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中华临床医师杂志(电子版) ›› 2023, Vol. 17 ›› Issue (02) : 202 -209. doi: 10.3877/cma.j.issn.1674-0785.2023.02.016

基础研究

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
张懿炜, 胡亚欣, 出良钊, 严昭, 曾茜, 蒲茜   
  1. 550025 贵阳,贵州医科大学临床学院
  • 收稿日期:2022-03-03 出版日期:2023-02-15

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

Yiwei Zhang, Yaxin Hu, Liangzhao Chu, Zhao Yan, Qian Zeng, Qian Pu   

  1. Clinical College of Guizhou Medical University, Guiyang 550025, China
  • Received:2022-03-03 Published:2023-02-15
引用本文:

张懿炜, 胡亚欣, 出良钊, 严昭, 曾茜, 蒲茜. 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[J]. 中华临床医师杂志(电子版), 2023, 17(02): 202-209.

Yiwei Zhang, Yaxin Hu, Liangzhao Chu, Zhao Yan, Qian Zeng, Qian Pu. Knockdown of CREB3 inhibits proliferation, migration, and invasion of glioma cells by decreasing phosphorylated FAK (Tyr397) expression[J]. Chinese Journal of Clinicians(Electronic Edition), 2023, 17(02): 202-209.

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.

