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目的:VEGF-VASH1(vasohibin-1)通路在血管新生过程的精细调节中发挥重要作用,转录因子锌指蛋白667(zinc finger protein 667,ZNF667)具有促进血管新生作用。本研究主要探讨ZNF667对上述通路中VEGF和VASH1表达的调控,以期阐明ZNF667促进血管新生的分子机制。方法:采用LAD建立慢性心肌缺血小鼠模型,采用HE染色和CD31免疫组化分析缺血心肌组织形态学变化及微血管形成。采用Matrigel、划痕和Transwell分析HUVEC管型形成和迁移;采用Western blot、ELISA和定量PCR分别检测ZNF667和VASH1蛋白质和m RNA表达;m RNA测序分析ZNF667过表达HUVEC的m RNA差异表达;染色质免疫沉淀(chromatin immunoprecipitation,Ch IP)检测ZNF667与VEGF和VASH1启动子区结合情况。结果:免疫组化和HE染色显示与假手术组相比,缺血心肌中组织损伤加重伴随有CD31~+微血管数目增加,同时心肌组织中VEGF和ZNF667蛋白和m RNA表达呈时间依赖性增加,而VASH1表达降低。m RNA测序、ELISA和定量PCR显示ZNF667过表达可促进HUVEC中VEGF表达而抑制VASH1表达。VASH1过表达可抑制VEGF和ZNF667的促HUVEC管型形成和迁移作用。Ch IP显示ZNF667与VEGF(-346~-350 bp;-265~-269 bp)和VASH1(-170~-175 bp)基因启动子区结合。结论:HUVEC中ZNF667靶向调节VEGF-VASH1通路促进管型形成和迁移作用,这一调节机制可能参与缺血心肌中微血管新生过程。
OBJECTIVE: The vasohibin-1 pathway plays an important role in the fine regulation of angiogenesis. The transcription factor zinc finger protein 667 (ZNF667) can promote angiogenesis. This study mainly discussed the regulation of ZNF667 on the expression of VEGF and VASH1 in the above pathways in order to clarify the molecular mechanism of ZNF667 promoting angiogenesis. Methods: The model of chronic myocardial ischemia was established by LAD. HE staining and CD31 immunohistochemistry were used to analyze the morphological changes of ischemic myocardium and the formation of microvessels. The formation and migration of HUVECs were detected by Matrigel, scratch and Transwell analysis. The protein and mRNA expression of ZNF667 and VASH1 were detected by Western blot, ELISA and quantitative PCR respectively. The m RNA expression of ZNF667 overexpression HUVEC was analyzed by m RNA sequencing. Chromatin immunoprecipitation (ChIP) was used to detect the binding of ZNF667 to the VEGF and VASH1 promoter regions. Results: Immunohistochemistry and HE staining showed that compared with the sham-operated group, the myocardial injury in the ischemic myocardium was accompanied by an increase in the number of CD31 + microvessels. At the same time, the expression of VEGF and ZNF667 protein and m RNA in myocardial tissue increased in a time-dependent manner, VASH1 expression decreased. m RNA sequencing, ELISA and quantitative PCR showed that overexpression of ZNF667 could promote VEGF expression in HUVEC and inhibit VASH1 expression. VASH1 overexpression can inhibit the VEGF and ZNF667 promote HUVEC tubular formation and migration. Ch IP showed that ZNF667 bound to the promoter region of VEGF (-346 ~ -350 bp; -265 ~ -269 bp) and VASH1 (-170 ~ -175 bp). CONCLUSIONS: ZNF667 targeting HUVECs regulates the VEGF-VASH1 pathway to promote tube formation and migration. This regulatory mechanism may be involved in the angiogenesis of ischemic myocardium.