摘要
目的:探讨骨肉瘤外泌体miR-21对骨肉瘤细胞侵袭能力的影响以及对血管生成的作用机制。方法:qRT-PCR法检测人正常骨细胞hFOB1.19与人骨肉瘤细胞系U2OS、Saos-2和MG-63中miR-21的表达,并确定表达水平高的作为本研究细胞系。通过差速离心提取细胞外泌体,并采用透射电镜和Western blot鉴定该外泌体;通过免疫荧光染色检测外泌体能否进入人血管内皮细胞(human vascular endothelial cells,HUVEC)发挥作用;利用脂质体2000分别转染Control、miR-21-inhibitor以及miR-21-mimic,通过Transwell实验检测外泌体miR-21对U2OS细胞侵袭能力的影响,并进一步采用Western blot检测miR-21对血管相关蛋白VEGF、HIF-1α以及PI3K/AKT通路蛋白的影响。结果:人骨肉瘤细胞系Saos-2、MG-63和U2OS中miR-21均呈高表达。透射电镜观察到,外泌体呈圆形或椭圆形泡状结构,且其特征蛋白CD9、CD63和CD81蛋白在外泌体中较U2OS细胞中表达高。免疫荧光结果显示,U2OS外泌体能成功进入HUVEC细胞中发挥作用。Transwell实验结果表明,外泌体miR-21可以促进骨肉瘤细胞侵袭。Western blot结果表明,与Control组相比,miR-21-mimic组血管生成蛋白VEGF和HIF-1α表达水平显著上升,PI3K/AKT通路蛋白表达水平亦显著升高,差异均有统计学意义(P<0.05)。结论:U2OS外泌体miR-21可以促进骨肉瘤细胞侵袭和血管生成,其作用机制可能与miR-21进入细胞激活PI3K/AKT信号通路有关。
Objective:To investigate the effect of osteosarcoma exosomes miR-21 on the invasion ability of osteosarcoma cells and the mechanism of action on angiogenesis.Methods:qRT-PCR method was used to detect the expression of miR-21 in human normal bone cells hFOB1.19 and human osteosarcoma cell lines U2OS,Saos-2 and MG-63,and the cell line of the high expression level was confirmed as the cell line of this study.Cell exosomes were extracted by differential centrifugation,and the exosomes were identified by transmission electron microscopy and Western blot.Detect whether exosomes can enter human vascular endothelial cells(HUVEC)to play a role by immunofluorescence staining.Use liposome 2000 to transfect Control,miR-21-inhibitor and miR-21-mimic,respectively.The effect of exosomal miR-21 on the invasion ability of U2OS cells was detected by Transwell experiment,and Western blot was further used to detect the effects of exosomal miR-21 on vascular-related proteins VEGF,HIF-1αand PI3K/AKT pathway proteins.Results:miR-21 was highly expressed in human osteosarcoma cell line Saos-2,MG-63 and U2OS.Transmission electron microscopy showed that the exosomes showed a round or elliptical bubble-like structure,and their characteristic proteins CD9,CD63 and CD81 were more highly expressed in exosomes than in U2OS cells.The results of immunofluorescence staining showed that U2OS exosomes can successfully enter HUVEC cells to play a role.The results of the Transwell experiment showed that exosomal miR-21 can promote the invasion of osteosarcoma cells.Western blot results showed that,compared with the Control group,the expression levels of angiogenic proteins VEGF and HIF-1αin the miR-21-mimic group increased significantly,and the expression levels of PI3K/AKT pathway proteins also increased significantly,and the differences were statistically significant(P<0.05).Conclusion:U2OS exosomes miR-21 can promote osteosarcoma cell invasion and angiogenesis,and its mechanism of action may be related to the activation of PI3K/AKT signaling path
作者
潘光银
杨家福
吴佳奇
瞿刚波
袁俊
PAN Guangyin;YANG Jiafu;WU Jiaqi;QU Gangbo;YUAN Jun(Bone Traumatology Department,Southwest Medical University,Sichuan Luzhou 646000,China;Trauma Surgery,Affiliated Hospital of Traditional Chinese Medicine,Southwest Medical University,Sichuan Luzhou 646000,China)
出处
《现代肿瘤医学》
CAS
北大核心
2022年第23期4221-4226,共6页
Journal of Modern Oncology
基金
国家自然科学基金委员会资助项目(编号:81373656)
四川省泸州市政府-西南医大联合课题(重点项目)(编号:2019LZXNYDZ13)。