图1 CREB3在胶质瘤组织中的表达。图a为CGGA网站(http://www.vgga.org.cn/index.jsp),基于癌症基因组图谱数据库中CREB3在不同级别胶质瘤样本中的表达情况;图b为基于CGGA数据库对胶质瘤患者CREB3总体存活率的Kaplan-Meier分析;图c为免疫组化检测CREB3和p-FAK蛋白在不同级别胶质瘤中的表达情况注:CREB3为环磷酸腺苷反应原件结合蛋白3;p-FAK(397)为磷酸化部黏着斑激酶;CGGA为中国胶质瘤基因组图谱
图2 病毒感染验证及WB验证CREB3敲低后相关蛋白表达。图a为荧光显微镜观察慢病毒敲低后U87和U251转染情况;图b为WB检测慢病毒感染后的U87和U251中CREB3的表达;图c为WB检测U87和U251细胞株中p-FAK、p-AKT、FAK、AKT和蛋白的表达水平;图d为WB检测U87和U251细胞系中BCL-2和BAX蛋白的表达水平(
x¯
±sn=3,*P<0.05;#P<0.05)
注:WB为蛋白质印迹实验;CREB3为环磷酸腺苷反应原件结合蛋白;p-FAK为磷酸化的局部黏着斑激酶;p-AKT为磷酸化的蛋白激酶B;BCL-2为抗凋亡蛋白;BAX为促凋亡蛋白
图3 CREB3敲低后对细胞增殖迁移和侵袭的影响。图a为细胞集落试验用于检测CREB3敲除后对U87和U251细胞系增殖的影响;图b为使用CCK-8绘制细胞增殖曲线用于检测CREB3敲除后对U87和U251细胞系增殖的影响;图c为慢病毒敲低CREB3后,使用细胞划痕验证U87和U251细胞的迁移能力;图d为Transwell实验检测U87和U251在敲低CREB3后的侵袭能力(
x¯
±sn=3,*P<0.05;#P<0.05;##P<0.01)
注:CREB3为环磷酸腺苷反应原件结合蛋白;CCK-8为细胞计数盒;Transwell为细胞侵袭实验
1
Schiff D, Van den Bent M, Vogelbaum MA, et al. Recent developments and future directions in adult lower-grade gliomas: Society for Neuro-Oncology (SNO) and European Association of Neuro-Oncology (EANO) consensus [J]. Neuro Oncol, 2019, 21(7): 837-853.
2
GBD 2016 Brain and Other CNS Cancer Collaborators. Global, regional, and national burden of brain and other CNS cancer, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016 [J]. Lancet Neurol, 2019, 18(4): 376-393.
3
Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, 2015 [J]. CA Cancer J Clin, 2016, 66(2): 115-132.
4
Wang X, Chen JX, Zhou Q, et al. Statistical report of central nervous system tumors histologically diagnosed in the Sichuan province of China from 2008 to 2013: A west China glioma center report [J]. Ann Surg Oncol, 2016, 23(Suppl 5): 946-953.
5
Saito T, Sugiyama K, Takeshima Y, et al. Prognostic implications of the subcellular localization of survivin in glioblastomas treated with radiotherapy plus concomitant and adjuvant temozolomide [J]. J Neurosurg, 2018, 128(3): 679-684.
6
Wang L, Lu M, Zhang R, et al. Inhibition of Luman/CREB3 expression leads to the upregulation of testosterone synthesis in mouse Leydig cells [J]. J Cell Physiol, 2019, 234(9): 15257-15269.
7
Xue H, Zhang J, Guo X, et al. CREBRF is a potent tumor suppressor of glioblastoma by blocking hypoxia-induced autophagy via the CREB3/ATG5 pathway [J]. Int J Oncol, 2016, 49(2): 519-528.
8
Hu Y, Chu L, Liu J, et al. Knockdown of CREB3 activates endoplasmic reticulum stress and induces apoptosis in glioblastoma [J]. Aging (Albany NY), 2019, 11(19): 8156-8168.
9
Demircioglu F, Wang J, Candido J, et al. Cancer associated fibroblast FAK regulates malignant cell metabolism [J]. Nat Commun, 2020, 11(1): 1290.
10
Wu HJ, Hao M, Yeo SK, et al. FAK signaling in cancer-associated fibroblasts promotes breast cancer cell migration and metastasis by exosomal miRNAs-mediated intercellular communication [J]. Oncogene, 2020, 39(12): 2539-2549.
11
Mitra SK, Schlaepfer DD. Integrin-regulated FAK-Src signaling in normal and cancer cells [J]. Curr Opin Cell Biol, 2006, 18(5): 516-523.
12
Wang X, Fu X, Zhao S, et al. Antiangiogenic properties of caudatin in vitro and in vivo by suppression of VEGF-VEGFR2-AKT/FAK signal axis [J]. Mol Med Rep, 2017, 16(6): 8937-8943.
13
Wang J, Cai C, Nie D, et al. FRK suppresses human glioma growth by inhibiting ITGB1/FAK signaling [J]. Biochem Biophys Res Commun, 2019, 517(4): 588-595.
14
Furnari FB, Cloughesy TF, Cavenee WK, et al. Heterogeneity of epidermal growth factor receptor signalling networks in glioblastoma [J]. Nat Rev Cancer, 2015, 15(5): 302-310.
15
Wen PY, Reardon DA. Neuro-oncology in 2015: Progress in glioma diagnosis, classification and treatment [J]. Nat Rev Neurol, 2016, 12(2): 69-70.
16
Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma [J]. N Engl J Med, 2005, 352(10): 987-996.
17
Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways [J]. Nature, 2008, 455(7216): 1061-1068.
18
Fox RM, Hanlon CD, Andrew DJ. The CrebA/Creb3-like transcription factors are major and direct regulators of secretory capacity [J]. J Cell Biol, 2010, 191(3): 479-492.
19
Ying Z, Zhang R, Verge VM, et al. Cloning and characterization of rat Luman/CREB3, a transcription factor highly expressed in nervous system tissue [J]. J Mol Neurosci, 2015, 55(2): 347-354.
20
Cao G, Ni X, Jiang M, et al. Molecular cloning and characterization of a novel human cAMP response element-binding (CREB) gene (CREB4) [J]. J Hum Genet, 2002, 47(7): 373-376.
21
Xue H, Zhang J, Guo X, et al. CREBRF is a potent tumor suppressor of glioblastoma by blocking hypoxia-induced autophagy via the CREB3/ATG5 pathway [J]. Int J Oncol, 2016, 49(2): 519-528.
22
Lee BY, Timpson P, Horvath LG, et al. FAK signaling in human cancer as a target for therapeutics [J]. Pharmacol Ther, 2015, 146: 132-149.
23
Lechertier T, Hodivala-Dilke K. Focal adhesion kinase and tumour angiogenesis [J]. J Pathol, 2012, 226(2): 404-412.
24
Zheng KB, Xie J, Li YT, et al. Knockdown of CERB expression inhibits proliferation and migration of glioma cells line U251 [J]. Bratisl Lek Listy, 2019, 120(4): 309-315.
25
Avizienyte E, Frame MC. Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition [J]. Curr Opin Cell Biol, 2005, 17(5): 542-547.
26
Gabarra-Niecko V, Schaller MD, Dunty JM. FAK regulates biological processes important for the pathogenesis of cancer [J]. Cancer Metastasis Rev, 2003, 22(4): 359-374.
27
Wörthmüller J, Blum W, Pecze L, et al. Calretinin promotes invasiveness and EMT in malignant mesothelioma cells involving the activation of the FAK signaling pathway [J]. Oncotarget, 2018, 9(91): 36256-36272.
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