Ⅱ型cGMP依赖性蛋白激酶抑制VEGFR2介导的人胃癌HGC-27细胞增殖

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摘要 ［摘要］目的: 探讨Ⅱ型cGMP依赖性蛋白激酶（type ⅡcGMP-dependent protein kinase，PKGⅡ）对血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）介导的人胃癌HGC-27细胞增殖的影响及其作用机制。方法: 应用编码PKGⅡcDNA的腺病毒（AdPKGⅡ）感染HGC27细胞，使其高表达PKGⅡ，并以特异性激动剂8-pCPT-cGMP激活PKGⅡ。应用血管内皮生长因子-A（vascular endothelial growth factor A，VEGFA）激活VEGFR2。MTT法检测细胞增殖；蛋白质印迹法检测p-VEGFR2、磷酸化蛋白激酶B（phosphorylated-protein kinase B, p-PKB/pAkt）和磷酸化细胞外信号调节激酶1/2（phosphorylatedextracellular signal-regulated kinase, p-ERK1/2）表达；免疫共沉淀法检测PKGⅡ与VEGFR2的结合；免疫沉淀法联合蛋白质印迹法检测PKGⅡ对VEGFR2丝氨酸/苏氨酸（Serine/Threonine,Ser/Thr）的磷酸化作用。结果： VEGF-A可促进HGC-27细胞增殖，并诱导胞内pVEGFR2、p-Akt和p-ERK1/2表达水平明显升高；以Ad-PKGⅡ感染HGC-27细胞使其高表达PKGⅡ并激活后，VEGF-A引起的细胞增殖受到明显抑制，pVEGFR2、pAkt和p-ERK1/2表达水平显著降低；PKGⅡ可与VEGFR2相互作用，使后者丝氨酸/苏氨酸磷酸化。结论：激活的PKGⅡ可通过使VEGFR2发生丝氨酸/苏氨酸磷酸化抑制人胃癌HGC-27细胞的VEGFR2酪氨酸磷酸化，进而抑制其下游的增殖相关信号转导，最终抑制该细胞的增殖。

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关键词 ：血管内皮生长因子受体-2, Ⅱ型cGMP依赖性蛋白激酶, 细胞增殖, 胃癌HGC-27细胞

Abstract：[Abstract]Objective: To investigate the influence of type Ⅱ cGMPdependent protein kinase(PKGⅡ)on proliferation mediated by vascular endothelial growth factor receptor 2(VEGFR2) and its related mechanism in human gastric cancer HGC27 cell line. Methods: The HGC27 cell line was transfected with adenoviral constructs encoding the cDNA of PKGⅡ(AdPKGⅡ) to increase the expression of PKGⅡ, and treated with 8pCPTcGMP to activate the PKGⅡ and treated with vascular endothelial growth factor A (VEGFA) to activate VEGFR2. MTT assay was applied to measure the proliferation activity; Western blotting was used to evaluate the phosphorylation of VEGFR2, Akt and ERK1/2; the interaction between VEGFR2 and PKGⅡwas detected by coimmunoprecipitation;the phosphorylation on serine/threonine(Ser/Thr) of VEGFR2 caused by PKGⅡwas detected by immunoprecipitation and Western blotting. Results: VEGFA treatment caused obvious increase of proliferation and phosphorylation(activation) of VEGFR2, Akt and ERK1/2 in HGC27 cells. In cells transfected with AdPKGⅡto highly express PKGⅡand treated with 8pCPTcGMP, the VEGFA induced increase of proliferation and level of phosphorylation of VEGFR2, Akt and ERK1/2 was significantly inhibited. PKGⅡcould bind with VEGFR2 and cause serine/threonine phosphorylation of the receptor. Conclusion: Activated PKGⅡinhibits the tyrosine phosphorylation(activation) of VEGFR2 through phosphorylating its serine/threonine, subsequently inhibits the proliferation related signal transduction, and finally inhibits the proliferation of human gastric cancer cell line HGC27.

收稿日期: 2015-11-23

基金资助:

国家自然科学基金资助项目（81272755，81201959）；江苏省高校自然科学研究计划资助项目（12KJB310001）；江苏大学高级专业人才科研启动基金资助项目（11JDG114）；江苏省博士后科研资助计划(1401144C)；中国博士后科学基金资助项目（2014M561599）

通讯作者: 陈永昌(通讯作者)，教授，博士生导师，E-mail:ycchen54@ujs.edu.cn

作者简介: 章伟慧(1990—)，女，硕士研究生；

引用本文:

章伟慧，伍敏，庞吉，蓝婷，陶燕，吴燕，陈永昌. Ⅱ型cGMP依赖性蛋白激酶抑制VEGFR2介导的人胃癌HGC-27细胞增殖[J]. 江苏大学学报:医学版, 2016, 26(02): 108-112,118. ZHANG Wei-hui, WU Min, PANG Ji, LAN Ting, TAO Yan, WU Yan, CHEN Yong-chang. Type ⅡcGMP-dependent protein kinase inhibits proliferation of gastric cancer cell line HGC-27 mediated by vascular endothelial growth factor receptor 2. Journal of Jiangsu University(Medicine Edition), 2016, 26(02): 108-112,118.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2016/V26/I02/108

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