerchenK, GreisKD, JiangX, WeiM, QuL, GuanJL, HeC, YangJ, ChenJ. Recognition of RNA N(6)-methyladenosine by IGF2BP proteins enhances mRNA stability and translation. Nature Cell Biology, 2018, 20: 285-295.

[16] JinS, Guerrero-JuarezCF, ZhangL, ChangI, RamosR, KuanCH, MyungP, PlikusMV, NieQ. Inference and analysis of cell-cell communication using cellchat. Nature Communications, 2021, 121088.

[17] JundeZ, TingtingL, LuZ, ShanC, DanY, YizhenZ. Lithium chloride promotes neural functional recovery after local cerebral ischaemia injury in rats through Wnt signalling pathway activation. Folia Morphol (Warsz), 2023, 82: 519-532.

[18] Lee, J. H., Lin, S. Y., Liu, J. W., Lin, S. Z., Harn, H. J., & Chiou, T. W. (2021). n-Butylidenephthalide modulates autophagy to ameliorate neuropathological progress of spinocerebellar ataxia type 3 through mTOR pathway. International Journal of Molecular Sciences, 22. https://doi.org/10.3390/ijms22126339

[19] LiR, LiuZ, WuX, YuZ, ZhaoS, TangX. Lithium chloride promoted hematoma resolution after intracerebral hemorrhage through GSK-3β-mediated pathways-dependent microglia phagocytosis and M2-phenotype differentiation, angiogenesis and neurogenesis in a rat model. Brain Research Bulletin, 2019, 152: 117-127.

[20] LiM, XiaM, ChenW, WangJ, YinY, GuoC, LiC, TangX, ZhaoH, TanQ, ChenY, JiaZ, LiuX, FengH. Lithium treatment mitigates white matter injury after intracerebral hemorrhage through brain-derived neurotrophic factor signaling in mice. Translational Research: the Journal of Laboratory and Clinical Medicine, 2020, 217: 61-74.

[21] LiQ, LanX, HanX, DurhamF, WanJ, WeilandA, KoehlerRC, WangJ. Microglia-derived interleukin-10 accelerates post-intracerebral hemorrhage hematoma clearance by regulating CD36. Brain, Behavior, and Immunity, 2021, 94: 437-457.

[22] LiY, LiuX, ChenS, WangJ, PanC, LiG, TangZ. Effect of antiplatelet therapy on the incidence, prognosis, and rebleeding of intracerebral hemorrhage. Cns Neuroscience & Therapeutics, 2023, 29: 1484-1496.

[23] Li, Y., Hu, K., Li, J., Yang, X., Wu, X., Liu, Q., Chen, Y., Ding, Y., Liu, L., Yang, Q., & Wang, G. (2024). Tetrahydroxy Stilbene glucoside promotes mitophagy and ameliorates neuronal injury after cerebral ischemia reperfusion via promoting USP10-mediated YBX1 stability. eNeuro, 11. https://doi.org/10.1523/eneuro.0269-24.2024

[24] LiuZ, LiR, JiangC, ZhaoS, LiW, TangX. The neuroprotective effect of lithium chloride on cognitive impairment through glycogen synthase kinase-3β Inhibition in intracerebral hemorrhage rats. European Journal of Pharmacology, 2018, 840: 50-59.

[25] LiuP, XuH, ShiY, DengL, ChenX. Potential molecular mechanisms of plantain in the treatment of gout and hyperuricemia based on network Pharmacology. Evid Based Complement Alternat Med, 2020, 20203023127.

[26] LiuJ, LiN, ZhuZ, KiangKM, NgACK, DongCM, LeungGK. Vitamin D enhances hematoma clearance and neurologic recovery in intracerebral hemorrhage. Stroke, 2022, 53: 2058-2068.

[27] LiuW, LiuY, LiH, WangS, ChenP, LiuZ, HuoX, TianJ. IGF2BP2 orchestrates global expression and alternative splicing profiles associated with glioblastoma development in U251 cells. Transl Oncol, 2025, 51102177.

[28] LoveMI, HuberW, AndersS. Moderated Estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 2014, 15550.

[29] Mazya, M., Bartek, J. Jr., & Hansen, B. (2023). [Treatment of intracerebral hemorrhage - An overview]. Lakartidningen 120.

