骨髓间充质干细胞外泌体miR-126-3p靶向CCR1抑制非小细胞肺癌细胞恶性增殖及转移

Bone marrow mesenchymal stem cells exosome miR-126-3p targets CCR1 to inhibit the malignant proliferation and metastasis of non-small cell lung cancer cells

目的探讨骨髓间充质干细胞(bone marrow stromal cells,BMSCs)来源的外泌体中miR-126-3p靶向趋化因子受体1(chemokine receptor 1,CCR1)对肺癌细胞增殖、迁移和侵袭的影响。方法培养BMSCs,提取外泌体并通过外泌体标志蛋白CD63和TSG101进行鉴定;外泌体共培养A549细胞不同时长(0、24、48、72 h)后,CCK-8检测细胞存活率,q RT-PCR检测miR-126-3p和CCR1 m RNA表达,Transwell实验检测细胞迁移和侵袭能力;Western blotting检测CCR1、上皮钙黏蛋白(E-cadherin,E-cad)、神经钙黏蛋白(N-cadherin,N-cad)和波形蛋白(Vimentin)相对表达。结果外泌体呈边缘高亮、中间灰暗的圆形或椭圆形杯状结构,粒径分布范围152 nm左右,有CD63、TSG101蛋白表达;外泌体中miR-126-3p表达高于A549细胞,A549细胞中CCR1 m RNA表达高于外泌体,而在A549细胞与外泌体共培养后,A549细胞中miR-126-3p表达升高,CCR1表达降低;A549细胞与外泌体共培养0、24、48、72 h,细胞的存活率、迁移和侵袭能力和CCR1、N-cad和Vimentin蛋白表达及CCR1 m RNA表达均随着培养时间的延长逐渐降低,而miR-126-3p水平和E-cad蛋白表达随着培养时间的延长逐渐升高。结论BMSCs来源的外泌体与A549细胞共培养可提高细胞中miR-126-3p的表达,miR-126-3p通过靶向抑制CCR1表达可降低A549细胞增殖、迁移和侵袭能力。

Objective To investigate the effects of miR-126-3p targeting chemokine receptor 1(CCR1)in exosomes derived from bone marrow mesenchymal stem cells(BMSC)on the proliferation,migration,and invasion of lung cancer cells.Methods BMSC cells were cultured;exosomes were extracted and identified by the exosomal marker proteins CD63 and TSG101.After exosome culture of A549 cells for different durations(0,24,48,and 72 h),cell survival rate was detected by CCK-8,m RNA levels of miR-126-3p and CCR1 were detected by q RTPCR,and cell migration and invasion abilities were detected by Transwell assay.The relative expressions of CCR1,epithelial cadherin(E-cad),neural cadherin(N-cadherin),and Vimentin were detected by Western blotting.Results Exosomes had round or oval cup-shaped structures with bright edges and dark middle,with a particle size distribution of about 152 nm,expressing CD63 and TSG101 proteins.The expression of miR-126-3p in exosomes was higher than that in A549 cells.The expression of miR-126-3p was low in A549 cells and that of CCR1 m RNA was high.However,after co-culture with exosomes,the expression of miR-126-3p in A549 cells was increased,while the expression of CCR1 was decreased.A549 cells were cocultured with exosomes for 0,24,48,and 72 h.The survival rate,migration and invasion abilities,CCR1 gene and protein expression levels,and N-cad and Vimentin protein expression levels of A549 cells decreased gradually with the extension of culture time.The level of miR-126-3p and the expression of E-cad protein increased gradually with the extension of culture time.Conclusion The co-culture of exosomes derived from bone marrow mesenchymal stem cells with A549 cells can increase the expression level of miR-126-3p,and miR-126-3p can reduce the proliferation,migration,and invasion of A549 cells by targeting the inhibition of CCR1 expression.