沉默Grb7对口腔鳞癌细胞Cal27生物学行为的影响

上海口腔医学 ›› 2016, Vol. 25 ›› Issue (6): 688-693.

沉默Grb7对口腔鳞癌细胞Cal27生物学行为的影响

刘秉尧, 曹罡, 董震, 陈伟, 徐金科, 张森林, 翁志强

南京军区南京总医院口腔科,江苏南京 210002

收稿日期:2016-03-29 出版日期:2016-12-25 发布日期:2016-12-29
通讯作者: 翁志强,E-mail:Wengzhiqiang9898@sina.com
作者简介:刘秉尧（1982-）,男,硕士,主治医师,E-mail: LBY820122@126.com

Knockdown of Grb7 inhibits growth of oral squamous cell carcinoma, cell proliferation and promoted apoptosis through ERK/FOXM1 pathway

LIU Bing-yao, CAO Gang, DONG Zhen, CHEN Wei, XU Jin-ke, ZHANG Sen-lin, WENG Zhi-qiang

Department of Stomatology, Nanjing General Hospital of Nanjing Military Command. Nanjing 210002, Jiangsu Province, China

Received:2016-03-29 Online:2016-12-25 Published:2016-12-29
Contact: 英文通讯作者
About author:英文作者简介
Supported by:
英文基金

摘要/Abstract

摘要： 目的研究生长因子受体结合蛋白7（growth factor receptor-bound 7,Grb7）对口腔鳞癌细胞生长及裸鼠移植瘤的作用。方法培养hNOK和Cal27细胞,实时定量PCR和Western印迹法检测Grb7的蛋白表达。采用siRNA沉默Grb7并转染至Cal27细胞48 h,MTT法检测细胞增殖,流失细胞仪检测细胞周期和凋亡率,Western印迹法检测Cyclin D1、Rb、E2F1、Caspase3、Bax和Bcl-2的蛋白表达;同时检测沉默Grb7对ERK1/2和FOXM1蛋白表达的影响。Cal27细胞经转染siGrb7后接种于裸鼠,建立口腔鳞癌移植瘤模型,测量移植瘤体积及重量。采用SPSS 11.0软件包对数据进行统计学分析。结果Grb7在Cal27细胞中高表达,沉默Grb7显著抑制细胞增殖、阻滞G1/S期转换、促进细胞凋亡。沉默Grb7下调Cyclin D1、Rb和Bcl-2表达,上调E2F1、c-caspase3和Bax表达。敲减Grb7显著抑制p-ERK1/2和FOXM1的表达。此外,沉默Grb7裸鼠移植瘤的体积及重量减少。结论Grb7通过ERK/FOXM1通路抑制Cal27细胞增殖、促进凋亡并减少移植瘤增长。

关键词: 口腔鳞癌, 生长因子受体结合蛋白7, 细胞增殖, 细胞凋亡, 移植瘤

Abstract: PURPOSE: To investigate the effect of growth factor receptor-bound 7 (Grb7) on oral squamous cell carcinoma growth and tumor xenografts. METHODS: Cal27 and hNOK cells were cultivated, real-time PCR and Western blotting were used to detect the expression of Grb7 in hNOK and Cal27. Cal27 was transfected with Grb7 siRNA for 48 h, cell proliferation was assayed using MTT. Flow cytometry was used to determine the cell cycle and apoptosis. Western blot was used to evaluate the expression of caspase3, Bax, bcl-2, Cyclin D1, Rb, E2F1, ERK and FOXM1. Grb7 siRNA and negative control were designed and injected subcutaneously into the mice, tumor volume and weight were measured. Statistical analysis was performed using SPSS 11.0 software package. RESULTS: Grb7 was highly expressed in Cal27 compared with hNOK. Depletion of Grb7 significantly inhibited cell proliferation, blocked G1/S phase transition, promoted cell apoptosis. Knockdown of Grb7 suppressed the expression of Cyclin D1 and Rb, upregulated E2F1 expression. Moreover, c-caspase 3 and Bax was also reduced after inhibition of Grb7. ERK/FOXM1 signaling pathway was also inhibited by Grb7. In addition, the volume and weight of tumor xenografts were reduced by siGrb7. CONCLUSIONS: Depletion of Grb7 inhibits cell proliferation, promotes apoptosis and reduces tumor xenografts through ERK/FOXM1 pathway.

Key words: Oral squamous cell carcinoma, Growth factor receptor-bound 7 (Grb7), Cell proliferation, Cell apoptosis, Tumor xenografts

中图分类号:

R739.8

刘秉尧, 曹罡, 董震, 陈伟, 徐金科, 张森林, 翁志强. 沉默Grb7对口腔鳞癌细胞Cal27生物学行为的影响[J]. 上海口腔医学, 2016, 25(6): 688-693.