[30] MeiY, WangX. RNA modification in mRNA cancer vaccines. Clin Exp Med, 2023, 23: 1917-1931.

[31] NamKN, KimKP, ChoKH, JungWS, ParkJM, ChoSY, ParkSK, ParkTH, KimYS, LeeEH. Prevention of inflammation-mediated neurotoxicity by butylidenephthalide and its role in microglial activation. Cell Biochemistry and Function, 2013, 31: 707-712.

[32] PengJ, HeJ, LinL, LiY, XiaY. Neural stem cell extracellular vesicles carrying YBX1 inhibited neuronal pyroptosis through increasing m6A-modified GPR30 stability and expression in ischemic stroke. Transl Stroke Res, 2025, 16: 262-279.

[33] PuyL, Parry-JonesAR, SandsetEC, DowlatshahiD, ZiaiW, CordonnierC. Intracerebral haemorrhage. Nat Rev Dis Primers, 2023, 914.

[34] RobinX, TurckN, HainardA, TibertiN, LisacekF, SanchezJC, MüllerM. pROC: An open-source package for R And S + to Analyze And compare ROC curves. Bmc Bioinformatics, 2011, 1277.

[35] SatijaR, FarrellJA, GennertD, SchierAF, RegevA. Spatial reconstruction of single-cell gene expression data. Nature Biotechnology, 2015, 33: 495-502.

[36] SeiffgeDJ, Fandler-HöflerS, DuY, GoeldlinMB, JolinkWMT, KlijnCJM, WerringDJ. Intracerebral haemorrhage - mechanisms, diagnosis and prospects for treatment and prevention. Nat Rev Neurol, 2024, 20: 708-723.

[37] SureshPS, TsutsumiR, VenkateshT. YBX1 at the crossroads of non-coding transcriptome, exosomal, and cytoplasmic granular signaling. European Journal of Cell Biology, 2018, 97: 163-167.

[38] TianM, MaoL, ZhangL. Crosstalk among N6-methyladenosine modification and RNAs in central nervous system injuries. Frontiers in Cellular Neuroscience, 2022, 161013450.

[39] TongM, HeZ, LinX, ZhouY, WangQ, ZhengZ, ChenJ, XuH, TianN. Lithium chloride contributes to blood-spinal cord barrier integrity and functional recovery from spinal cord injury by stimulating autophagic flux. Biochemical and Biophysical Research Communications, 2018, 495: 2525-2531.

[40] TsaiNM, ChenYL, LeeCC, LinPC, ChengYL, ChangWL, LinSZ, HarnHJ. The natural compound n-butylidenephthalide derived from Angelica sinensis inhibits malignant brain tumor growth in vitro and in vivo. Journal of Neurochemistry, 2006, 99: 1251-1262.

[41] TwissJL, KalinskiAL, SachdevaR, HouleJD. Intra-axonal protein synthesis - a new target for neural repair?. Neural Regen Res, 2016, 11: 1365-1367.

[42] WangW, van NiekerkE, WillisDE, TwissJL. RNA transport and localized protein synthesis in neurological disorders and neural repair. Developmental Neurobiology, 2007, 67: 1166-1182.

[43] WangJ, ChenL, QiangP. The role of IGF2BP2, an m6A reader gene, in human metabolic diseases and cancers. Cancer Cell International, 2021, 2199.

[44] WuX, LiuH, WangJ, ZhangS, HuQ, WangT, CuiW, ShiY, BaiH, ZhouJ, HanL, LiL, ZhaoT, WuY, LuoJ, FengD, GuoW, GeS, QuY. The m(6)A methyltransferase METTL3 drives neuroinflammation and neurotoxicity through stabilizing BATF mRNA in microglia. Cell Death and Differentiation, 2025, 32: 100-117.

[45] Xu, R., Feng, N., Li, Q., Wang, H., Li, L., Feng, X., Su, Y., & Zhu, W. (2024). Pectin supplementation accelerates post-antibiotic gut Microbiome reconstitution orchestrated with reduced gut redox potential. Isme J, 18. https://doi.org/10.1093/ismejo/wrae101

[46] XuZ, LiH, LiX, LuJ, CaoC, PengL, LiL, ZhangJ, ChenG. Fto-dependent Vdac3 m6A modification regulates neuronal ferroptosis induced by the Post-ICH mass effect and transferrin. Neuroscience Bulletin, 2025.

