骨桥蛋白剪接变体对胶质瘤细胞侵袭性的影响

The effects of osteopontin splicing variants on glioma cell invasion in vitro

目的探讨骨桥蛋白(OPN)剪接变体对胶质瘤细胞侵袭性的影响及其作用机制。方法采用荧光定量聚合酶链反应或逆转录-聚合酶链反应分别检测正常脑组织、WHOⅡ～Ⅳ级胶质瘤组织,以及人胶质瘤细胞系U251、U87、SHG44和TJ905骨桥蛋白各剪接变体表达水平;小干扰RNA(siRNA)技术沉默野生型骨桥蛋白,慢病毒质粒表达载体分别导入三种突变型剪接变体OPN-a、OPN-b和OPN-c;细胞体外侵袭实验检测U251和U87细胞体外侵袭力。整合素α_vβ_3受体抗体和磷脂酰肌醇3-激酶(PI_3K)抑制剂LY294002抑制骨桥蛋白的作用,阻断PI_3K/Akt信号转导通路。Western blotting法检测侵袭相关蛋白尿激酶型纤溶酶原激活物(uPA)、基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)表达水平,以及丝氨酸/苏氨酸蛋白激酶AKT磷酸化水平和核因子-κB(NF-κB)p65转录进入细胞核水平,明胶酶谱法检测MMP-2和MMP-9活性。结果 WHOⅢ～Ⅳ级胶质瘤组织,以及U251和U87细胞骨桥蛋白各剪接变体呈高表达,而正常脑组织、WHOⅡ级胶质瘤组织及SHG44和TJ905细胞呈低表达。OPN-a和OPN-c可升高U251和U87细胞AKT磷酸化水平,诱导NF-κB p65转录进入细胞核,提高uPA、MMP-2和MMP-9表达水平,增强MMP-2和MMP-9活性,从而增强胶质瘤细胞侵袭力;OPN-b对胶质瘤细胞侵袭力无明显作用。结论 OPN-a和OPN-c通过激活PI_3K/Akt/NF-κB信号转导通路而增强胶质瘤细胞侵袭力。

Objective To investigate the role and mechanism of osteopontin （OPN） splicing variants （OPN-a, OPN-b, OPN-c）on glioma cell invasion in vitro： Methods The relative expression of OPN splicing variants in normal brain tissue, World Health Organization （WHO） Ⅱ -Ⅳ glioma tissues, and human glioma cell lines U251, U87, SHG44 and TJ905 were determined by real- time fluorescence quantitative polymerase chain reaction （PCR） and reverse transcriptase （RT）-PCR. Wild OPN was knocked down by small interference RNA （siRNA） and transfected with OPN splicing variants, respectively. Additionally, anti-integrin α,β antibody and phosphoinostide 3-kinase （PLK） inhibitor LY294002 were used to block the binding of OPN and PI3K/Akt signal transduction pathway, respectively. Invasion related- proteins, urokinase, type plasminogen activator （uPA）, matrix metalloproteinase- 2 （MMP- 2）, matrix metalloproteinase-9 （MMP-9）, pAKT and nuclear factor-κB （NF-κB） were determined by Transwell assay, Western blotting and gelatin zymography. Results OPN-a, OPN-b and OPN-c high expression were seen in WHO Ⅲ-Ⅳ glioma tissues and U251 and U87 cells, while low expression in normal brain tissue, WHO Ⅱ glioma tissue and SHG44 and TJ905 cells. Over-expression OPN-a and OPN-c up-regulated uPA, MMP- 2, MMP-9, pAKT and NF-κB expression and promoted U251 and U87 cells invasion. Over-expression OPN- b had no obvious effect on invasion. Conclusion OPN-a and OPN-c, but not OPN-b promote U251 and U87 cells invasion by activation of PI3K/Akt/NF-κB signal transduction pathway.