Department of obstetrics and gynecology, East Hospital, Tongji University School of Medicine, Shanghai, China.
J Cell Biochem. 2018 Nov;119(10):8112-8122. doi: 10.1002/jcb.26736. Epub 2018 Jun 22.
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.
我们的研究旨在探讨位于 HOTAIR 中的 rs4759314 如何参与先天性心脏病的发病机制。通过荧光素酶报告基因检测评估 rs4759314 是否影响 HOTAIR 启动子的转录效率，并确认 miR-545 的靶基因。实时 PCR、Western-blot 和免疫组化分析用于研究 HOTAIR、EGFR、p-ERK、P-P38 MAPK 之间的关系。MTT 检测和流式细胞术分析用于检测 HOTAIR 对细胞活力和凋亡的影响。该多态性位于 HOTAIR 中的 G 等位基因促进 HOTAIR 启动子的转录活性。此外，HOTAIR 抑制 miR-545 的表达。生物信息学分析表明 EGFR 是 miR-545 的虚拟靶基因，miR-545 明显降低野生型 EGFR 3'UTR 的荧光素酶活性，而 miR-545 对突变型 EGFR 3'UTR 的荧光素酶活性没有影响。在 VSD 组中，HOTAIR 和 EGFR 表达水平较低，而 miR-545 表达水平较高。与 AA 组相比，AG 组中 HOTAIR 和 EGFR 水平较高，miR-545 水平较低。HOTAIR 和 miR-545 之间以及 EGFR 和 miR-545 之间的调控关系呈负相关，负相关系数分别为-0.49 和-0.46。HOTAIR 显著增加 EGFR p-ERK 和 P-P38 MAPK 表达水平，此外，HOTAIR 显著促进细胞活力，抑制细胞凋亡。在本研究中，我们提出了 rs4759314 多态性与先天性心脏病风险之间的可能关系，并探讨了 HOTAIR/miR-545/EGFR/MAPK 信号通路失调在先天性心脏病发病机制中的作用。
