分泌型cGMP依赖性蛋白激酶Ⅱ对胃癌细胞增殖相关基因表达的调控  

Regulation of secreted typeⅡcGMP-dependent protein kinase on proliferation-related gene expression of human gastric cancer cell line

作　　者：李帼瑞 庞吉 钱海[1] 吴燕[1] 陈永昌[1] LI Guo-rui;PANG Ji;QIAN Hai;WU Yan;CHEN Yong-chang(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 212013,China)

机构地区：[1]江苏大学医学院,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2020年第3期221-228,共8页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(31771564);江苏省卫生健康委科研项目(LGY2018025);333工程科研项目资助计划(BRA2019172)。

摘　　要：目的:初步探索分泌型cGMP依赖性蛋白激酶Ⅱ(typeⅡcGMP-dependent protein kinase,PKGⅡ)对人胃癌HGC-27和AGS细胞株基因表达的影响。方法:采用Illumina HiSeq 2000高通量测序平台对使用谷胱甘肽硫转移酶(glutathione S-transferases,GSTs)标签表达的重组全长人PKGⅡ处理前、后的人胃癌AGS细胞株转录组进行测序;从表达差异明显的基因中筛选与肿瘤细胞增殖相关的基因,采用qRT-PCR法验证在cGMP类似物8-pCPT-cGMP的激活下重组PKGⅡ对相关基因转录水平的影响,并通过蛋白质免疫印迹进一步验证其对相关蛋白表达水平的影响。结果:转录组测序结果显示,579个基因表达有明显差异,其中267个基因表达显著上调,312个显著下调;筛选9个与肿瘤细胞增殖相关的基因进行qRT-PCR检测,证实在GST-PKGⅡ和8-pCPT-cGMP共同作用下,EGF、DFNA5、DACT1、PSCA等mRNA水平在AGS和HGC-27两个细胞株中都表现出与转录组测序一致的趋势(与空白对照组相比P均<0.05);蛋白质免疫印迹结果显示,在GST-PKGⅡ和8-pCPT-cGMP共同作用下,表皮生长因子蛋白水平下调,与转录组测序和qRT-PCR结果一致。结论:分泌型PKGⅡ可从转录水平调控人胃癌HGC-27和AGS细胞中EGF等增殖相关基因的表达,并能够抑制EGF的翻译。Objective:To explore the effect of secreted typeⅡcGMP-dependent protein kinase(PKGⅡ)on gene expression in human gastric cancer cell lines HGC-27 and AGS.Methods:Illumina HiSeq 2000 high-throughput sequencing platform was used to sequence the transcriptome of AGS human gastric cancer cells treated with recombinant full-length human PKGⅡwhich was expressed by baculovirus in Sf9 insect cells using an N-terminal Glutathione S-transferases(GST)tag.Several genes associated with proliferation were selected from differentially expressed genes(DEGs)caused by recombinant PKGⅡtreatment,and the effect of recombinant PKGⅡon the transcription level of related genes under the activation of cGMP analog 8-pCPT-cGMP was verified by real-time quantitative PCR.The effect of GST-PKGⅡon the expression of related protein was verified by Western blotting.Results:Transcriptome sequencing results showed a significant difference in expression of 579 genes,of which 267 up-regulated genes and 312 down-regulated genes with the treatment of GST-PKGⅡ.Among the 9 cell proliferation related genes selected by RNA-Sequencing,there was a consistent trend in the mRNA levels of EGF,DFNA5,DACT1,and PSCA with the RNA-Sequencing result both in HGC-27 and AGS human gastric cell line under the treatment of GST-PKGⅡand 8-pCPT-cGMP(P<0.05),compared with blank control group.The protein level of EGF was down-regulated with the treatment of GST-PKGⅡand 8-pCPT-cGMP,which was consistent with the results of RNA-Sequencing and qRT-PCR.Conclusion:Secreted PKGⅡcould affect the expression of various proliferation-related genes included EGF in AGS and HGC-27 gastric cancer cells from the transcription level,and inhibit the translation of EGF.

关 键 词：分泌型cGMP依赖性蛋白激酶Ⅱ 胃癌细胞 基因表达 转录组测序 表皮生长因子 

分 类 号：R735.2[医药卫生—肿瘤]