[47] YangB, ChenQ. Cross-Talk between oxidative stress and m(6)A RNA methylation in cancer. Oxid Med Cell Longev, 2021, 20216545728.

[48] YangH, XieC, WuYF, ChengY, ZhuDS, GuanYT. N(6)-Methyladenosine (m(6)A) methylation is associated with the immune microenvironments in acute intracerebral hemorrhage (ICH). Molecular Neurobiology, 2024, 61: 1781-1793.

[49] YeL, TangX, ZhongJ, LiW, XuT, XiangC, GuJ, FengH, LuoQ, WangG. Unraveling the complex pathophysiology of white matter hemorrhage in intracerebral stroke: A single-cell RNA sequencing approach. Cns Neuroscience & Therapeutics, 2024, 30e14652.

[50] YuG, WangLG, HanY, HeQY. ClusterProfiler: An R package for comparing biological themes among gene clusters. Omics, 2012, 16: 284-287.

[51] YuX, XuX, ZhangJ, LiX. Batch alignment of single-cell transcriptomics data using deep metric learning. Nature Communications, 2023, 14960.

[52] ZhangH, MeltzerP, DavisS. RCircos: An R package for circos 2D track plots. Bmc Bioinformatics, 2013, 14244.

[53] ZhangQ, ChenZW, ZhaoYH, LiuBW, LiuNW, KeCC, TanHM. Bone marrow stromal cells combined with sodium ferulate and n-Butylidenephthalide promote the effect of therapeutic angiogenesis via advancing Astrocyte-Derived trophic factors after ischemic stroke. Cell Transplantation, 2017, 26: 229-242.

[54] ZhangM, ZhangX, NiuJ, HuaC, LiuP, ZhongG. Integrated analysis of single-cell RNA sequencing and bulk RNA data reveals gene regulatory networks and targets in dilated cardiomyopathy. Scientific Reports, 2024, 1413942.

[55] ZhangP, GaoC, GuoQ, YangD, ZhangG, LuH, ZhangL, ZhangG, LiD. Single-cell RNA sequencing reveals the evolution of the immune landscape during perihematomal edema progression after intracerebral hemorrhage. J Neuroinflammation, 2024, 21140.

[56] ZhaoYH, LiuNW, KeCC, LiuBW, ChenYA, LuoC, ZhangQ, XiaZY, LiuRS. Combined treatment of sodium ferulate, n-butylidenephthalide, and ADSCs rehabilitates neurovascular unit in rats after photothrombotic stroke. Journal of Cellular and Molecular Medicine, 2019, 23: 126-142.

[57] ZhengY, GaoW, ZhangQ, ChengX, LiuY, QiZ, LiT. Ferroptosis and Autophagy-Related genes in the pathogenesis of ischemic cardiomyopathy. Front Cardiovasc Med, 2022, 9906753.

[58] ZhengM, HuY, LiuO, LiS, WangY, LiX, LiuJ, YangQ, LiX, LinB. Oxidative stress response biomarkers of ovarian cancer based on Single-Cell and bulk RNA sequencing. Oxid Med Cell Longev, 2023, 20231261039.

[59] ZhouT, PanJ, XuK, YanC, YuanJ, SongH, HanY. Single-cell transcriptomics in MI identify Slc25a4 as a new modulator of mitochondrial malfunction and apoptosis-associated cardiomyocyte subcluster. Scientific Reports, 2024, 149274.

Funding

The Foundation of Shenzhen Science and Technology Innovation Committee(JCYJ20240813144017023)

The National Natural Science Foundation of China(82374567)

The Nature Science Foundation of Guangdong Province(2023A1515012779)

Sanming Project of Medicine in Shenzhen(SZZYSM202411004)

RIGHTS & PERMISSIONS

Shenzhen University School of Medicine; Fondazione Istituto FIRC di Oncologia Molecolare

Just Accepted

This article has successfully passed peer review and final editorial review, and will soon enter typesetting, proofreading and other publishing processes. The currently displayed version is the accepted final manuscript. The officially published version will be updated with format, DOI and citation information upon launch. We recommend that you pay attention to subsequent journal notifications and preferentially cite the officially published version. Thank you for your support and cooperation.