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应癌旁组织以及骨肉瘤U2-OS、Saos-2、MG-63和143B细胞(以正常成骨细胞hfoBI-19为对照)中PFKFB3 mRNA及蛋白的表达水平。采用慢病毒感染的方法将携带特异性针对PFKFB3基因的shRNA(shPFKFB3)转入U2-OS细胞,将携带有PFKFB3全基因的重组慢病毒载体转入Saos-2细胞。分别采用CCK-8法及EdU实验检测沉默或上调PFKFB3基因表达对骨肉瘤细胞增殖能力的影响,FCM法检测对骨肉瘤细胞的凋亡率的影响,划痕愈合实验和Transwell小室侵袭实验检测对骨肉瘤细胞迁移及侵袭能力的影响,并进一步通过蛋白质印迹法检测对波形蛋白(Vimentin)、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和E盒结合锌指蛋白2(E-box binding zinc finger protein 2,ZEB2)以及PI3K/AKT信号通路中PI3K、AKT和其磷酸化蛋白表达水平的影响。用PI3K抑制剂LY294002处理PFKFB3过表达的Saos-2细胞,再用蛋白质印迹法、CCK-8法、FCM法和Transwell小室侵袭实验检测对PI3K/AKT信号通路,Saos-2细胞增殖、凋亡及侵袭能力的影响。结果:PFKFB3 mRNA及蛋白在骨肉瘤组织中的表达水平均明显上调(P值均<0.01)。免疫组织化学法检测结果证实,25例骨肉瘤组织样本中PFKFB3蛋白高表达为19例(19/25,76%),而癌旁正常组织仅有4例(4/25,16%)。成功构建沉默PFKFB3基因表达的U2-OS细胞,PFKFB3基因过表达的Saos-2细胞。沉默PFKFB3基因表达后,U2-OS细胞的增殖、迁移及侵袭能力被明显抑制(P值均<0.05),而细胞的凋亡率被明显提高(P<0.01),转移相关蛋白Vimentin、N-cadherin和ZEB2的表达水平被明显下调,而E-cadherin蛋白的表达水平明显上调;相反上调PFKFB3基因表达后,Saos-2细胞的增殖、迁移及侵袭能力被明显提高(P值均<0.05),而细胞的凋亡率被明显抑制(P<0.05),Vimentin、N-cadherin和ZEB2蛋白的表达水平均明显上调,而E-cadherin蛋白的表达水平明显下调。PI3K抑制剂LY294002处理PFKFB3过表达的Saos-2细胞后,PI3K、AKT以及上调的磷酸化PI3K(phospho-PI3K,p-PI3K)和p-AKT蛋白的表达水平均被明显抑制,Saos-2细胞的增殖及侵袭能力被抑制(P<0.05和P<0.01),而细胞的凋亡率被提高(P<0.05),提示PFKFB3可能通过影响PI3K/AKT信号通路的磷酸化进而促进骨肉瘤生长和转移。结论:PFKFB3可能是骨肉瘤和抗肿瘤靶向治疗的潜在生物标志物。

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.