微小RNA-1268在先天性心脏病发病中的作用及机制研究

TBX1, a major pathogenicity factor of Congenital Heart Disease (CHD), has not been identified in its mechanism. Previous researches indicate that the insG SNP site in its 3' UTR region is related to paroxysm of CHD. Bioinformatics analysis reveals that in thc coding region of wild-type TBX1, the target site of miR-1268 is identified, and SNP site in 3' UTR region of insG TBX1 would shape into an other target site of miR-1268. Cell test confirms that miR-1268 respectively distinguishes TBX1 coding region and 3'UTR region of insG TBX1, holding negative regulation. The investigation of patients displays that expression level of miR-1268 in blood serum of patients with G/G genotype is comparatively higher than that of patients with -/- genotype, while expression levels of TBX1 in lymphocyte and of downstream target genes decline. miR-1268, the wild-type TBX1 regulator, hypothetically existing in natural situation, strengthens remarkably its regulation when double regulatory site appears in the coding region and 3' UTR region with insG SNP taking place, and restrains expression of TBX1, stunts heart growth, and therefore increases the morbidity of CHD. This research aims to discuss the effect and mechanism of miR-1268 in CHD, and provides a new target for clinical prognosis and therapy of CHD.

TBX1是先天性心脏病（CHD）的重要致病因子，其机制未明。前期工作提示其3'UTR区insG SNP位点与CHD发病相关，生物信息学分析显示野生型TBX1编码区有miR-1268结合位点，insG的3'UTR区SNP位点会形成另一个miR-1268结合位点。细胞实验证实miR-1268分别识别TBX1编码区及insG的3'UTR区，发挥负性调控作用。对病人调查显示：G/G基因型CHD患者血清miR-1268表达水平较-/-基因型正常对照上升，而淋巴细胞中TBX1及其下游靶基因表达下调。我们假设，自然情况下存在miR-1268调节野生型TBX1，当发生insG SNP后，编码区与3'UTR区出现双调节位点，miR-1268调控作用显著放大，抑制TBX1表达，影响心脏发育，从而提高CHD发病率。本研究旨在探讨miR-1268在CHD发病中的作用及机理，为临床预测及治疗CHD提供新的靶点。

先天性心脏病（CHD）是一种严重威胁患儿健康的常见出生缺陷，也是出生后一年内非感染性死亡的主要原因，TBX1是CHD的重要致病因子，机制未明。我们发现TBX1 3’UTR区的rs41298842位点（ins G）与CHD的发病率有明显相关性。生物信息学分析显示野生型TBX1编码区有miR-1268结合位点，insG的3’UTR区SNP位点会形成另一个miR-1268结合位点。我们的研究明确miR-1268识别insG的3'UTR区SNP位点，负性调控TBX1及其下游的NKX2.6。但不调节TBX1编码区的miR-1268预测调节位点。使用H2O2诱导H9c2心肌细胞凋亡模型进行研究，结果显示，miR-1268促进心肌细胞凋亡，H2O2诱导凋亡的H9c2心肌细胞中miR-1268表达显著上升。并且，超表达miR-1268的H9c2细胞中促凋亡蛋白Bax表达显著上升。提示，miR-1268在心肌细胞凋亡中发挥重要作用。并且，我们发现，TBX1抑制H2O2诱导的H9c2心肌细胞凋亡，miR-1268识别insG SNP的TBX1，负性调控TBX1，反逆其抑制凋亡效应。此外，研究显示，miR-1268上游有转录因子STAT3、FOXC1结合靶点，STAT3、FOXC1负性调控miR-1268。我们的研究初步阐明了miR-1268对TBX1的调控及对心肌细胞凋亡的调节机制，为临床预测及治疗CHD提供新的靶点和方向。