微生物通过巨噬细胞RIG⁃I乳酸化修饰促进结直肠癌肝转移的机制研究
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国家自然科学基金(82171759)


The mechanism of microbes promoting colorectal cancer liver metastases via macrophage RIG⁃I lactylation
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    摘要:

    目的:探讨微生物在结直肠癌肝转移(colorectal cancer liver metastases,CRLM)的作用及潜在机制。方法:对结直肠癌肝转移患者的结肠肿瘤及肝脏肿瘤行16S rDNA测序分析。建立小鼠结直肠癌肝转移模型,分别用抗生素、大肠杆菌、乳酸脱氢酶抑制剂、氯膦酸盐脂质体处理,24 d后获取肝脏肿瘤组织行HE染色、免疫荧光染色、乳酸浓度及微生物丰度检测。将大肠杆菌与MC38细胞按100∶1共培养,CCK8检测细胞增殖能力,Seahorse检测细胞外酸化率,比色法检测乳酸浓度。分别用乳酸、乳酸化修饰抗体、肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)处理小鼠骨髓来源巨噬细胞,免疫荧光检测其CD206、 iNOS的表达水平,qPCR检测其iNOS、CD86、CD163、ARG的水平,Western blot检测乳酸化修饰水平及核因子κB(nuclear factor kappa-B,NF-κB)的表达情况。结果:CRLM患者中结肠及肝脏肿瘤中存在微生物,优势物种相似。微生物促进小鼠CRLM,并发现肿瘤内巨噬细胞呈M2样极化,而清除巨噬细胞后肝转移瘤减少。微生物不能增强肿瘤细胞的增殖能力,但可升高肿瘤糖酵解及乳酸水平,同时抑制乳酸脱氢酶后小鼠肝转移瘤减少。乳酸可诱导巨噬细胞向M2极化及乳酸化修饰,并发现视黄酸诱导基因(I retinoic acid-inducible gene I,RIG-I)的去乳酸化可抑制乳酸对巨噬细胞的极化作用;RIG-I下游NF-κB的表达在乳酸处理后减弱;NF-κB激动剂TNF-α可抑制巨噬细胞向M2极化。结论:微生物促进瘤内乳酸形成,通过对巨噬细胞RIG-Ilys852乳酸化修饰诱导其极化为M2,调控结直肠癌肝转移。

    Abstract:

    Objective:To investigate the role of microbes in colorectal cancer liver metastases(CRLM)and the underlying mechanisms. Methods:16S rDNA sequencing was performed on colon tumors and liver tumors in CRLM patients. The mouse model of CRLM was established and treated with antibiotics,Escherichia coli(E.coli),lactate dehydrogenase inhibitor and clodronate liposome. After 24 days,the liver tumor tissues were obtained for HE staining,immunofluorescence staining and detection of lactate concentration and microbial abundance. E. coli and MC38 cells were co - cultured at 100∶1. The cell proliferation,the extracellular acidification rate(ECAR)and concentration of lactate was determined by CCK8,Seahorse and colorimetry respectively. Macrophages (MØ)derived from mouse bone marrow were treated with lactate,lactylation antibody and TNF-α respectively. The expression levels of CD206 and iNOS were detected by immunofluorescence;the mRNA levels of iNOS,CD86,CD163 and ARG were detected by qPCR; The lactylation level and NF-κB expression were detected by Western Blot. Results:There were microbes in colon and liver tumors in CRLM patients and the dominant species were similar. Microbiota promoted liver metastasis of colorectal cancer in mice and MØ was found to be M2 - like polarized,while the liver metastases decreased after depletion of MØ. Microbiota could not enhance the proliferation of tumor cells,but could increase the level of tumor glycolysis and lactate and inhibition of lactate dehydrogenase could reduce liver metastases in mice.Lactate could induce MØ polarization to M2 and lactylation. RIG - Ilys852 was found to inhibit the polarization of MØ induced by lactate;the expression of NF -κB was decreased after macrophage treated with lactate;TNF -α could inhibit MØ polarization to M2. Conclusion:Microbes enhance the level of glycolysis and lactate in tumor and promote liver metastasis of colorectal cancer via macrophage RIG-I lactylation.

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朱晓文,岳磊,赵文虎,古鉴,钱晓峰.微生物通过巨噬细胞RIG⁃I乳酸化修饰促进结直肠癌肝转移的机制研究[J].南京医科大学学报(自然科学版),2022,42(12):1664-1672

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