PFKFB3通过调控PI3K/AKT信号通路促进骨肉瘤细胞的增殖及转移

目的:探讨6-磷酸果糖2-激酶/果糖-2,6-二磷酸酶3(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFB3)对骨肉瘤细胞增殖、凋亡、迁移及侵袭的影响,及可能的作用机制.方法:采用实时荧光定量PCR法、蛋白质印迹法及免疫组织化学法检测骨肉瘤组织及其相...

PFKFB3 promotes the growth and metastasis of osteosarcoma through PI3K/AKT signal pathway

Objective:To investigate the effects of 6-phosphofructos2 kinase/fructose-2,6-diphosphatase 3(PFKFB3)on proliferation,apoptosis,migration and invasion of osteosarcoma cells and its possible mechanism.Methods:Real-time fluorescent quantitative PCR,Western blotting and immunohistochemistry were used to detect the expression levels of PFKFB3 mRNA and protein in osteosarcoma tissues and its corresponding adjacent tissues,as well as osteosarcoma U2-OS,Saos-2,MG-63 and 143B cells(osteoblast hfoBI-19 cells as the control).The recombinant lentiviral vector carrying shRNA specific targeting PFKFB3 gene(shPFKFB3)was infected U2-OS cells,the recombinant lentiviral vector carrying the full PFKFB3 gene was infected Saos-2 cells.The CCK-8 and EdU assays to detect the effect of up-regulation or down-regulation of PFKFB3 expression on the proliferation of osteosarcoma cells,FCM method to analyze the apoptosis rate of osteosarcoma cells,the wound healing assay and Transwell chamber invasion test to detect the impact on the migration and invasion of osteosarcoma cells.The expression levels of Vimentin,E-cadherin,N-cadherin,E-box binding zinc finger protein 2(ZEB2)proteins and phosphoinositide3-kinase(PI3K)/protein kinase B(PBK,also known as AKT)signaling pathway-related protein PI3K,phospho-PI3K(p-PI3K),AKT and p-AKT were detected by Western blotting.The Saos-2 cells PFKFB3 gene overexpressing were treated with PI3K inhibitor LY294002,the PI3K/AKT signaling pathway related protein expression levels,proliferation,apoptosis and invasion were detected by Western blotting,CCK-8 method,FCM method and Transwell chamber invasion test.Results:The results of real-time fluorescent quantitative PCR and Western blotting showed that the expression levels of PFKFB3 mRNA and protein in osteosarcoma tissues were significantly increased(both P<0.01).And the immunohistochemistry also confirmed in 25 cases of osteosarcoma tissue samples,19 cases showed high expression(19/25,76%),while only 16%of the adjacent tissues showed high expression(4/25).The U2-OS cells PFKFB3 gene silencing and Saos-2 cells PFKFB3 gene overexpressing were successfully constructed.After the PFKFB3 expression of U2-OS cells was silenced,the proliferation,migration and invasion were inhibited(all P<0.05),the apoptosis rate was increased significantly(P<0.01),the expression levels of Vimentin,N-cadherin and ZEB2 proteins were significantly up-regulated,while the expression level of E-cadherin protein was significantly down-regulated.On the contrary,the proliferation,migration and invasion of Saos-2 cells that had up-regulated PFKFB3 expression were enhanced(all P<0.05),but cell apoptosis was inhibited(P<0.05);the expression levels of Vimentin,N-cadherin and ZEB2 proteins were significantly up-regulated,while the expressions of E-cadherin protein were significantly down-regulated.Mechanically,PFKFB3 promotes the growth and metastasis of osteosarcoma by affecting the phosphorylation of PI3K/AKT signal pathway.Conclusion:PFKFB3 may be a potential biomarker for osteosarcoma and anti-tumor targeting genes.