PDF(Pubmed)
Abstract：
OBJECTIVE: The main focus of this study is to explore the molecular mechanism of IRF7 regulation on RPS18 transcription in M1-type macrophages in pancreatic adenocarcinoma (PAAD) tissue, as well as the transfer of RPS18 by IRF7 via exosomes to PAAD cells and the regulation of ILF3 expression.
METHODS: By utilising single-cell RNA sequencing (scRNA-seq) data and spatial transcriptomics (ST) data from the Gene Expression Omnibus database, we identified distinct cell types with significant expression differences in PAAD tissue. Among these cell types, we identified those closely associated with lipid metabolism. The differentially expressed genes within these cell types were analysed, and target genes relevant to prognosis were identified. Flow cytometry was employed to assess the expression levels of target genes in M1 and M2 macrophages. Cell lines with target gene knockout were constructed using CRISPR/Cas9 editing technology, and cell lines with target gene knockdown and overexpression were established using lentiviral vectors. Additionally, a co-culture model of exosomes derived from M1 macrophages with PAAD cells was developed. The impact of M1 macrophage-derived exosomes on the lipid metabolism of PAAD cells in the model was evaluated through metabolomics analysis. The effects of M1 macrophage-derived exosomes on the viability, proliferation, division, migration and apoptosis of PAAD cells were assessed using MTT assay, flow cytometry, EdU assay, wound healing assay, Transwell assay and TUNEL staining. Furthermore, a mouse PAAD orthotopic implantation model was established, and bioluminescence imaging was utilised to assess the influence of M1 macrophage-derived exosomes on the intratumoural formation capacity of PAAD cells, as well as measuring tumour weight and volume. The expression of proliferation-associated proteins in tumour tissues was examined using immunohistochemistry.
RESULTS: Through combined analysis of scRNA-seq and ST technologies, we discovered a close association between M1 macrophages in PAAD samples and lipid metabolism signals, as well as a negative correlation between M1 macrophages and cancer cells. The construction of a prognostic risk score model identified RPS18 and IRF7 as two prognostically relevant genes in M1 macrophages, exhibiting negative and positive correlations, respectively. Mechanistically, it was found that IRF7 in M1 macrophages can inhibit the transcription of RPS18, reducing the transfer of RPS18 to PAAD cells via exosomes, consequently affecting the expression of ILF3 in PAAD cells. IRF7/RPS18 in M1 macrophages can also suppress lipid metabolism, cell viability, proliferation, migration, invasion and intratumoural formation capacity of PAAD cells, while promoting cell apoptosis.
CONCLUSIONS: Overexpression of IRF7 in M1 macrophages may inhibit RPS18 transcription, reduce the transfer of RPS18 from M1 macrophage-derived exosomes to PAAD cells, thereby suppressing ILF3 expression in PAAD cells, inhibiting the lipid metabolism pathway, and curtailing the viability, proliferation, migration, invasion of PAAD cells, as well as enhancing cell apoptosis, ultimately inhibiting tumour formation in PAAD cells in vivo. Targeting IRF7/RPS18 in M1 macrophages could represent a promising immunotherapeutic approach for PAAD in the future.
METHODS: By utilising single-cell RNA sequencing (scRNA-seq) data and spatial transcriptomics (ST) data from the Gene Expression Omnibus database, we identified distinct cell types with significant expression differences in PAAD tissue. Among these cell types, we identified those closely associated with lipid metabolism. The differentially expressed genes within these cell types were analysed, and target genes relevant to prognosis were identified. Flow cytometry was employed to assess the expression levels of target genes in M1 and M2 macrophages. Cell lines with target gene knockout were constructed using CRISPR/Cas9 editing technology, and cell lines with target gene knockdown and overexpression were established using lentiviral vectors. Additionally, a co-culture model of exosomes derived from M1 macrophages with PAAD cells was developed. The impact of M1 macrophage-derived exosomes on the lipid metabolism of PAAD cells in the model was evaluated through metabolomics analysis. The effects of M1 macrophage-derived exosomes on the viability, proliferation, division, migration and apoptosis of PAAD cells were assessed using MTT assay, flow cytometry, EdU assay, wound healing assay, Transwell assay and TUNEL staining. Furthermore, a mouse PAAD orthotopic implantation model was established, and bioluminescence imaging was utilised to assess the influence of M1 macrophage-derived exosomes on the intratumoural formation capacity of PAAD cells, as well as measuring tumour weight and volume. The expression of proliferation-associated proteins in tumour tissues was examined using immunohistochemistry.
RESULTS: Through combined analysis of scRNA-seq and ST technologies, we discovered a close association between M1 macrophages in PAAD samples and lipid metabolism signals, as well as a negative correlation between M1 macrophages and cancer cells. The construction of a prognostic risk score model identified RPS18 and IRF7 as two prognostically relevant genes in M1 macrophages, exhibiting negative and positive correlations, respectively. Mechanistically, it was found that IRF7 in M1 macrophages can inhibit the transcription of RPS18, reducing the transfer of RPS18 to PAAD cells via exosomes, consequently affecting the expression of ILF3 in PAAD cells. IRF7/RPS18 in M1 macrophages can also suppress lipid metabolism, cell viability, proliferation, migration, invasion and intratumoural formation capacity of PAAD cells, while promoting cell apoptosis.
CONCLUSIONS: Overexpression of IRF7 in M1 macrophages may inhibit RPS18 transcription, reduce the transfer of RPS18 from M1 macrophage-derived exosomes to PAAD cells, thereby suppressing ILF3 expression in PAAD cells, inhibiting the lipid metabolism pathway, and curtailing the viability, proliferation, migration, invasion of PAAD cells, as well as enhancing cell apoptosis, ultimately inhibiting tumour formation in PAAD cells in vivo. Targeting IRF7/RPS18 in M1 macrophages could represent a promising immunotherapeutic approach for PAAD in the future.
