EFEMP1促进骨肉瘤侵袭及转移能力的研究

Role of EFEMP1 in promoting invasion and migration of osteosarcoma

目的检测EFEMP1在骨肉瘤组织及细胞株F4和F5M2中的表达,探讨EFEMP1在骨肉瘤细胞中的表达,明确其对骨肉瘤细胞侵袭和迁移的影响。方法采用免疫组织化学SP法检测45例临床骨肉瘤及12例骨软骨瘤病例肿瘤组织中EFEMP1表达;采用免疫印迹法(Western blot)及逆转录定量PCR(qRT-PCR)检测具有不同转移潜能的骨肉瘤细胞株(F4、F5M2)中EFEMP1的表达水平;Tanswell实验检测重组人EFEMP1对F5M2细胞侵袭和迁移的影响。结果免疫组化结果显示在45例骨肉瘤标本中,有42例EFEMP1阳性(93.3%),其中强阳性为29例(64.4%),3例(6.7%)为阴性。骨软骨瘤中EFEMP1表达均为阴性。qRT-PCR检测以及Western-blot结果显示,在mRNA和蛋白水平上,骨肉瘤细胞株F4和F5M2中均有EFEMP1的表达,F5M2细胞株EFEMP1的表达高于F4细胞株,Western-blot显示EFEMP1在F4和F5中均有表达,且在F5M2中EFEMP1表达水平高于F4(P<0.05),这一结果与qRT-PCR检测结果一致。Tanswell实验结果显示,与阳性对照组和空白对照组相比,实验组F5M2细胞的侵袭和迁移能力显著增加,重组人全长蛋白EFEMP1可促进骨肉瘤细胞的侵袭和迁移能力。结论EFEMP1在人骨肉瘤组织中表达水平增高,骨肉瘤细胞株F4及F5M2中EFEMP1表达阳性,且高转移骨肉瘤细胞株F5M2中EFEMP1表达量较低转移细胞株F4表达水平明显增高,重组人EFEMP1能够促进骨肉瘤细胞侵袭及转移。EFEMP1可为骨肉瘤患者早期诊断和治疗提供一个新的策略。

Objective To detect the expression of EGF containing fibulin-like extracellular matrix protein 1（ EFEMP1） in thetissues and cell lines（ F4 and F5M2） of osteosarcoma,and explore the its role in promoting the invasion and migrationof human osteosarcoma. Methods The expression of EFEMP1 in tumor tissue of 45 cases of osteosarcoma and 12 cases of osteochondroma were detected by using immunohistochemical SP method; the expression of EFEMP1 protein and mRNA in F4 and F5M2 cell lines was detected by using Western-blot and quantitative Real Time PCR,respectively. The effects of EFEMP1 on the invasion and migration of F5M2 cells were investigated by Transwell methods. Results EFEMP1 was expressed in 93. 3%（ 42 /45） of osteosarcoma tissues,strongly positive in 64. 4%（ 29 /45）,3（ 6. 7%） negative cases,and negative in all osteochondroma tissues. Western-blot and quantitative Real Time PCR showed that EFEMP1 was expressed differently in the human osteosarcoma cell line F4 and F5M2,F5M2 cells had a higher level（ P 〈 0. 05）. Transwell test showed that the invasion and migration of F5M2 cells in the experimental group were increased,prompting that recombinant human EFEMP1 protein can stimulate the migration and invasion of F5M2 in vitro（ P 〈 0. 05）. Conclusion EFEMP1 highly expressed in the human osteosarcoma tissues and cell lines F5M2 and F4（ expression levels in F5M2 exceed F4）. The recombinant human EFEMP1 protein can stimulate the invasion and migration of human osteosarcoma. To detect the expression of EFEMP1 will provide a new strategy for early diagnosis and therapy for osteosarcoma patients.