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
惠琪 1高万鹏 2李欢 1赵琦 1王珺 1魏建华 1杨新杰 1杨子桧1
作者信息
- 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. 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;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
- 折叠
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1 595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.
Abstract
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 1 595 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-21-5pKey words
Salivary gland/Adenoid cystic carcinoma/Tumor-associated macrophages/Exosomes/miRNAs/hsa-miR-21-5p引用本文复制引用
基金项目
国家自然科学基金青年基金(82002867)
国家自然科学基金面上项目(82173165)
国家自然科学基金面上项目(81973114)
陕西省重点研发计划(2023-YBSF-230)
出版年
2024
NETL
NSTL
万方数据