摘要：
目的：本研究的主要重点是探讨IRF7调控胰腺腺癌（PAAD）M1型巨噬细胞RPS18转录的分子机制，以及IRF7通过外泌体将RPS18转移到PAAD细胞和调节ILF3表达。
方法：通过利用来自基因表达综合数据库的单细胞RNA测序（scRNA-seq）数据和空间转录组学（ST）数据，我们鉴定了PAAD组织中具有显著表达差异的不同细胞类型.在这些细胞类型中，我们确定了与脂质代谢密切相关的那些。分析了这些细胞类型中差异表达的基因，并确定了与预后相关的靶基因。流式细胞术用于评估M1和M2巨噬细胞中靶基因的表达水平。利用CRISPR/Cas9编辑技术构建靶基因敲除细胞系,使用慢病毒载体建立具有靶基因敲低和过表达的细胞系。此外,建立了M1巨噬细胞与PAAD细胞来源的外泌体共培养模型。通过代谢组学分析评估了M1巨噬细胞来源的外泌体对模型中PAAD细胞脂质代谢的影响。M1巨噬细胞来源的外泌体对生存力的影响，扩散,司,使用MTT法评估PAAD细胞的迁移和凋亡，流式细胞术,EdU分析，伤口愈合试验，Transwell测定和TUNEL染色。此外，建立小鼠PAAD原位植入模型，和生物发光成像被用来评估M1巨噬细胞来源的外泌体对PAAD细胞的肿瘤内形成能力的影响，以及测量肿瘤的重量和体积。使用免疫组织化学检查肿瘤组织中增殖相关蛋白的表达。
结果：通过对scRNA-seq和ST技术的联合分析，我们发现PAAD样本中的M1巨噬细胞与脂质代谢信号密切相关，以及M1巨噬细胞和癌细胞之间的负相关。预后风险评分模型的构建将RPS18和IRF7确定为M1巨噬细胞中的两个预后相关基因，呈现负相关和正相关，分别。机械上,研究发现，M1巨噬细胞中的IRF7可抑制RPS18的转录，减少RPS18通过外泌体向PAAD细胞的转移，从而影响PAAD细胞中ILF3的表达。M1巨噬细胞中的IRF7/RPS18还可以抑制脂质代谢，细胞活力，扩散,迁移,PAAD细胞的侵袭和肿瘤内形成能力，同时促进细胞凋亡。
结论：在M1巨噬细胞中过表达IRF7可能抑制RPS18转录，减少RPS18从M1巨噬细胞来源的外泌体转移到PAAD细胞，从而抑制PAAD细胞中ILF3的表达，抑制脂质代谢途径，限制生存能力，扩散,迁移,PAAD细胞的侵袭，以及增强细胞凋亡，最终抑制体内PAAD细胞中的肿瘤形成。在M1巨噬细胞中靶向IRF7/RPS18可能代表未来PAAD的有希望的免疫治疗方法。
方法：通过利用来自基因表达综合数据库的单细胞RNA测序（scRNA-seq）数据和空间转录组学（ST）数据，我们鉴定了PAAD组织中具有显著表达差异的不同细胞类型.在这些细胞类型中，我们确定了与脂质代谢密切相关的那些。分析了这些细胞类型中差异表达的基因，并确定了与预后相关的靶基因。流式细胞术用于评估M1和M2巨噬细胞中靶基因的表达水平。利用CRISPR/Cas9编辑技术构建靶基因敲除细胞系,使用慢病毒载体建立具有靶基因敲低和过表达的细胞系。此外,建立了M1巨噬细胞与PAAD细胞来源的外泌体共培养模型。通过代谢组学分析评估了M1巨噬细胞来源的外泌体对模型中PAAD细胞脂质代谢的影响。M1巨噬细胞来源的外泌体对生存力的影响，扩散,司,使用MTT法评估PAAD细胞的迁移和凋亡，流式细胞术,EdU分析，伤口愈合试验，Transwell测定和TUNEL染色。此外，建立小鼠PAAD原位植入模型，和生物发光成像被用来评估M1巨噬细胞来源的外泌体对PAAD细胞的肿瘤内形成能力的影响，以及测量肿瘤的重量和体积。使用免疫组织化学检查肿瘤组织中增殖相关蛋白的表达。
结果：通过对scRNA-seq和ST技术的联合分析，我们发现PAAD样本中的M1巨噬细胞与脂质代谢信号密切相关，以及M1巨噬细胞和癌细胞之间的负相关。预后风险评分模型的构建将RPS18和IRF7确定为M1巨噬细胞中的两个预后相关基因，呈现负相关和正相关，分别。机械上,研究发现，M1巨噬细胞中的IRF7可抑制RPS18的转录，减少RPS18通过外泌体向PAAD细胞的转移，从而影响PAAD细胞中ILF3的表达。M1巨噬细胞中的IRF7/RPS18还可以抑制脂质代谢，细胞活力，扩散,迁移,PAAD细胞的侵袭和肿瘤内形成能力，同时促进细胞凋亡。
结论：在M1巨噬细胞中过表达IRF7可能抑制RPS18转录，减少RPS18从M1巨噬细胞来源的外泌体转移到PAAD细胞，从而抑制PAAD细胞中ILF3的表达，抑制脂质代谢途径，限制生存能力，扩散,迁移,PAAD细胞的侵袭，以及增强细胞凋亡，最终抑制体内PAAD细胞中的肿瘤形成。在M1巨噬细胞中靶向IRF7/RPS18可能代表未来PAAD的有希望的免疫治疗方法。
