Our study was aimed to investigate the mechanism of rs4759314 located in HOTAIR involved in congenital heart disease. Luciferase assay was performed to evaluate whether rs4759314 affected the transcription efficiency of HOTAIR promoter, and confirm miR-545 target gene. Real-time PCR, Western-blot and IHC were carried out to investigate the relationship among HOTAIR, EGFR, p-ERK, P-P38 MAPK. MTT assay and flow cytometry analysis were performed to detect the effect of HOTAIR on cell viability and apoptosis. The presence of G allele of the polymorphism located in HOTAIR promoted transcription activity of HOTAIR promoter. Furthermore, HOTAIR inhibited miR-545 expression. EGFR was identified as a virtual target gene of miR-545 using bioinformatics analysis, and miR-545 apparently decreased luciferase activity of wild-type EGFR 3'UTR, while miR-545 had no effect on luciferase activity of mutant EGFR 3'UTR. HOTAIR and EGFR were lowly expressed, while miR-545 was highly expressed in VSD group. Higher levels of HOTAIR and ECFR, while a lower level of miR-545 were observed in AG than AA groups. Regulatory relationships between HOTAIR and miR-545, as well as EGFR and miR-545 were found to be negatively correlated, with the negative correlation coefficient being -0.49 and -0.46, respectively. HOTAIR evidently increased EGFR p-ERK and P-P38 MAPK expression levels, moreover HOTAIR substantially promoted cell viability, and inhibited cell apoptosis. In this study, we suggested the possible relation between the rs4759314 polymorphism and the risk of congenital heart disease, and explored the deregulation of HOTAIR/miR-545/EGFR/MAPK signaling pathway in the pathogenesis of congenital heart disease.
Keywords: EGFR; HOTAIR; MAPK; Rs4759314; congenital heart disease; miR-545.
© 2018 Wiley Periodicals, Inc.