冠心病候选基因β2肾上腺素能受体基因相关微小核糖核酸let-7i的实验研究

Preliminary Investigation of MicroRNAs Related to β_2 Adrenergic Receptor Gene in Rats

目的:β2肾上腺素能受体(ADRB2)基因是冠心病最重要的候选基因之一,本研究探讨微小核糖核酸(miRNA)对ADRB2基因的调控及其在冠心病中的意义。方法:根据"种子区"原理预测对ADRB2具有调节作用的miRNA,按种系恒定性以及与冠心病的关系程度进一步筛选。对候选miRNA在大鼠心肌细胞H9c2(2-1)中进行增强和抑制处理,验证候选miRNA对ADRB2基因的调控。增强处理为加入相应miRNA的前体分子以提高其功能水平,抑制处理采用经化学修饰的变异体作为竞争性拮抗剂以抑制其活性水平。增强和抑制实验分别以miRNA自身序列随机化所产生的阴性分子作对照。前体或抑制剂经转染导入细胞,48 h后对ADRB2蛋白进行免疫印迹杂交分析。结果:研究结果表明,let-7i是冠心病背景下对ADRB2最具调控意义的miRNA之一。ANOVA统计分析显示,以let-7i增强剂处理心肌细胞48 h后使ADRB2蛋白水平降低了61.83%(P=0.0012),同时,以let-7i拮抗剂处理心肌细胞48 h导致ADRB2蛋白水平升高了48.49%(P=0.0071)。结论:let-7i对心肌细胞ADRB2水平具有显著的调节作用,并与之呈负相关,研究提示ADRB2的miRNA调控机制在冠心病病因学及治疗学中具有重要意义。

Objective： β2 adrenergic receptor（ADRB2）gene is one of the important coronary artery disease（CAD） candidate genes, our research investigated its related microRNAs for ADRB2 regulation with its significances for CAD occurrence in rats. Methods：The ADRB2 related microRNAs were predicted by ＂seed-region＂ principle, and further selected with their conser- vation properties and relevance to CAD. Rats＇ myocardial cell line H9c2 （2-1）was used for microRNAs enhancing and inhibiting tests. For enhancing experiment, microRNA precursor molecules were added to increase its function;and for inhibiting experiment, chemically modified microRNA molecules were sued as competitive antagonists to decrease its activity. The control materials were the corresponding scrambled negative molecules by randomized microRNA sequencing. The precursor and inhibitor were transfect- ed into H9c2（2-1 ）cells and ADRB2 protein level was examined by immunoblotting analysis after 48 hours of treatment. Results：MicroRNA let-7i was one of the most prominent candidate regulators for CAD. ANOVA statistic analysis indicated that with 48 hour enhancing treatment of let-7i, the cellular protein level of ADRB2 decreased at 61.83%, P = 0. 0012; mean- while, with 48 hours antagonists treatment of let-7i, the cellular protein of ADRB2 increased at 48.49%, P = 0. 0071. Conclusion ： MicroRNA let-7i could significantly and negatively regulate myocardial ADRB2 protein level in H9c2 （2-1）cells,the microRNA regulation for ADRB2 may play an important role for CAD occurrence in rats.