摘要
目的探讨艾地骨化醇(eldecalcitol,ED-71)对骨肉瘤细胞增殖、转移和富含半胱氨酸蛋白61(CYR61)表达的影响。方法首先,使用Lipofectamine 2000将CYR61过表达重组pcDNA3.1质粒(pcDNA3.1-CYR61组)及阴性对照pcDNA3.1质粒(pcDNA3.1-NC组)转染到人骨肉瘤细胞系(U2OS)中,通过RT-PCR和Western blot验证转染效率。然后,将U2OS细胞分为对照组、ED-71组、ED-71+pcDNA3.1-NC组和ED-71+pcDNA3.1-CYR61组,对照组和ED-71组细胞未进行转染,ED-71+pcDNA3.1-NC组细胞转染pcDNA3.1-NC,ED-71+pcDNA3.1-CYR61组细胞转染pcDNA3.1-CYR61。转染后,对照组细胞不做药物处理,其余3组细胞用含有5 nmol/L艾地骨化醇的1640培养基培养24 h。CCK-8法和集落形成实验测定U2OS细胞的增殖能力,流式细胞术检测细胞凋亡,Transwell实验检测细胞迁移和侵袭,RT-PCR检测细胞中CYR61 mRNA水平,Western blot检测细胞中CYR61、Bax、Bcl-2、MMP-2、MMP-9、Fibronectin、E-cadherin和N-cadherin的蛋白表达水平。结果与pc-DNA3.1-NC组比较,pcDNA3.1-CYR61组细胞中CYR61的mRNA和蛋白表达水平升高(P<0.001)。与对照组比较,ED-71组中CYR61的mRNA和蛋白表达水平降低(P<0.001),OD_(450 nm)值降低(P<0.001),集落数量降低(P<0.001),细胞凋亡率升高(P<0.001),Bax蛋白表达水平升高(P<0.001),Bcl-2蛋白表达水平降低(P<0.001),迁移和侵袭细胞数降低(P<0.001),MMP-2、MMP-9和Fibronectin蛋白表达水平降低(P<0.001),E-cadherin蛋白表达水平升高(P<0.001),N-cadherin蛋白表达水平降低(P<0.001)。与ED-71+pcDNA3.1-NC组比较,ED-71+pcDNA3.1-CYR61组U2OS细胞中CYR61的mRNA和蛋白表达水平升高(P<0.001),集落数量升高(P<0.001),OD_(450 nm)值升高(P<0.001),细胞凋亡率降低(P<0.001),Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高(P<0.001),迁移和侵袭细胞数升高(P<0.001),MMP-2、MMP-9和Fibronectin蛋白相对表达水平升高(P<0.001),E-cadherin蛋白表达水平降低(P<0.001),N-cadherin蛋白表达水平升高(P<0.001)。结论艾地骨化�
Objective To reveal the effects of eldecalcitol(ED-71)on proliferation,metastasis and CYR61 expression of osteosarcoma cells.Methods Firstly,CYR61 overexpression recombinant pcDNA3.1 plasmid(pcDNA3.1-CYR61 group)and negative control pcDNA3.1 plasmid(pcDNA3.1-NC group)were transfected into human osteosarcoma cell line(U2OS)with Lipofectamine 2000.The transfection efficiency was verified by RT-PCR and Western blot.Then,U2OS cells were divided into control group,ED-71 group,ED-71+pcDNA3.1-NC group and ED-71+pcDNA3.1-CYR61 group.Cells in control group and ED-71 group were not transfected,cells in ED-71+pcDNA3.1-NC group were transfected with pcDNA3.1-NC,and cells in ED-71+pcDNA3.1-CYR61 group were transfected with pcDNA3.1-CYR61.After transfection,cells in control group were not treated with drugs,while cells in the other three groups were cultured in 1640 medium containing 5 nmol/L ED-71 for 24 h.CCK-8 method and colony formation experiment were used to determine the proliferation ability of U2OS cells.Cell apoptosis was detected by flow cytometry,cell migration and invasion were detected by Transwell test,CYR61 mRNA levels in cells were detected by RT-PCR,and the protein levels of CYR61,Bax,Bcl-2,MMP-2,MMP-9,Fibronectin,E-cadherin and N-cadherin in cells were detected by Western blot.Results Compared with pcDNA3.1-NC group,the mRNA and protein expression levels of CYR61 were increased in pcDNA3.1-CYR61 group(P<0.001).Compared with control group,the mRNA and protein expression levels of CYR61 were decreased in ED-71 group(P<0.001),the OD_(450 nm)value was decreased(P<0.001),the colony number was decreased(P<0.001),the apoptosis rate was increased(P<0.001),the expression level of Bax protein was increased(P<0.001),the expression level of Bcl-2 protein was decreased(P<0.001),the numbers of migratory and invasive cells were decreased(P<0.001),the expression levels of MMP-2,MMP-9 and Fibronectin protein were decreased(P<0.001),the expression level of E-cadherin protein was increased(P<0.001),and the expression level of
作者
王海鹏
陈明
黄韬
杨彤涛
廖博
韩先伟
WANG Haipeng;CHEN Ming;HUANG Tao;YANG Tongtao;LIAO Bo;HAN Xianwei(Department of Orthopaedics,Second Affiliated Hospital of Air Force Medical University,Tangdu Hospital,Xi’an 710038,China)
出处
《山西医科大学学报》
CAS
2023年第1期38-47,共10页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(82174266)。
