幽门螺旋杆菌毒力因子CagA介导的ERK信号通路活化对胃癌细胞增殖和凋亡的影响

doi:10.13481/j.1671-587x.20190516

摘要/Abstract

摘要： 目的：探讨幽门螺旋杆菌毒力因子细胞毒素相关基因A蛋白（CagA）对细胞外调节蛋白激酶（ERK）信号通路的作用，阐明CagA的致癌机制。方法：构建pcDNA3.1/CagA真核表达载体，将胃癌AGS细胞分为空白对照组（空载体转染）、CagA转染组（GZ7/CagA转染）和CagA+ERKi组（ERK1/2抑制剂预处理+GZ7/CagA转染），采用Western blotting法测定各组细胞中CagA、磷酸化ERK（p-ERK）、总ERK（T-ERK）、B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）和激活型caspase-3（cleaved caspase-3）蛋白表达水平，采用CCK-8法检测各组胃癌AGS细胞活性，采用流式细胞术检测胃癌AGS细胞凋亡率。结果：与空白对照组比较，CagA转染组胃癌细胞中CagA、p-ERK和Bcl-2蛋白表达水平明显升高（P<0.01），Bax和cleaved caspase-3蛋白表达水平明显降低（P<0.01）；与CagA转染组比较，CagA+ERKi组胃癌细胞中p-ERK和Bcl-2蛋白表达水平明显降低（P<0.01），Bax和cleaved caspase-3蛋白表达水平明显升高（P<0.01）；与CagA转染组比较，CagA+ERKi组各时间点胃癌细胞活性明显降低（P<0.01）；与CagA转染组比较，CagA+ERKi组胃癌细胞凋亡率明显升高（P<0.05）。结论：幽门螺旋杆菌毒力因子CagA可抑制胃癌细胞凋亡，促进胃癌细胞增殖，其机制可能与CagA活化ERK信号通路有关。

关键词: 幽门螺旋杆菌, 细胞毒素相关基因A蛋白, 细胞外调节蛋白激酶, 胃癌细胞, 细胞增殖, 细胞凋亡

Abstract: Objective:To explore the effect of Helicobacter pylori virulence factor cytotoxin-associated gene A protein (CagA) on the extracellular regulated protein kinase (ERK) signaling pathway, and to elucidate the carcinogenesis mechanism of CagA. Methods:The pcDNA3.1/CagA eukaryotic expression vector was constructed, and the gastric cancer AGS cells were divided into blank control group (blank vector transfection), CagA transfection group (GZ7/CagA transfection), and CagA + ERKi group (ERK1/2 inhibitor pretreatment + GZ7/CagA transfection).The expression levels of CagA, phosphorylated ERK (p-ERK), total ERK (T-ERK), B-lymphocytoma-2 (Bcl-2), Bcl-2-related X protein (Bax) and cleaved caspase-3 proteins in the cells in various groups were determined by Western blotting method. The activities of AGS cells in various groups were determined by CCK-8 method, and the apoptotic rates of AGS cells were determined by flow cytometry. Results:Compared with blank control group, the expression levels of CagA, p-ERK, and Bcl-2 proteins in CagA transfection group were significantly increased(P<0.01), and the expression levels of Bax and cleaved caspase-3 proteins were significantly decreased (P<0.01). Compared with CagA transfection group, the expression levels of p-ERK and Bcl-2 proteins in CagA+ERKi group were significantly decreased (P<0.01), and the expression levels of Bax and cleaved caspase-3 proteins were significantly increased (P<0.01). Compared with CagA transfection group, the activities of gastric cancer cells in CagA + ERKi group at different time points were significantly decreased (P<0.01). Compared with CagA transfection group, the apoptotic rate of gastric cancer cells in CagA + ERKi group was significantly increased (P<0.05). Conclusion:Helicobacter pylori virulence factor CagA can inhibit the proliferation of gastric cancer cells and promote the apoptosis of gastric cancer cells, and its mechanism may be related to the activation of ERK signaling pathway by CagA.

Key words: Helicobacter pylori, cytotoxin-related gene A protein, extracellular regulated protein kinases, gastric cancer cells, cell proliferation, apoptosis

中图分类号:

R735.2

刘粉霞, 陈丽, 孙宁, 吴亚薇. 幽门螺旋杆菌毒力因子CagA介导的ERK信号通路活化对胃癌细胞增殖和凋亡的影响[J]. 吉林大学学报(医学版), 2019, 45(05): 1063-1068.

LIU Fenxia, CHEN Li, SUN Ning, WU Yawei. Effect of Helicobacter pylori virulence factor CagA-mediated activation of ERK signaling pathway on proliferation and apoptosis of gastric cancer cells[J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1063-1068.

