To Investigate The Effects Of ZNF580 Overexpression On The Expression Profile Of Endothelial Cell Line Using Gene Microarray

Objective: ZNF580,a novel gene associated with LDL stimulation in vascular endothelial cells,was cloned from the human aorta tissue cDNA library in 1999.The ZNF580 gene localized to human chromosome19q13.42 encodes a protein of 171 amino acids containing three repeated tandem C₂H₂-type zinc finger motifs at its carboxyl terminus.C₂H₂-zinc finger genes constitute the largest class of transcription factors within the human genome. They usually play an essential role in altering gene expression involved in the crucial cell functions such as growth and differentiation.Moreover,the data of Gene Expression Omnibus (GEO)show that C₂H₂-zinc finger gene ZNF580 is upregulated in the injured artery,chronic hepatitis,anoxia,etc.These results reveal that ZNF580 may be involved in transcriptional regulation of angiogenesis.In this study,we aimed to investigate the effects of ZNF580 on the expression profile of endothelial cell line EA.hy926 by DNA microarray.Methods:1 The human endothelial hybrid cell line EA.hy926 were cultured in a modified Eagle's Minimum Essential Medium containing with 10% Fetal calf serum(FCS).Cell cultures were maintained in 5% CO₂ atmosphere and saturated humidity at 37°C.2 The eukaryotie expression vector pEGFP-C1 and pEGFP-ZNF580 were transfected into EA.hy926 cells by liposomal transfection with LipofectamineTM2000,respectively.Successfully transfected cells were screened out by G418.The positive clones were identified by RT-PCR and Western blot.3 MTT colorimetric assay were designed to investigate the effects of ZNF580 on endothelial cell growth. 4 DNA microarray was used to sereen the effects of ZNF580 on the expression profile of endothelial cell line EA.hy926.5 Nineteen of different expression genes of EA.hy926 respectively transfected pEGFP-C1 and pEGFP-ZNF580 were validated by Real Time PCR.6 The pathway analysis for upregulated genes and downregulated genes detected by DNA microarray.Results:1 The endothelial cell line EA.hy926 cells that can steadily overexpressing ZNF580 gene were acquired after liposome transfection,G418 screening and RT-PCR and Western blot verification.2 The results of gene chip inspect shows that there were more changes in gene-expressing in pEGFP-ZNF580 transfected EA.hy926 cells than that in pEGFP-C1 transfected EA.hy926 cells;some genes are expressed higher,such as ANGPTL4 and FGF1;some are expressed lower,such as PDE3A and ABCA8.The most important altered genes belonged to functional categories of cell cycle,enzyme regulator activity,cell signal transducer activity,cell translation regulator activity,cell transporter activity and so on.Among those changes,IL8, ANGPTL4, FGF2, FGF1, PDE3A and ABCA8 were validated by Real Time-PCR.3 MEME predicted the consistency of the nine nucleotide sequence CCAG [G/C]CTGG and the E-value of the sequence is smallest, 1.1^e-531.Conclusion:1 The stable transfected celll ines EA.hy926_pEGFP-C1 and EA.hy926_pEGFP-ZNF580were established.2 DNA microarray was used to screen the effects of ZNF580on the expression profile of endothelial cell line EA.hy926.Verified results suggested the results of DNA microarray were reliable.3 The influence of ZNF580 on the expression profile of endothelial cell line EA.hy926 was wide.4 Some altered genes may belong to many important signal pathways.The results suggested ZNF580 may play an important role in vascular endothelial cell.