LIU Bing-yao, CAO Gang, DONG Zhen, CHEN Wei, XU Jin-ke, ZHANG Sen-lin, WENG Zhi-qiang. Knockdown of Grb7 inhibits growth of oral squamous cell carcinoma, cell proliferation and promoted apoptosis through ERK/FOXM1 pathway[J]. Shanghai Journal of Stomatology, 2016, 25(6): 688-693.

参考文献

[1] Rivera C, Venegas B. Histological and molecular aspects of oral squamous cell carcinoma (Review) [J]. Oncol Lett, 2014, 8(1): 7-11.
[2] 赵世禄, 程福强, 史繁华. 口腔鳞状细胞癌临床流行病学研究现状 [J]. 中国营养保健(上旬刊), 2013, 23(7): 1648-1649.
[3] Nadler Y, Gonzalez A, Camp R, et al. Growth factor receptor-bound protein-7 (Grb7) as a prognostic marker and therapeutic target in breast cancer [J]. Ann Oncol, 2010, 21(3): 466-473.
[4] 张丹, 高友鹤. 生长因子受体结合蛋白 7 (Grb7) 研究进展 [J]. 基础医学与临床, 2011, 31(4): 467-470.
[5] Yamano Y, Uzawa K, Shinozuka K, et al. Hyaluronan-mediated motility: a target in oral squamous cell carcinoma [J]. Int J Oncol, 2008, 32(5): 1001-1009.
[6] Chu PY, Li TK, Ding ST, et al. EGF-induced Grb7 recruits and promotes Ras activity essential for the tumorigenicity of Sk-Br3 breast cancer cells [J]. J Biol Chem, 2010, 285(38): 29279-29285.
[7] Kong FF, Zhu YL, Yuan HH, et al. FOXM1 regulated by ERK pathway mediates TGF-β1-induced EMT in NSCLC [J]. Oncol Res, 2014, 22(1): 29-37.
[8] Gooi Z, Chan JY, Fakhry C. The epidemiology of the human papillomavirus related to oropharyngeal head and neck cancer [J]. Laryngoscope, 2016, 126(4): 894-900.
[9] 巩斌, 熊燊源, 李良远, 等. GRB7 家族蛋白的功能及研究进展 [J]. 生物学杂志, 2015, 32(5): 80-83.
[10] Tanaka S, Pero SC, Taguchi K, et al. Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis [J]. J Natl Cancer Inst, 2006, 98(7): 491-498.
[11] Giricz O, Calvo V, Pero SC, et al. GRB7 is required for triple-negative breast cancer cell invasion and survival [J]. Breast Cancer Res Treat, 2012, 133(2): 607-615.
[12] Wang Y, Chan DW, Liu VW, et al. Differential functions of growth factor receptor-bound protein 7 (GRB7) and its variant GRB7v in ovarian carcinogenesis[J]. Clin Cancer Res, 2010, 16(9): 2529-2539.
[13] Sawada G, Niida A, Hirata H, et al. An integrative analysis to identify driver genes in esophageal squamous cell carcinoma [J]. PloS One, 2015, 10(10): e0139808.
[14] Giacinti C, Giordano A. RB and cell cycle progression [J]. Oncogene, 2006, 25(38): 5220-5227.
[15] Dick FA, Rubin SM. Molecular mechanisms underlying RB protein function [J]. Nat Rev Mol Cell Biol, 2013, 14(5): 297-306.
[16] Brentnall M, Rodriguez-Menocal L, De Guevara RL, et al. Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis [J]. BMC Cell Biol, 2013, 14: 32.
[17] Czabotar PE, Lessene G, Strasser A, et al. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy [J]. Nat Rev Mol Cell Biol, 2014, 15(1): 49-63.
[18] Calvo VA, Giricz O, Kenny PA. Defining the GRB7 interactome in triple-negative breast cancer [J]. Cancer Res, 2013, 73(8 Suppl): 4311.
[19] Bai T, Luoh SW. GRB-7 facilitates HER-2/Neu-mediated signal transduction and tumor formation [J]. Carcinogenesis, 2008, 29(3): 473-479.
[20] 李永峰, 朱世泽. Foxm1 在细胞周期和肿瘤中的研究进展 [J]. 国际肿瘤学杂志, 2011, 38(5): 332-335.
[21] Huang PY, Li Y, Luo DH, et al. Expression of Aurora-B and FOXM1 predict poor survival in patients with nasopharyngeal carcinoma [J]. Strahlenther Onkol, 2015, 191(8): 649-655.
[22] Myatt SS, Lam EW. The emerging roles of forkhead box (Fox) proteins in cancer [J]. Nat Rev Cancer, 2007, 7(11): 847-859.
[23] Wang IC, Chen YJ, Hughes DE, et al. FoxM1 regulates transcription of JNK1 to promote the G1/S transition and tumor cell invasiveness [J]. J Biol Chem, 2008, 283(30): 20770-20778.
[24] 江虹,郑建华. GRB7/ERK/FOXM1信号通路与卵巢癌关系的研究进展 [J]. 中华临床医师杂志(电子版), 2013, 7(21): 9736-9739.