参考文献

[1] HUANG X, WANG C, SUN J,et al.Clinical value ofCagA, c-Met, PI3K and Beclin-1 expressed in gastric cancer and their association with prognosis[J].Oncol Lett,2018,15(1):947-955.
[2] YANG D, LI R D, WANG H L,et al.Clinical implications of progranulin in gastric cancer and its regulation via a positive feedback loop involving AKT and ERK signaling pathways[J].Mol Med Rep,2017,16(6):9685-9691.
[3] CHEN S Y, DUAN G C, ZHANG R G,et al.Helicobacter pylori cytotoxin-associated gene A protein upregulates α-enolase expression via Src/MEK/ERK pathway:implication for progression of gastric cancer[J].Int J Oncol,2014,45(2):764-770.
[4] LAN K H, LEE W P, WANG Y S,et al.Helicobacter pylori CagA protein activates Akt and attenuates chemotherapeutics-induced apoptosis in gastric cancer cells[J].Oncotarget,2017,8(69):113460-113471.
[5] LEE D Y, JUNG D E, YU S S,et al.Regulation of SIRT3 signal related metabolic reprogramming in gastric cancer by Helicobacter pylori oncoprotein CagA[J].Oncotarget,2017,8(45):78365-78378.
[6] HATAKEYAMA M.Structure and function of Helicobacter pylori CagA, the first-identified bacterial protein involved in human cancer[J].Proc Jpn Acad Ser B Phys Biol Sci,2017,93(4):196-219.
[7] 龙妮娅,熊林,赵艳,等.幽门螺杆菌东、西方株CagA的序列差异及其对胃癌细胞生长与凋亡的影响[J].微生物学通报,2018,45(4):848-855.
[8] WANG Y Z, XIAO H J, WU H T,et al.G protein subunit α q regulates gastric cancer growth via the p53/p21 and MEK/ERK pathways[J].Oncol Rep,2017,37(4):1998-2006.
[9] CAIH,CHEN X,ZHANG J,et al.18β-glycyrrhetinic acid inhibits migration and invasion of human g astric cancer cells via the ROS/PKC-α/ERK pathway[J].J Nat Med,2018,72(1):252-259.
[10] PEI Y F, ZHANG Y J, LEI Y,et al.Hypermethylation of the CHRDL1 promoter induces proliferation and metastasis by activating Akt and Erk in gastric cancer[J].Oncotarget,2017,8(14):23155-23166.
[11] 孟令超,张娜,单粉粉,等.胃癌组织细胞外信号调节激酶1/2表达及其与幽门螺杆菌关系的初步分析[J].临床与实验病理学杂志,2016,32(2):131-135.
[12] 苏连明,庄彦华,颜彬,等.幽门螺杆菌感染与胃癌组织细胞外调节蛋白激酶1基因表达的相关性研究[J].中华实用诊断与治疗杂志,2014,28(8):750-751,754.
[13] WATANABE T, TAKAHASHI A, SUZUKI K,et al.Epithelial-mesenchymal transition in human gastric cancer cell lines induced by TNF-nsition in humanbmed/Helicobacter pylori[J].Int J Cancer,2014,134(10):2373-2382.
[14] NGO H K, LEE H G, PIAO J Y,et al.Helicobacter pylori induces Snail expression through ROS-mediated activation of Erk and inactivation of GSK-3throuhuman gastric cancer cells[J].Mol Carcinog,2016,55(12):2236-2246.
[15] 魏晓东, 唐艳萍. 胃癌中幽门螺旋杆菌感染对DNA甲基化影响的研究进展[J].中国中西医结合外科杂志, 2018, 24(6):803-805.
[16] 奚经巧, 林枝, 陈建欧, 等. 幽门螺旋杆菌阳性慢性萎缩性胃炎患者血清CagA抗体浓度评估黏膜病变程度的价值[J]. 中国卫生检验杂志, 2018, 28(17):2128-2131.
[17] 刘凤霞,刘文娟,李建勇,等.ERK1/2在食管鳞状细胞癌中促进细胞增殖、抑制凋亡及其调控机制的研究[J].生物技术通报,2015,31(10):242-248.
[18] 王希明,张晔,刘云鹏,等.MAPK/ERK通路及泛素连接酶c-Cbl调控埃克替尼抑制肺癌HCC827细胞增殖和诱导细胞凋亡的研究[J].现代肿瘤医学,2014,22(5):1024-1027.
[19] 谢琼,胡桂明,王洪涛.miRNA-223-3p调控JAK2/STAT3信号通路对胃癌细胞增殖、凋亡、迁移的影响[J].郑州大学学报:医学版,2018,53(5):629-634.
[20] 檀碧波, 李勇, 赵群, 等.胃癌组织中miR-218和凋亡相关蛋白Bcl-2、Bax、xIAP、Survivin mRNA的表达观察[J]. 山东医药, 2018,58(47):20-23.
[21] 王玲, 周巧直, 王晓燕, 等. 阿托伐他汀调控MMP-9、Cleaved Caspase-3、Bcl-2和Bax蛋白表达对胃癌细胞增殖、周期和凋亡的影响[J]. 胃肠病学和肝病学杂志, 2018,27(11):1206-1210.