分化抑制因子1通过调控VEGF-A表达促进骨肉瘤血管生成  

Inhibitor of differentiation-1 promotes angiogenesis of human osteosarcoma by regulating VEGF-A

作　　者：赵郭盛 晏铮剑[1] 高自然 曹亚 郭乔楠[2] 秦晋[1] 陈富[1] ZHAO Guosheng;YAN Zhengjian;GAO Ziran;CAO Ya;GUO Qiaonan;QIN Jin;CHEN Fu(Department of Spine Surgery,the Second Affiliated Hospital of Chongqing Medical University,Chongqing,400010;Department of Pathology,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400037,China)

机构地区：[1]重庆医科大学附属第二医院脊柱外科,重庆400010 [2]陆军军医大学(第三军医大学)附属第二医院病理科,重庆400037

出　　处：《第三军医大学学报》2021年第9期813-821,共9页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81672653)。

摘　　要：目的观察分化抑制因子1(inhibitor of DNA binding 1,ID1)通过调控血管内皮生长因子A(vascular endothelial growth factor A,VEGF-A)表达对骨肉瘤143B细胞血管生成影响,并验证人骨肉瘤组织中ID1与血管生成和VEGF-A表达相关性。方法收集33例人骨肉瘤石蜡组织,利用免疫组化分析ID1表达与微血管密度(microvessel density,MVD)相关性,并结合生物信息学分析,探讨骨肉瘤中ID1与血管生成相关通路及VEGF-A表达的相关性。利用慢病毒和小干扰RNA建立差异表达ID1的143B细胞株,制备相应细胞条件培养基(conditional medium,CM),通过Transwell迁移、小管形成实验,比较各组CM对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVCEs)迁移和成管能力的影响;利用PCR、Western blot、ELISA检测差异表达ID1后143B细胞中VEGF-A的表达。结果生信分析提示ID1参与多条骨肉瘤血管生成相关通路,且VEGF-A与ID1的mRNA表达呈正相关(r=0.2441,P=0.0057)。33例人体骨肉瘤组织中ID1阳性表达组中MVD较ID1阴性组明显增高(P<0.05),且ID1与VEGF-A蛋白表达呈正相关(r=0.4643,P=0.0065)。体外实验证实过表达ID1组较对照组的CM能显著促进内皮细胞迁移和成管能力(P<0.05);与之对应,敲低ID1表达组较无效干扰组的CM能抑制内皮细胞迁移和成管能力(P<0.05)。过表达ID1的143B细胞中VEGF-A表达和分泌显著升高,而敲低ID1表达后143B细胞中VEGF-A表达和分泌降低(P<0.05)。结论ID1可能通过上调骨肉瘤细胞中的VEGF-A表达来促进骨肉瘤血管生成。Objective To investigate the effect of inhibitor of differentiation-1(ID1)on angiogenesis in osteosarcoma cell line 143B by regulating vascular endothelial growth factor A(VEGF-A),and verify the correlation of ID1 expression with angiogenesis and VEGF-A expression in human osteosarcoma tissues.Methods We investigated the expression patterns of ID1 and microvessel density(MVD)in 33 human osteosarcoma tissues by immunohistochemistry(IHC)and further analyzed the correlation between ID1 and VEGF-A expression in osteosarcoma by IHC and bioinformatics analysis.Lentiviral vector and siRNA transfection were employed to make ID1 differential expressed in osteosarcoma 143B cells.And the conditional medium(CM)were collected.Transwell assay and tube formation assay were used to determine the effect of CM collected from ID1 differential expressed 143B cells on abilities of the migration and tube formation in human umbilical vein endothelial cells(HUVECs)in vitro.PCR,ELISA and Western blot assay were adopted to detect the responses of VEGF-A expression in 143B cells in vitro,with or without ID1 differential expression.Results Bioinformatics analysis indicated that ID1 was involved in many pathways related to the angiogenesis in osteosarcoma,and the expression of VEGF-A was positively correlated with the mRNA level of ID1.In the human osteosarcoma tissues,these with positive ID1 expression had higher MVD than those with negative expression(P<0.05),and the protein level of ID1 had a positive correlation with that of VEGF-A(r=0.4643,P=0.0065).In vitro study showed that the CM collected from ID1 overexpression 143B cells significantly enhanced the abilities of migration and tube formation in HUVECs than the CM from control cells(P<0.05),and the CM from ID1-silencing 143B cells obviously suppressed the abilities(P<0.05).The expression and secretion of ID1 was enhanced in the ID1 overexpression 143B cells,but reduced in the ID1-silencing 143B cells(P<0.05).Conclusion ID1 promotes angiogenesis in human osteosarcoma by up-regulating V

关 键 词：分化抑制因子1 骨肉瘤 VEGF-A 血管生成 

分 类 号：R364.33[医药卫生—病理学] R730.23[医药卫生—基础医学]