OX-LDL Inhibiting Human CREG Expression Via Increasing DNA Methylation In HUVECs

ObjectiveAtherosclerosis(AS)is a vascular disease characterized by intramural inflammation and plaque formation.At present,AS is considered to be the result of interaction between risk factors such as serum lipids,serum glucose,inflammation and genetic susceptible genes.However,the traditional genetranscriptional regulation can not effectively explain the interaction between cardiovascular diseases and environmental variables.The role of epigenetic regulation in cardiovascular diseases has attracted more and more attention.Epigenetics refers to the genetic changes in gene expression and normal DNA sequence,which often occur at the level of transcriptional or post-transcriptional regulation.In recent years,it has been found that epigenetics is a bridge between genes and environment,and DNA methylation is the most important epigenetic marker that leads to the continuous change of gene expression.DNA methylation is the most well-defined and most stable epigenetic marker in current research leading to sustained changes in gene expression.Recent studies have confirmed that the degree of methylation of whole genome DNA is closely related to the occurrence of AS.Local DNA hypermethylation regulation is involved in the occurrence and evolution of AS by altering the expression of AS-regulated genes,but the specific targets and mechanisms of action remain unclear.Gill's study identified that Cell repressor of E1A-stimulated genes(CREG)is a transcriptional factor cloned from Hela cells.Our previous study implicated that CREG gene is an important homeostastic regulator of tissue and cell differentiation.CREG is highly expressed in human normal vascular endothelial cells,but significantly decreased in atherosclerotic vascular endothelial cells.Further studies have confirmed that over-expression of CREG gene can resist the occurrence of AS,but the molecular mechanism of its down-regulation has not been elucidated.Using bioinformatics software,we found that there were a large number of CpG islands in the first exon of hCREG gene,which suggested that DNA methylation might be involved in the negative regulation of CREG gene expression in AS vascular endothelial cells.Based on these findings,we propose a scientific hypothesis that repressor of CREG expression is involved in atherosclerosis through DNA methylation regulation.Therefore,the aim of this study is to explore the methylation regulation mechanism of CREG in OX-LDL induced HUVECs,and to provide a new theoretical basis and therapeutic target for the prevention and treatment of AS.Methods(1)The effects of oxidized low density lipoprotein(OX-LDL)and methyltransferase inhibitor(5-Aza-2'-deoxycytidine,5-Aza-CdR)on primary human umbilical vein endothelial cells(HUVEC)were studied by Western blot and real-time PCR.The correlation between human CREG gene expression and methyltransferase DNMTs was determined preliminarily.(2)By bioinformatics software prediction and luciferase reporter gene activity detection,the location of CpG islands of human CREG gene and the regulatory role of CpG islands on the expression of CREG gene were identified.(3)Primary HUVEC cells were treated with OX-LDL and 5-Aza-CdR to extract DNA and sequence pyrophosphate.The methylation sites of the key CpG islands of CREG gene in each group were observed.To construct a luciferase reporter gene vector containing different methylation sites of key CpG islands of CREG gene and detect luciferase activity.It was confirmed that the change of methylation affected the expression of CREG gene.Results(1)OX-LDL(50 and 100 ?g/mL)treated the primary HUVEC cells.Western blot and real-time PCR showed that with the increase of OX-LDL concentration,the expression of DNMT1,DNMT3 a and DNMT3 b elevated,while the expression of CREG is decreased.After inhibition of the expression of DNMT1,DNMT3 a and DNMT3 b in primary HUVEC cells by 5-Aza-CdR treatment at different concentrations(10,50 and 100 micromole),CREG transcription and expression increased.These results suggest that the transcriptional expression of human CREG gene is regulated by the methylation level of HUVEC cells.(2)By bioinformatics,CpG islands(+1/+588)were found in the first exon of human CREG gene.Further luciferase reporter gene vector with different promoter fragments confirmed that CpG island had negative transcriptional regulation effect on CREG expression,and +79/+255 was the key negative transcriptional regulatory region.(3)OX-LDL(50,100?g/mL)and 5-Aza-CdR(10,50,100?M)acted on primary HUVEC cells respectively.Pyrophosphate sequencing of CREG promoter +79/+255 region showed that methylation level of key CpG islands of CREG gene in cells treated with OX-LDL was higher than that in control group,while methylation level of key CpG islands of CREG gene in cells treated with 5-Aza-CdR was lower than that in control group.These results suggest that OX-LDL can promote the methylation of CpG island(+201/+255)fragment in promoter region of CREG gene.Subsequently,luciferase reporter gene vectors with 8 CpG Island mutations in the +201/+255 fragment were constructed to detect luciferase activity and determine the key methylation regulatory site as +201/+202.These results suggest that the expression of CREG gene is affected by methylation.ConclusionsOX-LDL,as an important pathological factors of AS,regulated the CpG island of CREG gene hypermethylated and the expression of CREG is down-regulated,which leads to atherosclerosis.This may be one of the key regulatory mechanisms of CREG protein deletion in endothelial cells during AS initiation.