背景:LncRNA结直肠肿瘤差异表达(CRNDE)被发现是许多癌症的重要调节因子。本项目主要研究CRNDE对巨噬细胞代谢重编程和肝细胞癌(HCC)的功能。
方法:采用qRT-PCR和免疫荧光方法分析HCC组织和巨噬细胞中Arg-1、IL-10、CD163、CCL-18、CD206和CRNDE的表达。Western印迹用于分析ERK和p-ERK表达。Edu测定,进行transwell测定和异种移植实验以研究细胞活力,迁移和侵入能力。免疫组织化学染色用于评估Ki67表达。进行液相色谱-串联质谱(LC-MS/MS)用于巨噬细胞代谢物分析。
结果:Arg-1、IL-10、CD163、CD206和CRNDE在HCC组织中显著上调,M2巨噬细胞和M0巨噬细胞与CRNDE过表达(OV-CRNDE-M0),在CRNDE敲低的M0巨噬细胞中下调(sh-CRNDE-M0)。M2细胞和OV-CRNDE-M0细胞的条件培养基(CM)促进细胞活力,入侵,和肝癌细胞的迁移,sh-CRNDE-M0细胞CM逆转了这种作用。OV-CRNDE-M0细胞促进肿瘤生长,Ki67和CD206在异种移植模型中的表达。检测到61种代谢物,其中18种代谢产物在OV-CRNDE-M0组与M0组相比发生显著变化,9上调,9下调。KEGG分析显示富集途径为生物合成,乙醛酸和二羧酸代谢。SMPDB分析显示富集途径为低乙酰天冬氨酸,卡纳文病,和天冬氨酸代谢.
结论:CRNDE通过ERK通路调节M2巨噬细胞的代谢重编程,从而促进肝癌的增殖,迁移,和入侵。
BACKGROUND: LncRNA colorectal neoplasia differentially expressed (
CRNDE) was found to be an important regulator in many cancers. This project focuses on the function of
CRNDE on macrophage metabolic reprogramming and Hepatocellular carcinoma (HCC).
METHODS: qRT-PCR and Immunofluorescence were used to analyze Arg-1, IL-10, CD163, CCL-18, CD206, and CRNDE expression in HCC tissues and macrophages. Western Blotting was used to analyze ERK and p-ERK expression. Edu assay, transwell assay and xenograft experiments were carried out to study cell viability, migrated and invasive capability. Immunohistochemical staining was used to evaluate Ki67 expression. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed for macrophages metabolites analysis.
RESULTS: Arg-1, IL-10, CD163, CD206, and CRNDE were significantly up-regulated in HCC tissues, M2 macrophage and M0 macrophage with
CRNDE overexpressed (OV-
CRNDE-M0), which downregulated in M0 macrophage with
CRNDE knockdown (sh-
CRNDE-M0). The conditioned medium (CM) of M2 cells and OV-CRNDE-M0 cells promoted cell viability, invasion, and migration of HCC cells, the effect was reversed by sh-
CRNDE-M0 cells CM. OV-CRNDE-M0 cells promoted tumor growth, Ki67 and CD206 expression in xenograft model. 61 metabolites were detected, of which 18 metabolites changed significantly in OV-CRNDE-M0 group compared to M0 group, with 9 upregulated and 9 downregulated. KEGG analysis showed the enrichment pathways were biosynthesis, glyoxylate and dicarboxylate metabolism. SMPDB analysis showed the enrichment pathways were hypoacetylaspartia, canavan disease, and aspartate metabolism.
CONCLUSIONS: CRNDE regulated the metabolic reprogramming of M2 macrophage via ERK pathway, which thereby contributed to HCC proliferation, migration, and invasion.