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中国临床药理学与治疗学 ›› 2023, Vol. 28 ›› Issue (6): 624-632.doi: 10.12092/j.issn.1009-2501.2023.06.004

• 基础研究 • 上一篇    下一篇

肺癌细胞外泌体来源的miR-718通过靶向PTEN诱导血管新生的研究

付 婷,李 芳,潘大燕,夏园园,张秋园   

  1. 湖北医药学院附属十堰市人民医院肿瘤科,十堰  442000,湖北

  • 收稿日期:2023-04-17 修回日期:2023-05-19 出版日期:2023-06-26 发布日期:2023-07-12
  • 通讯作者: 张秋园,女,本科,研究方向:肺癌的治疗相关研究。 E-mail:380441933@qq.com
  • 作者简介:付婷,女,本科,研究方向:胸部肿瘤的治疗研究。 E-mail:2095570937@qq.com

miR-718 from exosomes of lung cancer cells induces angiogenesis by targeting PTEN

FU Ting, LI Fang, PAN Dayan, XIA Yuanyuan, ZHANG Qiuyuan   

  1. Department of Oncology, Shiyan People's Hospital Affiliated to Hubei Medical College, Shiyan 442000, Hubei, China
  • Received:2023-04-17 Revised:2023-05-19 Online:2023-06-26 Published:2023-07-12

摘要:

目的:探究肺癌细胞外泌体(exosome,exo)对血管新生的影响及机制。方法:提取人肺上皮细胞HFL-1和人肺癌细胞A549、H522、H460细胞分泌的外泌体,记为HFL-1 exo、A549 exo、H522 exo、H460 exo,将上述外泌体以及等体积的PBS缓冲液与人脐静脉内皮细胞(HUVEC)共孵育,记为HFL-1 exo组、A549 exo组、H522 exo组、H460 exo组和PBS组,PBS组作为对照组。HUVEC转染PTEN siRNA和Negative control,记为si-PTEN组和si-NC组。提取转染miR-NC、miR-718 mimic、miR-718 inhibitor的A549细胞所分泌的外泌体,记为A549/miR-NC exo、A549/miR-718 mimic exo、A549/miR-718 inhibitor exo,将上述外泌体与HUVEC细胞共孵育,记为A549/miR-NC exo组、A549/miR-718 mimic exo组、A549/miR-718 inhibitor exo组,HUVEC细胞与A549/miR-718 mimic exo孵育后转染PTEN过表达质粒,记为A549/miR-718 mimic exo+PTEN组。实时荧光定量聚合酶链反应(qRT-PCR)检测各组细胞及外泌体miR-718的表达;Transwell小室法和小管形成实验检测各组细胞的迁移能力和小管形成情况;Western blot检测PTEN蛋白的表达。结果:本文所提取的HFL-1 exo、A549 exo、H522 exo、H460 exo均符合外泌体的结构特征;与PBS组比较,A549 exo、H522 exo、H460 exo组HUVEC细胞迁移数和小管形成数显著升高(P均<0.05)。与HFL-1 细胞比较,A549、H522、H460细胞中miR-718表达显著升高(P均<0.05)。与HFL-1 exo比较,A549 exo、H522 exo、H460 exo中miR-718表达显著升高(P均<0.05),并且miR-718可靶向调控PTEN的表达。相比于si-NC组,si-PTEN组HUVEC细胞小管形成和细胞迁移数显著升高(P均<0.05)。相比于A549/miR-NC exo组,A549/miR-718 mimic exo组细胞迁移和小管形成数显著增加(P均<0.05),A549/miR-718 inhibitor exo组细胞迁移和小管形成数显著减少(P均<0.05);相比于A549/miR-NC exo组,A549/miR-718 mimic exo+PTEN组细胞迁移和小管形成数显著升高(P均<0.05)。结论:肺癌细胞外泌体miR-718能够通过靶向抑制PTEN的表达而促进血管生成。

关键词: 肺癌, 外泌体, miR-718, PTEN, 血管新生

Abstract:

AIM: To explore the effect and mechanism of lung cancer cell exosomes on angiogenesis. METHODS: The exosomes secreted by human lung epithelial cells HFL-1 and human lung cancer cells A549, H522, and H460 cells were extracted and denoted as HFL-1 exo, A549 exo, H522 exo, and H460 exo. Above exosomes and a volume of PBS buffer were incubated with HUVEC cells of vascular endothelial cells, which were denoted as HFL-1 exo group, A549 exo group, H522 exo group, H460 exo group, and PBS group, the PBS group was used as the control group. HUVECs were transfected with PTEN siRNA and Negative control, denoted as si-PTEN group and si-NC group. Extract exosomes secreted by A549 cells transfected with miR-NC, miR-718 mimic, miR-718 inhibitor, and denote them as A549/miR-NC exo, A549/miR-718 mimic exo, A549/ miR-718 inhibitor exo, the above exosomes were incubated with HUVEC cells, and they were marked as A549/miR-NC exo group, A549/miR-718 mimic exo group, A549/miR-718 inhibitor exo group. After incubated with A549/miR-718 mimic exo group, the PTEN overexpression plasmid was transfected into HUVECs, and recorded as the A549/miR-718 mimic exo+PTEN group. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-718 in each group of cells and exosomes, Transwell chamber method and tubule formation experiment were used to detect the migration ability and tubule formation of each group of cells. Western blot was used to detect the expression of PTEN protein. The LSD-t test was used to compare the two groups, and P<0.05 indicated that the difference was statistically significant. RESULTS: The HFL-1 exo, A549 exo, H522 exo, and H460 exo extracted in this paper are in line with the structural characteristics of exosomes. Compared with the PBS group, the number of HUVEC cell migration in the A549 exo, H522 exo, and H460 exo groups and the number of tubule formation were significantly increased (P<0.05). Compared with HFL-1 cells, the expression levels of miR-718 in A549, H522, and H460 cells were significantly higher (P<0.05). Compared with HFL-1 exo, the expression levels of miR-718 in A549 exo, H522 exo, and H460 exo were significantly increased (P<0.05), and miR-718 could target and regulate the expression of PTEN. Compared with the si-NC group, the HUVEC cell tubule formation in the si-PTEN group and the number of cell migration were significantly increased (P<0.05). Compared with the A549/miR-NC exo group, the cell migration ability and the numbers of tubule formation in A549/miR-718 mimic exo group were significantly increased (P<0.05), while those were decreased in the A549/miR-718 inhibitor exo group. Compared with A549/miR-NC exo group, the cell migration ability and the number of tubule formation were significantly higher in A549/miR-718 mimic exo+PTEN group (P<0.05). CONCLUSION: Lung cancer cell exosomal miR-718 could promote angiogenesis by targeting to inhibit the expression of PTEN.

Key words: lung cancer, exosomes, miR-718, PTEN, angiogenesis

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