EPHB4对骨肉瘤细胞恶性进展的调控作用及机制研究OA
Regulation of EPHB4 on Malignant Progression of Osteosarcoma Cells
目的 探讨肝配蛋白B4(erythropoietin-producing hepatocyte receptor B4,EPHB4)对骨肉瘤恶性进展的调控与作用机制.方法 蛋白质印迹法检测U2OS、SaOS2-LM7、SaOS2、143B中EPHB4 的表达变化.将SaOS2-LM7 分为对照组、si-NC 组、si-EPHB4 组、sEPHB4(可溶性 EPHB4 封闭抗体)组,利用CCK-8 法和EdU染色检测细胞增殖,利用流式细胞术检测细胞凋亡,利用Transwell实验和细胞划痕实验检测细胞侵袭迁移能力.用红色荧光蛋白(red fluorescent protein,RFP)标记的 SaOS2-LM7 构建稳定沉默EPHB4 的细胞(EPHB4-si-RNA-RFP).在体内实验中,将 24 只荷瘤裸鼠分为 2 组,即 control-RFP 组和EPHB4-siRNA-RFP组.通过小动物活体成像仪检测1～4 周后癌细胞的肺转移情况.免疫组织化学法分析4周后肿瘤组织中EPHB4、分化簇33(cluster of differentiation 33,CD33)、细胞周期蛋白D1(CYCLIN D1)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)、MMP-14、β-CATENIN的表达.结果 与U2OS比较,EPHB4 在SaOS2-LM7、SaOS2、143B中的表达增加(P均<0.05).SaOS2-LM7 细胞实验中,与对照组比较,sEPHB4 组和si-EPHB4 组的EPHB4 表达、细胞的增殖活力和EdU阳性率显著降低,凋亡率显著增加,迁移率和侵袭率显著降低(P均<0.05).此外,与对照组比较,sEPHB4 组和si-EPHB4 组的β-CATE-NIN、CYCLIN D1、MMP-9、MMP-14 的相对表达水平也显著降低(P均<0.05).在体内实验中,EPHB4-siRNA-RFP组在3 周和4 周的肺部转移率显著低于control-RFP组(P均<0.05).EPHB4-siRNA-RFP组的肿瘤组织中EPHB4、CD33、CYCLIN D1、MMP-9、MMP-14、β-CATENIN的表达均下调(P均<0.05).结论 沉默EPHB4 能显著抑制骨肉瘤细胞中WNT/β-CATENIN信号通路的激活,抑制细胞的增殖、迁移和侵袭,促进细胞凋亡.
Objective To explore the effect of erythropoietin-producing hepatocyte receptor B4(EPHB4)on the malignant progression of osteosarcoma and its mechanism.Methods The expres-sion level of EPHB4 in U2OS,SaOS2-LM7,SaOS2,and 143B was detected by Western blotting.SaOS2-LM7 cells was divided into 4 groups:control group,si-NC group,si-EPHB4 group,and sEPHB4(soluble EPHB4 blocking antibody)group.Cell proliferation was detected by CCK-8 assay and EdU staining,cell apoptosis was detected by flow cytometry,cell invasion and migration were detected by Transwell assay and Wound healing assay.A SaOS2-LM7 cell line la-beled with red fluorescent protein(RFP)which was stably silenced of EPHB4 was constructed(EPHB4-si-RNA-RFP).Twenty-four tumor-bearing nude mice were divided into 2 groups:the con-trol-RFP group and the EPHB4-si-RNA-RFP group.The lung metastasis was detected 1-4 weeks later by using small animal in vivo imaging system.Immunohistochemistry was used to analyze the expres-sion of EPHB4,cluster of differentiation 33(CD33),CYCLIN D1,matrix metalloproteinase 9(MMP-9),MMP-14,and β-CATENIN in tumor tissues 4 weeks later.Results The expression level of EPHB4 in the SaOS2-LM7,SaOS2,and 143B was significantly higher than that in the U2OS(all P<0.05).Compared with those in the control group,the expression level of EPHB4,the proliferation activity,and the EdU positive rate of SaOS2-LM7 in the sEPHB4 group and the si-EPHB4 group were significantly decreased,the apoptosis rate was significantly increased,and the migration rate and invasion rate were significantly decreased(all P<0.05).In addition,the rela-tive expression levels of β-CATENIN,CYCLIN D1,MMP-9,and MMP-14 in the sEPHB4 group and the si-EPHB4 group were also significantly decreased when compared with those in the control group(all P<0.05).The lung metastasis at 3 weeks and 4 weeks was less in the EPHB4-siRNA-RFP group than in the control-RFP group(both P<0.05).The protein expression of EPHB4,CD33,CYCLIN D1,MMP-9,MMP-14,and β-CATENIN in the tumor tissues of the EPHB4-siR-NA-RFP group was down-regulated(all P<0.05).Conclusion Silencing EPHB4 significantly in-hibits the activation of the WNT/β-CATENIN signaling pathway in osteosarcoma cells,suppresses cell proliferation,migration,and invasion,and promotes cell apoptosis.
张飞;陆红祥;买买提克里木·吐松江;杨童磊;刘秋宏;许刚
新疆军区总医院创伤骨科(关节组),乌鲁木齐市,830000新疆军区总医院创伤骨科(关节组),乌鲁木齐市,830000新疆军区总医院创伤骨科(关节组),乌鲁木齐市,830000新疆军区总医院创伤骨科(关节组),乌鲁木齐市,830000新疆军区总医院影像科普放室,乌鲁木齐市,830000新疆军区总医院创伤骨科(关节组),乌鲁木齐市,830000
医药卫生
肝配蛋白B4骨肉瘤迁移侵袭凋亡WNT/β-CATENIN信号通路
erythropoietin-producing hepatocyte receptor B4osteosarcomamigrationinva-sionapoptosisWNT/β-CATENIN signaling pathway
《医学分子生物学杂志》 2026 (1)
51-59,9
新疆军区总医院院内课题-喀喇昆仑人才基金拔尖项目(No.2022BJ001) This work was supported by a grant from the Internal Research Project of Xinjiang Military Region General Hospital-"Karakoram"Talent Fund Top-notch Project(No.2022BJ001)
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