摘要
目的:肿瘤细胞获得对内分泌治疗药物的耐受性是ER+乳腺癌治疗失败的关键因素。肿瘤相关巨噬细胞是乳腺癌微环境中的重要成分,然而内分泌治疗药物对巨噬细胞的调控作用尚不清楚,本研究旨在探讨内分泌治疗药物氟维司群对单核-巨噬细胞极化的影响。方法:借助PMA诱导获得THP-1细胞来源的巨噬细胞,通过RT-qPCR、Western blot、流式细胞术验证氟维司群刺激后的巨噬细胞表面marker变化;通过RNA-Seq技术对氟维司群刺激后的巨噬细胞的转录组改变进行分析,筛选组间差异表达基因;结合GO功能与KEGG通路富集分析差异基因的功能;构建差异基因的蛋白相互作用网络并筛选Hub基因;最后通过RT-qPCR和趋化因子阵列实验验证氟维司群对巨噬细胞Hub基因中5种趋化因子表达的影响。结果:RT-qPCR、Western blot、流式细胞术分析结果表明氟维司群刺激后的巨噬细胞M2相关标志分子表达升高而M1相关标志分子表达降低;RNA-Seq结果显示,氟维司群刺激后的巨噬细胞出现118个差异表达基因;GO与KEGG富集分析结果显示差异基因富集于趋化因子相关功能和信号通路;蛋白互作网络和Hub基因筛选确定了CXCL8、CCL20、CCL22、CXCL10、CCL7 5个关键的趋化因子;对5个关键趋化因子进行RT-qPCR验证表明其表达趋势与RNA-Seq结果一致;趋化因子阵列实验证实氟维司群刺激后的巨噬细胞分泌的CXCL8、CCL20、CCL22增多,而CXCL10、CCL7减少。结论:内分泌治疗药物氟维司群使巨噬细胞M2相关分子CD206表达升高而M1相关分子CD86表达降低,同时使巨噬细胞M2相关趋化因子分泌增多而M1相关趋化因子分泌减少,提示氟维司群促进巨噬细胞向M2极化,抑制其向M1极化,可能与临床内分泌治疗耐药形成有关。
Objective:In ER+breast cancer.Tumour⁃associated macrophages(TAMs)are important components of the breast cancer microenvironment.However,the effects of endocrine therapeutic agents on TAMs is unclear.To investigate the effect of fulvestrant on the polarization of macrophages.Methods:Using THP⁃1 cell⁃derived macrophages,we observed changes in macro⁃phage following fulvestrant stimulation through RT⁃qPCR,Western blot,and flow cytometry.RNA⁃Seq was employed to ana⁃lyze the transcriptomic changes in macrophages induced by fulvestrant.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted to assess the functional roles of these differentially expressed genes.A protein⁃protein interaction network was constructed to identify hub genes.Finally,the effects of fulvestrant on the expres⁃sion of five key chemokines among the hub genes were validated using RT⁃qPCR and a chemokine array.Results:RT⁃qPCR,Western blot,and flow cytometry analyses revealed that fulvestrant stimulation increased the expression of M2⁃related markers while decreasing the expression of M1⁃related markers in macrophages.RNA⁃Seq results revealed that 118 differentially expressed genes appeared in macrophages stimulated by fulvestrant.GO and KEGG enrichment analyses showed that the differentially ex⁃pressed genes were enriched in the chemokine⁃related functions and signalling pathways.Protein⁃protein interaction network analy⁃sis and hub gene screening identified five key chemokines(CXCL8,CCL20,CCL22,CXCL10 and CCL7).RT⁃qPCR showed that the expression trends of these key chemokines were consistent with the RNA⁃Seq results.Additionally,chemokine array ex⁃periments confirmed that the secretion of CXCL8,CCL20,and CCL22 by macrophages after fulvestrant stimulation was in⁃creased,while that of CXCL10 and CCL7 was decreased.Conclusions:The endocrine therapeutic agent fulvestrant was found to promote the expression of the M2⁃associated macrophage marker CD206 while inhibiting the expression of the M1⁃associated marker CD86.Additionally,fulvestrant treatment enhanced the secretion of M2⁃related chemokines and reduced the secretion of M1⁃related chemokines by macrophages.These findings suggest that fulvestrant treatment promotes macrophage polarization to⁃wards the M2 phenotype and inhibits polarization towards the M1 phenotype.This shift in macrophage polarization may be associ⁃ated with the development of resistance to endocrine therapy in clinical settings.
作者
刘宇婷
叶敬文
刘泊含
刘鷖雯
何怡青
杜艳
张国良
郭倩
高锋
杨翠霞
LIU Yuting;YE Jingwen;LIU Bohan;LIU Yiwen;HE Yiqing;DU Yan;ZHANG Guoliang;GUO Qian;GAO Feng;YANG Cuixia(Shanghai Jiao Tong University Affiliated Sixth People's Hospital Central Laboratory,Shanghai 200233,China;Shanghai Jiao Tong University Affiliated Sixth People's Hospital Department of Clinical Laboratory,Shanghai 200233,China)
出处
《海南医科大学学报》
北大核心
2025年第14期1041-1049,1056,共10页
Journal of Hainan Medical University
基金
国家自然科学基金资助项目(81974445、82273462)。
