摘要
目的探讨外泌体来源的circ_0003759在肝细胞癌(HCC)中的表达情况,及其诱导巨噬细胞M2极化对HCC预后的影响。方法基于GSE97332联合HCC患者血清外泌体环状RNA(cirRNAs)测序数据筛选出circ_0003759。透射电镜及蛋白质免疫印迹法鉴定及检测外泌体,荧光定量PCR检测外泌体circ_0003759表达水平。PKH26标记HCC外泌体与人巨噬细胞共培养,流式细胞术检测巨噬细胞M2型极化标记物CD206和CD209。构建sh_circ_0003759慢病毒并转染至肝癌HCCLM3细胞,抽提外泌体与人巨噬细胞共培养,检测巨噬细胞M2型极化情况。CIBERSORT分析HCC微环境免疫细胞浸润丰度,Kaplan-Meier法和Cox比例风险回归模型分析M2型巨噬细胞对HCC预后的影响。基因集富集分析(GSEA)巨噬细胞M2型极化的相关信号通路。结果circ_0003759在HCC组织和HCC血清外泌体中表达显著增加(P<0.05)。将HCC外泌体与人巨噬细胞共同培养后,巨噬细胞CD206和CD209阳性细胞比率显著上升(P<0.05)。肝癌HCCLM3细胞转染sh_circ_0003759,将其外泌体与人巨噬细胞共同培养后,巨噬细胞CD206和CD209阳性细胞比率显著下降(P<0.05)。此外,HCC组织中M2型巨噬细胞的浸润相对丰度明显高于对照组(P<0.001),生存分析结果表明M2型巨噬细胞相对丰度高的HCC患者预后较差(P<0.05)。GSEA分析显示巨噬细胞M2型极化与补体、炎性反应、干扰素γ及抗肿瘤坏死因子α/细胞核因子κB免疫信号通路的抑制相关。结论HCC外泌体高表达circ_0003759并诱导巨噬细胞M2型极化,与HCC预后差、免疫抑制状态相关。
Objective To investigate the expression of circ_0003759 derived from exosomes in hepatocellular carcinoma(HCC),and its impact on the prognosis of HCC patients through inducing M2 polarization of macrophages.Methods Circ_0003759 was selected based on GSE97332 combined with sequencing data of exosomal circRNAs in serum of HCC patient.Transmission electron microscopy and western blotting were used to detect exosomal molecular markers CD63 and CD9,while real-time fluorescene quantitavie PCR(qRCR)was utilized to examine the expression level of circ_0003759.HCC-derived exosomes labeled with PKH26 were co-cultured with human macrophages,and flow cytometry was used to detect CD206 and CD209,surface markers of M2 polarized macrophages.Lentiviral vector circ_0003759 knockdown was established,and HCCLM3 cells were transfected.Exosomes were extracted and co-cultured with human macrophages,and the polarization status of M2 macrophages was analyzed by flow cytometry.CIBERSORT was used to analyze the immune cell infiltration abundance of the HCC microenvironment,and Kaplan-Meier and Cox proportional risk model was utilized to analyze the impact of M2 macrophages on the prognosis of HCC patients.Gene set enrichment analysis(GSEA)was performed to explore the molecular pathways relevant to M2 polarization in macrophages.Results The expression of circ_0003759 was significantly elevated in HCC tissues and HCC serum-derived exosomes(P<0.05).Co-culturing HCC exosomes with human macrophages led to a significant increase in the percentage of CD206+and CD209+macrophages(P<0.05).Transfection of HCCLM3 cells with lentivirus carrying a circ_0003759 knockdown construct resulted in a significant decrease in the percentage of CD206+and CD209+macrophages when co-cultured with human macrophages(P<0.05).Furthermore,there was a significantly higher relative abundance of M2-type macrophage infiltration in the HCC group compared to the control group(P<0.001),and survival analysis revealed a significantly lower overall survival in HCC patients with a higher relative abundance of M2-type macrophages(P<0.05).Gene set enrichment analysis indicated that M2 polarization of macrophages was associated with the suppression of complement,inflammatory response,IFN-γ,and TNFα/NF-κB immune signaling pathways.Conclusion The high expression of circ_0003759 in HCC-derived exosomes induces M2 polarization of macrophages,which is associated with poor prognosis and an immunosuppressive state in HCC.
作者
杜艾浓
唐妮
陈旭祥
周爽
曾江正
DU Ainong;TANG Ni;CHEN Xuxiang;ZHOU Shuang;ZENG Jiangzheng(Department of Medical Oncology,the First Affiliated Hospital of Hainan Medical University,Haikou 570102,China)
出处
《临床肿瘤学杂志》
CAS
2024年第5期417-423,共7页
Chinese Clinical Oncology
基金
海南省科技厅高层次人才研究课题(820RC761)。
