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

doi:10.19438/j.cjoms.2024.03.001

中国口腔颌面外科杂志 ›› 2024, Vol. 22 ›› Issue (3): 209-215.doi: 10.19438/j.cjoms.2024.03.001

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

惠琪¹, 高万鹏^1,2, 李欢¹, 赵琦¹, 王珺¹, 魏建华¹, 杨新杰¹, 杨子桧¹

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

收稿日期:2022-10-08 修回日期:2024-01-26 出版日期:2024-05-20 发布日期:2024-06-11
通讯作者: 杨子桧,E-mail: 493830859@qq.com
作者简介:惠琪（2001-）,女,本科,E-mail: 2511611029@qq.com
基金资助:
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）

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

XI Qi¹, GAO Wan-peng^1,2, LI Huan¹, ZHAO Qi¹, WANG Jun¹, WEI Jian-hua¹, YANG Xin-jie¹, YANG Zi-hui¹

1. 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;
2. College of Stomatology & Affiliated Stomatological Hospital, Jiamusi University; Heilongjiang Provincial Key Laboratory of Oral Biomedical Materials and Clinical Applications. Jiamusi 154000, Heilongjiang Province, China

Received:2022-10-08 Revised:2024-01-26 Online:2024-05-20 Published:2024-06-11

摘要/Abstract

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。

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

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 according 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 expression 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 validate 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 significantly expressed(P<0.05). Enrichment analysis showed that the target genes of TAMs-derived exosomes were mainly involved 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.

Key words: Salivary gland, Adenoid cystic carcinoma, Tumor-associated macrophages, Exosomes, miRNAs, hsa-miR-21-5p

中图分类号:

R739.8

惠琪, 高万鹏, 李欢, 赵琦, 王珺, 魏建华, 杨新杰, 杨子桧. 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[J]. 中国口腔颌面外科杂志, 2024, 22(3): 209-215.

XI Qi, GAO Wan-peng, LI Huan, ZHAO Qi, WANG Jun, WEI Jian-hua, YANG Xin-jie, YANG Zi-hui. Screening and validation of miRNAs differentially expressed by exosomes of tumor-associated macrophages in microenvironment of salivary gland adenoid cystic carcinoma[J]. China J Oral Maxillofac Surg, 2024, 22(3): 209-215.

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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

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