唾液腺腺样囊性癌微环境中肿瘤相关巨噬细胞外泌体差异表达miRNAs的筛选及验证  

Screening and validation of miRNAs differentially expressed by exosomes of tumor-associated macrophages in microenvironment of salivary gland adenoid cystic carcinoma

作　　者：惠琪 高万鹏 李欢 赵琦 王珺 魏建华 杨新杰 杨子桧 XI Qi;GAO Wan-peng;LI Huan;ZHAO Qi;WANG Jun;WEI Jian-hua;YANG Xin-jie;YANG Zi-hui(State Key Laboratory of Oral&Maxillofacial Reconstruction and Regeneration,National Clinical Research Center for Oral Diseases,Shaanxi Clinical Research Center for Oral Diseases,Department of Maxillofacial Oncology,School of Stomatology,Fourth Military Medical University.Xi'an 710032,Shaanxi Province;College of Stomatology&Affiliated Stomatological Hospital,Jiamusi University,Heilongjiang Provincial Key Laboratory of Oral Biomedical Materials and Clinical Applications.Jiamusi 154000,Heilongjiang Province,China)

机构地区：[1]口颌系统重建与再生全国重点实验室,国家口腔疾病临床医学研究中心,陕西省口腔疾病临床医学研究中心,空军军医大学第三附属医院颌面肿瘤科,陕西西安710032 [2]佳木斯大学口腔医学院·附属口腔医院,黑龙江省口腔生物医学材料及临床应用重点实验室,黑龙江佳木斯154000

出　　处：《中国口腔颌面外科杂志》2024年第3期209-215,共7页China Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金青年项目(82002867);国家自然科学基金面上项目(82173165,81973114);陕西省重点研发计划(2023-YBSF-230)。

摘　　要：目的:探讨唾液腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)微环境中肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)外泌体miRNAs表达特征,分析其在SACC进展中的作用。方法:将SACC细胞与巨噬细胞共培养,获得SACC相关TAMs。应用超速离心法分别提取以上细胞外泌体,采用透射电镜、Western免疫印迹、外泌体纳米微粒追踪检测进行验证。对TAMs外泌体与对照组巨噬细胞外泌体进行RNA-seq测序,分析差异表达的miRNAs。利用miRanda和RNAhybrid软件对差异miRNAs进行靶基因预测,再对差异miRNAs作用的靶基因的集合分别进行GO和KEGG富集分析。利用qRT-PCR、CCK-8、细胞划痕实验、Transwell实验验证TAMs外泌体miRNAs表达及功能。采用SPSS 22.0软件包对数据进行统计学分析。结果:筛选出1595个差异表达的miRNAs,其中15个miRNAs表达差异变化具有统计学意义(P<0.05)。差异miRNAs作用靶基因富集分析发现,TAMs外泌体miRNAs靶基因参与调控癌症相关信号通路。qRT-PCR结果显示,TAMs外泌体has-miR-21-5p表达上调最显著。CCK-8、细胞划痕实验和Transwell实验发现,TAMs外泌体hsa-miR-21-5p促进SACC细胞增殖、迁移与侵袭能力。结论:TAMs外泌体hsa-miR-21-5p促进SACC细胞恶性进展。TAMs外泌体miRNAs可能在SACC进展中发挥重要作用,有望为SACC的诊治提供新的策略。PURPOSE:To investigate the expression patterns of exosomal miRNAs in tumor-associated macrophages(TAMs)and analyze the potential function in progression of salivary adenoid cystic carcinoma(SACC).METHODS:SACC cells and macrophages were co-cultured to obtain TAMs.Exosomes of both macrophages and TAMs were isolated accord-ing to the ultracentrifugation protocol,and then the exosomes were identified using transmission electron microscope,Western blot,and nanoparticle tracking analysis(NTA).RNA-seq analysis was performed to compare the differential ex-pression of miRNAs in TAMs-derived exosomes and the control macrophages-derived exosomes.The target genes of the differential miRNAs were predicted by miRanda and RNAhybrid database.Then GO and KEGG enrichment analysis were performed on the set of target genes.Assays of qRT-PCR,CCK-8,Wound healing,and Transwell were performed to vali-date the expression patterns and functions of TAMs-derived exosomes.SPSS 22.0 software package was used for data analysis.RESULTS:A total of 1595 differentially expressed miRNAs were screened out,among which 15 were signifi-cantly expressed(P<0.05).Enrichment analysis showed that the target genes of TAMs-derived exosomes were mainly in-volved in the regulation of cancer-related signaling pathways.Results of qRT-PCR showed that TAMs-derived exosomes carried higher levels of hsa-miR-21-5p than control macrophages derived exosomes.Results of CCK-8,Wound healing,and Transwell assay showed that TAMs-derived exosomal hsa-miR-21-5p promoted proliferation,motility,migration,and invasion of SACC cells.CONCLUSIONS:TAMs-derived exosomal hsa-miR-21-5p promoted malignant progression of SACC cells.TAMs-derived exosomal miRNAs may play important roles in the progression of SACC,and may provide a potential strategy for the diagnosis and treatment of SACC.

关 键 词：唾液腺 腺样囊性癌 肿瘤相关巨噬细胞 外泌体 MIRNAS hsa-miR-21-5p 

分 类 号：R739.8[医药卫生—肿瘤]