Expression of circ_0003759 derived from exosomes in hepatocellular carcinoma and its clinical significance in inducing M2 polarization of macrophages
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.
Hepatocellular carcinomaExosomescirc_0003759M2 polarization of macrophagesTumor microen-vironment
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