［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（AdPKGⅡ）感染HGC27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，VEGFA）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/pAkt）和磷酸化细胞外信号调节激酶1/2（phosphorylatedextracellular 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pVEGFR2、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，pVEGFR2、pAkt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。
Abstract
[Abstract]Objective: To investigate the influence of type Ⅱ cGMPdependent protein kinase(PKGⅡ)on proliferation mediated by vascular endothelial growth factor receptor 2(VEGFR2) and its related mechanism in human gastric cancer HGC27 cell line. Methods: The HGC27 cell line was transfected with adenoviral constructs encoding the cDNA of PKGⅡ(AdPKGⅡ) to increase the expression of PKGⅡ, and treated with 8pCPTcGMP to activate the PKGⅡ and treated with vascular endothelial growth factor A (VEGFA) 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 coimmunoprecipitation;the phosphorylation on serine/threonine(Ser/Thr) of VEGFR2 caused by PKGⅡwas detected by immunoprecipitation and Western blotting. Results: VEGFA treatment caused obvious increase of proliferation and phosphorylation(activation) of VEGFR2, Akt and ERK1/2 in HGC27 cells. In cells transfected with AdPKGⅡto highly express PKGⅡand treated with 8pCPTcGMP, the VEGFA 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 HGC27.
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
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脚注
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基金
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）
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