CREB3通过下调FAK磷酸化水平抑制胶质瘤细胞增殖及侵袭转移的体外实验研究

Knockdown of CREB3 inhibits proliferation, migration, and invasion of glioma cells by decreasing phosphorylated FAK (Tyr397) expression

目的研究环磷酸腺苷反应原件结合蛋白3(CREB3)基因与胶质瘤患者的生存预后相关性,以及其在胶质瘤细胞增殖及侵袭转移等恶性生物学行为中的功能作用及发生机制,为寻找有效的胶质瘤分子治疗靶点提供理论依据。方法CREB3的mRNA表达水平及其存活率来自中国胶质瘤基因组图谱(CGGA)数据库。免疫组化(IHC)验证在不同级别的人脑胶质瘤组织中CREB3和磷酸化部黏着斑激酶397位点(p-FAK397)蛋白的表达情况。慢病毒构建U87、U251的CREB3的敲低模型。利用蛋白质印记技术(WB)验证p-FAK和其通路上相关蛋白以及相关凋亡蛋白的表达情况。使用细胞增殖实验(CCK-8)计算细胞在450 nm出的光密度(OD)及平板克隆实验验证敲低慢病毒敲低CREB3后细胞的增殖能力,利用细胞划痕,Transwell实验验证敲低细胞系的迁移和侵袭的能力。结果根据CGGA数据库,发现CREB3在高级别胶质瘤中表达上调,提示预后不良(P<0.05)。IHC表明随着胶质瘤级别的增高CREB3表达随之增加,而p-FAK(397)则在Ⅳ级胶质瘤中明显表达。构建慢病毒敲低CREB3的U87、U251细胞系通过WB表明可以使p-FAK(397)的表达下降,促凋亡蛋白(BAX)表达增加,抗凋亡蛋白(Bcl-2)表达减少(P<0.05),并且可以在体外抑制胶质瘤的增殖、迁移和侵袭(P<0.05)。结论CREB3在高级别胶质瘤组织中表达增高,且与患者生存预后密切相关。敲低CREB3下调胶质瘤细胞中FAK的磷酸化水平,进而介导胶质瘤细胞的增殖及侵袭迁移能力变弱。

Objective To investigate the relationship between cAMP response binding protein 3(CREB3)gene and the survival prognosis of glioma patients,and its functional role and mechanism in malignant biological behaviors of glioma cells,such as proliferation,invasion,and metastasis,so as to provide a theoretical basis for finding effective molecular therapeutic targets for glioma.Methods The data on the mRNA expression level of CREB3 and survival rate were obtained from the Chinese Glioma Genome Atlas(CCGA)database.Immunocytochemistry(IHC)was performed to verify the expression of CREB3 and phosphorylated focal adhesion kinase 397(p-FAK)protein in different grades of human glioma tissues.Lentiviruses were used to construct CREB3 knockdown models in the glioma cell lines U87 and U251.Western blot(WB)was used to verify the expression of p-FAK and related proteins in the FAK signaling pathway,as well as related apoptotic proteins.Cell Counting Kit-8 was used to calculate the optical density(OD)of cells at 450 nm,plate cloning assay was used to verify the proliferation ability of cells after knockdown of CREB3,and cell scratch and Transwell assays were used to verify the migration and invasion ability of knockdown cell lines.Results According to the CGGA database,CREB3 expression was found to be up-regulated in high-grade gliomas,indicating a poor prognosis(P<0.05).IHC showed that CREB3 expression increased with increasing glioma grade,while p-FAK(397)was significantly expressed in grade IV gliomas.Knockdown of CREB3 decreased the expression of p-FAK(397),increased the expression of the pro-apoptotic protein BCL2-associated X(BAX),decreased the expression of the anti-apoptotic protein B-cell lymphoma-2(Bcl-2)(P<0.05),and inhibited the proliferation,migration,and invasion of glioma cells in vitro(P<0.05).Conclusion CREB3 expression is increased in high-grade glioma and is closely related to the survival prognosis of patients.Knockdown of CREB3 downregulates the phosphorylation of FAK in glioma cells,which in turn reduces the proliferation,invasion,and migration of glioma cells.