AS-tRF Regulates Glycosylation Of Proteins By Targeting B4GALNT4,Thereby Affecting Endothelial Cell Inflammation And Function

ObjectiveThis study aims to explore whether tRNA-derived segment(tRF)regulates glycoylation of proteins by targeting B4GALNT4 in atherosclerosis(AS),and thus regulates endothelial cell inflammation and function and its mechanism,and provide a potential target for the prevention and treatment of atherosclerosis.Methods(1)Through high-throughput sequencing of clinical atherosclerosis samples in the early stage,a tRNA-derived fragment with significantly different expression was screened,namely,tRF-1:29-Gly-GCC-1(hereafter referred to AS-tRF).Pathological artery and normal artery samples of AS patients were collected clinically,and the expression of AS-tRF was detected by RT-q PCR and quantified.In addition,a rat model of atherosclerosis was constructed,and the expression of AS-tRF in atherosclerotic mice model was detected by RT-q PCR.(2)Mimics and inhibitors of AS-tRF were constructed and transferred into human umbilical vein endothelial cells(HUVECs).The transfection efficiency was verified by RT-q PCR.Then RT-q PCR was used to detect the expression of related adhesion factors(MCP1,ICAM1 and VCAM1)and inflammatory factors(IL-6,IL-8 and TNF-α).The adhesion function and migration ability of cells were detected by adhesion test and scratch assay respectively.The expression of marker proteins in the pathway related to adhesion and inflammation(p-ERK,p-p38,p-JNK,p-c-Jun,p-AKT,p-p65 and p-Iκbα)was detected by Western Blot.(3)Bioinformatics websites were used to predict the potential downstream target of AS-tRF.Beta-1,4-N-Acetyl-Galactosaminyltransferase 4(B4GALNT4)was predicted as one of candidates.Subsequently,RNA Pull down experiments were conducted to confirm that it was indeed the downstream target of AS-tRF which might be involved in the pathogenesis of AS.The regulation of AS-tRF on B4GALNT4 expression was verified by RT-q PCR.Meanwhile,Western blot was used to detect the effect of AS-tRF on protein glycosylation in HUVEC in vitro.Results(1)The expression of AS-tRF was significantly higher in vascular tissue samples from human atherosclerosis patients,AS model rat vascular tissue samples and LPS treated endothelial cells than that of the control group.(2)The transfection of AS-tRF mimics and inhibitors into HUVEC showed good overexpression or knockdown efficiency.Overexpression of AS-tRF promoted endothelial cell adhesion,inflammation,and migration compared with the control group.Knockdown of AS-tRF exhibited opposite results.Western Blot assay confirmed that AS-tRF could promote the expression of some proteins in the pathway related to adhesion and inflammation,whereas knocking down AS-tRF inhibited those proteins.At the same time,in LPS-induced endothelial cells,AS-tRF overexpression or knockdown had the consistent regulatory function.(3)B4GALNT4 was screened and identified as the downstream target of AS-tRF by RNA Pull down and RT-q PCR combined with bioinformatics methods.AS-tRF can up-regulate the m RNA expression of B4GALNT4.The glycosylation level of AS-tRF cells increased after AS-tRF overexpression.ConclusionsOur study reveals for the first time that AS-tRF regulates HUVEC function of adhesion,inflammation,and migration by targeting B4GALNT4.This study may provide a novel theoretical mechanism for endotheila function,and indicate a potential therapeutic target for atherosclerosis.