NO-sGC-cGMP信号通路通过cGK调控PAI-2基因表达  

NO-sGC-cGMP signaling pathway regulates PAI-2 gene expression by cGK

作　　者：韩敬丽 邹丽辉[2] 金军华[2] 李文卿 李英[2] 肖飞[1] HAN Jing-li;ZOU Li-hui;JIN Jun-hua;LI Wen-qing;LI Ying;XIAO Fei(The Fifth Medical College of Peking University, Beijing 100730 , China;Key Laboratory of Geriatrics,Beijing Institute of Geriatrics, Beijing Hospital of the Ministry of Health, Beijing 100730 , China)

机构地区：[1]北京大学第五临床医学院,北京100730 [2]北京医院卫生部北京老年医学中心老年医学重点实验室,北京100730

出　　处：《中国病理生理杂志》2019年第6期961-967,共7页Chinese Journal of Pathophysiology

基　　金：国家重大新药创制国家科技重大专项资助项目(No.2017ZX09304026);国家自然科学基金资助项目(No.81571384);北京市自然科学基金资助项目(No.7172193)

摘　　要：目的:体外分析环磷酸鸟苷是否(cGMP)通过cGMP依赖性蛋白激酶(cGK)调控人纤溶酶原激活物抑制剂2(PAI-2)基因启动子活性,阐明PAI-2基因表达的分子调控机制。方法:构建cGK的真核过表达载体及其小干扰RNA(siRNA)敲减体系,以及PAI-2基因启动子缺失突变序列双萤光素酶报告载体,瞬时转染人肺动脉平滑肌细胞(HPASMCs),在可溶性鸟苷酸环化酶(sGC)特异性刺激剂BAY 41-2272处理前后,real-time PCR检测PKG1(cGK编码基因)和PAI-2基因的mRNA水平。结果:cGK过表达能显著上调细胞内PKG1的mRNA水平和蛋白表达水平,cGK siRNA则能显著降低其表达(P<0.01);BAY 41-2272处理HPASMCs后,PKG1基因和PAI-2基因的mRNA水平均明显升高,而cGK siRNA则能抑制该上调作用(P<0.01);双萤光素酶报告基因实验显示,cGK过表达显著上调PAI-2基因启动子活性,而cGK敲减组其活性与对照组比较差异无统计学显著性;BAY 41-2272处理后PAI-2基因启动子缺失突变体PAI-2-Prom-M1组和PAI-2-Prom-M2组的萤光素酶相对活性显著增强,其它启动子缺失突变体组其活性与对照组比较差异无统计学显著性。结论:NO-sGC-cGMP信号通路通过cGK调控PAI-2表达,PAI-2基因转录起始位点5’侧翼区的-1 000 bp^-800 bp是其表达调控的关键区域之一。AIM: To analyze the expression level of human plasminogen activator inhibitor 2(PAI-2) gene and the activity of PAI-2 gene promoter regulated by cyclic guanosine monophosphate(cGMP)-dependent protein kinase(cGK) in vitro. METHODS: The eukaryotic expression vector of cGK, cGK siRNA knockdown systems and dual-luciferase reporter vector of PAI-2 gene promoter were transiently transfected into human pulmonary arterial smooth muscle cells(HPASMCs). The mRNA levels of both PAI-2 and cGK encoding gene PKG1 were detected by real-time PCR before and after soluble guanylate cyclase(sGC) specific stimulator BAY 41-2272 treatment. RESULTS: The PKG1 expression at mRNA and protein levels was significantly up-regulated following cGK over-expression and was significantly reduced after cGK siRNA treatment(P<0.01). The mRNA levels of PKG1 and PAI-2 were remarkably increased after BAY 41-2272 treatment, while the up-regulation was suppressed by cGK siRNA(P<0.01). Dual-luciferase reporter assay results demonstrated that the activity of PAI-2 gene promoter was dramatically up-regulated following the over-expression of cGK in HPASMCs, while its activity in cGK knockdown group showed no significant difference compared with control group. The activity of PAI-2-Prom-M1 group and PAI-2-Prom-M2 group was significantly up-regulated after BAY 41-2272 treatment, while the activity of other promoter variants revealed no significant difference compared with control group. CONCLUSION: cGMP up-regulates the expression of PAI-2 dependent on cGK via modulating PAI-2 gene promoter. The key regulatory promoter region is-1 000 bp to-800 bp of the 5’ flanking region upstream of the transcription initiation site.

关 键 词：纤溶酶原激活物抑制剂2 cGMP依赖性蛋白激酶 NO-sGC-cGMP信号通路 BAY41-2272 

分 类 号：R319[医药卫生—基础医学] R363