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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 effect of osteopontin (OPN) splice variant on the invasiveness of glioma cells and its mechanism. Methods The normal brain tissue, WHO grade Ⅱ ~ Ⅳ glioma tissues and the osteopontin of human glioma cell lines U251, U87, SHG44 and TJ905 were respectively detected by fluorescence quantitative polymerase chain reaction or reverse transcription polymerase chain reaction The expression of OPN-a, OPN-b and OPN-c were detected by Western blot. Small interfering RNA (siRNA) technique was used to silence wild-type osteopontin and lentiviral vector. U251 and U87 cells in vitro invasiveness. The integrin α_vβ_3 receptor antibody and phosphatidylinositol 3-kinase (PI_3K) inhibitor LY294002 inhibited osteopontin and blocked the PI_3K / Akt signal transduction pathway. Western blotting was used to detect the expressions of uPA, MMP-2 and MMP-9, and the levels of serine / threonine AKT phosphorylation and nuclear factor-kappa B (NF-κB) p65 transcription into the nucleus, gelatin zymography detected MMP-2 and MMP-9 activity. Results The expression of osteopontin variants in WHO grade Ⅲ-Ⅳ gliomas and in U251 and U87 cells was highly expressed, while in normal brain tissues, WHO grade Ⅱ gliomas and SHG44 and TJ905 cells. OPN-a and OPN-c increased the phosphorylation of AKT in U251 and U87 cells and induced the transcription of NF-κB p65 into the nucleus, increased the expression of uPA, MMP-2 and MMP-9, and enhanced the activity of MMP-2 and MMP- , Thereby enhancing the invasiveness of glioma cells; OPN-b on glioma cell invasion no significant effect. Conclusion OPN-a and OPN-c can enhance the invasiveness of glioma cells by activating PI_3K / Akt / NF-κB signal transduction pathway.