外泌体微小RNA-let-7c靶向结合AGO1调控多发性骨髓瘤血管生成  

Exosomal microRNA-let-7c can regulate angiogenesis in multiple myeloma by targeting AGO1

作　　者：田翔宇[1] 孙淼淼[2] 杨琛擘 陈超[4] 王帅元 舒姣 陈奎生[2] Tian Xiangyu;Sun Miaomiao;Yang Chenbo;Chen Chao;Wang Shuaiyuan;Shu Jiao;Chen Kuisheng(Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450001,China;Department of Pathology,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450001,China;School of Medical Sciences,Zhengzhou University,Zhengzhou 450001,China;Department of Neurosurgery,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450001,China)

机构地区：[1]郑州大学第一附属医院骨科,郑州450000 [2]郑州大学第一附属医院病理科,郑州450000 [3]郑州大学医学科学院,郑州450000 [4]郑州大学第五附属医院神经外科,郑州450000

出　　处：《中华实验外科杂志》2023年第12期2541-2547,共7页Chinese Journal of Experimental Surgery

基　　金：国家自然基金项目资助(82370208、82070222、81873455);河南省自然科学基金青年项目(232300420245);河南省中青年卫生健康科技创新领军人才(YXKC2022015);河南省博士后科研启动基金(298449)。

摘　　要：目的探究多发性骨髓瘤外泌体中微小RNA(miR)-let-7c对血管内皮细胞生物学功能的影响及机制。方法从多发性骨髓瘤初治患者的骨髓活检标本中原代培养出骨髓瘤内皮细胞,慢病毒感染后建立miR-let-7c过表达或抑制内皮细胞稳染株,使用细胞计数试剂盒(CCK-8)、Transwell迁移实验及小管形成实验检测感染后内皮细胞的生物学行为改变,并采用生物学信息技术预测miR-let-7c与argonaute1(AGO1)蛋白的靶向调控区,双荧光素酶报告基因实验论证两者之间的靶向调控作用。构建多发性骨髓瘤移植瘤小鼠模型,采用实时定量聚合酶链式反应(qRT-PCR)检测各组移植瘤组织中miR-let-7c的表达水平,免疫组织化学标记内皮细胞表面CD31来计算各组肿瘤微血管密度(MVD);采用蛋白印迹法检测各组移植瘤组织中缺氧诱导因子1α(HIF-1α)、AGO1及血管内皮生长因子(VEGF)的蛋白表达;采用qRT-PCR检测各组移植瘤组织中HIF-1α、AGO1及VEGF的mRNA表达。两组均数间用独立样本t检验,两组以上均数的比较用方差分析。结果用CCK-8试剂盒检测发现,接种后2 d LV3-miR-let-7c mimics组细胞增殖能力较NC组出现明显增强(t=25.185,P<0.05),而LV3-miR-let-7c inhibitor组细胞增殖能力较NC组明显减慢(t=8.708,P<0.05)。通过Transwell迁移实验证实,Mimics组细胞迁移能力较NC组明显增强(t=30.261,P<0.001),Inhibitor组则较对照组明显下降(t=9.459,P<0.01),小管形成实验结果显示,Mimics组细胞体外成管长度较NC组明显增强[(17.861±0.550)mm比(13.632±0.500)mm,t=5.685,P<0.01],而Inhibitor组体外成管长度则较NC组明显下降[(7.180±0.144)mm比(13.632±0.500)mm,t=12.514,P<0.001]。采用蛋白印迹法及qRT-PCR发现,Mimics组VEGF蛋白及mRNA相对水平较NC组明显增加(VEGF蛋白:t=12.720,P<0.001;mRNA:t=12.493,P<0.01),而AGO1蛋白及mRNA水平则较NC组降低(AGO1蛋白:t=6.544,P<0.001;mRNA:t=5.433,P<0.05),Inhibitor组中AGO1蛋白及mRNA水平明显升高Objective To explore the effect and mechanism of microRNA(miR)-let-7c in multiple myeloma exosomes on the biological functions of vascular endothelial cells.Methods Myeloma endothelial cells were primary cultured from bone marrow biopsy specimens of newly treated patients with multiple myeloma.After lentiviral infection,stable endothelial cell lines overexpressing or inhibiting miR-let-7c were established.The cell counting kit(CCK-8),Transwell cell migration assay and tuber formation assay were used to detect the biological behavior changes of endothelial cells after infection.Bioinformatics analysis was used to predict the target regulatory regions of miR-let-7c with argonaute1(AGO1),and Dual-Luciferase reporter gene experiments were used to demonstrate the targeted regulatory relationship.A mouse model of multiple myeloma transplanted tumors was constructed,and quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the expression level of miR-let-7c in the transplanted tumor tissues of each group.Immunohistochemical labeling of endothelial cell surface CD31 was used to calculate the microvessel density(MVD)of each group;Western blotting was used to detect the expression of hypoxia-inducible factor 1α(HIF-1α),AGO1 and vascular endothelial growth factor(VEGF)protein in the transplanted tumor tissues of each group;qRT-PCR was used to detect the expression of HIF-1α,AGO1 and VEGF mRNA in the transplanted tumor tissues.The independent samples t-test was used to compare the means of two groups,and the analysis of variance(ANOVA)was used to compare the means of more than two groups.Results By using the CCK-8 kit,it was found that the cell proliferation ability of the LV3-miR-let-7c mimics group was significantly enhanced compared with the NC group 2 days after inoculation(t=25.185,P<0.05),while the cell proliferation of the LV3-miR-let-7c inhibitor group was significantly slowed down(t=8.708,P<0.05).The Transwell migration experiment confirmed that the migration ability of cells in the Mimics g

关 键 词：多发性骨髓瘤 微小RNA-let-7c 外泌体 血管生成 

分 类 号：R733.3[医药卫生—肿瘤]