狭缝引导配体13′UTR通过miR-34a-5p/SIRT1轴调节内皮细胞表型

SLIT13′UTR Modulates the Phenotype of Endothelial Cells Through MiR-34a-5p/SIRT1 Axis

在高等生物的进化过程中,mRNA的3′非翻译区(3′untranslated region,3′UTR)序列显著增加,提示3′UTR在生物功能调节中可能发挥重要作用。我们发现狭缝引导配体1(slit guidance ligand 1,SLIT1)3′UTR在肥厚型心肌病(HCM)患者心肌中水平降低,但其对肥厚心肌中血管功能调节的作用机制不清楚。利用腺病毒介导在主动脉内皮细胞(human aortic endothelial cells,HAECs)中过表达SLIT13′UTR,检测HAECs中一氧化氮合酶(endothelial nitric oxide synthases,eNOS)和血管内皮生长因子A(vascular endothelial growth factor,VEGFA)表达。分别采用划痕实验和基于Matrigel胶的管腔形成实验检测HAECs的迁移和成管腔能力。结果发现,过表达SLIT13′UTR会升高HAECs中p-eNOS、eNOS和VEGFA水平(P<0.01),并促进HAECs迁移和成管腔能力(P<0.01)。生物信息学预测提示,SLIT13′UTR上有多个微RNA(miRNA)的潜在结合位点,反义RNA纯化技术(RNA antisense purification,RAP)筛选和利用Ago2抗体进行RNA结合蛋白质免疫沉淀(RNA binding protein immunoprecipitation,RIP)结果显示,SLIT13′UTR与miR-34a-5p存在结合作用,而过表达SLIT13′UTR的HAECs中miR-34a-5p水平显著降低(P<0.05)。Western印迹结果证实,miR-34a-5p可逆转过表达SLIT13′UTR对去乙酰化酶SIRT1的上调作用(P<0.05)。在HAECs中转染miR-34a-5p和si-SIRT1,可一致地抑制过表达SLIT13′UTR引起的p-eNOS、eNOS和VEGFA增加(P<0.05,P<0.01),及促进HAECs迁移和成管腔的能力(P<0.01,P<0.001)。因此,SLIT13′UTR可以特异性吸附miR-34a-5p,通过miR-34a-5p/SIRT1轴发挥促进内皮细胞迁移和管腔生成的作用。

In the evolutionary process of higher organisms,the 3′untranslated region(3′UTR)sequence of mRNA markedly increase,which indicates a potential important role of 3′UTR in regulating biological functions.The 3′UTR of slit guidance ligand 1(SLIT1)mRNA was markedly decreased in the myocardium of hypertrophic cardiomyopathy(HCM)patients,however,the potential role and mechanism of SLIT13′UTR in regulating vascular function in the hypertrophic myocardium remains unclear.The expression of endothelial nitric oxide synthase(eNOS)and vascular endothelial growth factor A(VEGFA)was detected in aortic endothelial cells(HAECs)with overexpression of SLIT13′UTR.The migration and tube formation activities of HAECs were examined by using wound-healing assay and matrigel-based tube formation assay,respectively.Overexpression of SLIT13′UTR could significantly increase the levels of p-eNOS,eNOS and VEGFA in HAECs(P<0.01,respectively),and promote the migration and tube formation activities of HAECs(P<0.01,respectively).Bioinformatic analysis indicated multiple potential binding sites of microRNAs(miRNAs)exist in SLIT13′UTR.The results of RNA antisense purification(RAP)assay,as well as Ago2-based RNA binding protein immunoprecipitation(RIP)assay,verified the interaction between SLIT13′UTR and miR-34a-5p.Additionally,a significant reduction of miR-34a-5p was observed in HAECs with overexpression of SLIT13′UTR(P<0.05),and transfection of miR-34a-5p mimic could reverse the increase of SIRT1 expression by SLIT13′UTR in HAECs(P<0.05).Transfection of miR-34a-5p and SIRT1 siRNA could consistently suppress the increase of p-eNOS,eNOS and VEGFA,and the enhancement of migration and tube formation activities of HAECs with overexpression of SLIT13′UTR(P<0.05,P<0.01,respectively).In conclusion,SLIT13′UTR can sponge miR-34a-5p,and promote the endothelial cell migration and tube formation by targeting miR-34a-5p/SIRT1 axis.