破骨细胞外泌体miR-183-5p靶向程序性细胞死亡因子4促进肺腺癌细胞增殖及侵袭

王厚磊; 王会仁

doi:10.12025/j.issn.1008-6358.2023.20231336

破骨细胞外泌体miR-183-5p靶向程序性细胞死亡因子4促进肺腺癌细胞增殖及侵袭

Osteoclast-derived exosome miR-183-5p targeting programmed cell death 4 promotes proliferation and invasion of lung adenocarcinoma cells

摘要

摘要:
目的探讨破骨细胞来源外泌体中miR-183-5p对肺腺癌细胞侵袭和增殖的影响及其可能的机制。
方法采用RAW264.7细胞诱导分化破骨细胞并提取外泌体，通过透射电子显微镜及激光粒度仪进行鉴定。将外泌体与肺腺癌细胞A549细胞共培养，采用qRT-PCR检测A549细胞中miR-183-5p表达水平。采用划痕实验和Transwell侵袭实验检测miR-183-5p对A549细胞迁移和侵袭能力的影响；采用CCK-8检测共培养A549细胞增殖活性，Annexin Ⅴ/PI染色法检测共培养A549细胞凋亡率。采用Western印迹法检测共培养A549细胞中程序性细胞死亡因子4（programmed cell death 4，PDCD4）、细胞周期蛋白D1（cyclin D1，CCND1）和细胞周期蛋白依赖性激酶4（cyclin-dependent kinase 4，CDK4）相对表达水平。
结果外泌体呈边缘透亮清晰的椭圆形囊泡状结构，粒径30~200 nm；荧光探针发现外泌体能较快被A549摄取。共培养后A549细胞中miR-183-5p表达水平显著升高（P < 0.01）。划痕实验和Transwell实验显示，共培养72 h时A549细胞的迁移和侵袭能力高于共培养24 h时（P < 0.01）；CCK-8实验、Annexin Ⅴ/PI染色法显示，共培养A549细胞活性高于对照组（P < 0.01）、细胞凋亡率低于对照组（P < 0.05）。Western印迹法显示，A549细胞中miR-183-5p过表达后，PDCD4表达降低，CCND1、CDK4表达增加（P < 0.05）。
结论破骨细胞来源外泌体与A549细胞共培养可提高细胞中miR-183-5p的表达；miR-183-5p可能通过靶向抑制PDCD4表达等促进肺腺癌细胞侵袭、增殖和迁移能力。

Abstract:
Objective To explore the effect of osteoclast-derived exosome miR-183-5p on the invasion and proliferation of lung adenocarcinoma cells and its possible mechanisms.
Methods Osteoclasts were induced by RAW264.7 cells and the exosomes were extracted and identified by transmission electron microscope and dynamic light scattering. The exosomes were co-cultured with lung adenocarcinoma cell line A549 cells, and the expression of miR-183-5p in A549 cells was detected by qRT-PCR. Scratch test and Transwell invasion test were used to observe the effect of miR-183-5p on the migration and invasion of A549 cells. CCK-8 was used to detect the proliferation activity of co-cultured A549 cells, and Annexin Ⅴ/PI staining was used to detect the apoptosis of A549 cells. Western blotting was used to detect the relative expression of programmed cell death 4 (PDCD4), cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) in co-cultured A549 cells.
Results The exosome showed a clear oval vesicle-like structure with a clear edge, and the particle size was 30-200 nm. Fluorescence probe showed that the exosome was quickly absorbed by A549 cells, and the expression level of miR-183-5p in A549 cells increased significantly after co-culture (P < 0.01). The results of scratch test and Transwell assay showed that the migration and invasion ability of co-cultured A549 cells at 72 h was significantly higher than that at 24 h (P < 0.01). The CCK-8 and Annexin Ⅴ/PI tests showed that the activity of co-cultured A549 cells was significantly higher than that in the control group (P < 0.01), and the apoptosis rate of co-cultured A549 cells was significantly lower than that in the control group (P < 0.05). Western blotting showed that after overexpression of miR-183-5p, the expression of PDCD4 decreased, and the expression of CCND1 and CDK4 increased in A549 cells (P < 0.05).
Conclusions Osteoclast-derived exosomes co-cultured with A549 cells can increase the expression of miR-183-5p, and miR-183-5p can promote the invasion, proliferation and migration of lung adenocarcinoma cells by targeting inhibition of PDCD4 expression, etc..

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