房颤射频消融对冠状窦血miRNA表达的影响

Effect of radiofrequency ablation on mi RNA expression in coronary sinus blood of patients with atrial fibrillation

目的探讨房颤射频消融手术(RFA)对房颤患者冠状窦血mi RNAs表达的影响,以揭示房颤发生发展的机制并寻找可能的mi RNAs干预靶点。方法选择30例行房颤射频消融术的患者(阵发性、持续性和永久性房颤各10例),以同期10例健康体检者作为正常对照组。射频消融术前分别取冠状窦血和外周血,术后3个月取外周血,应用mi RNAs芯片进行全基因组mi RNAs表达谱微阵列分析,Real-time PCR对冠状窦血差异表达的主要调控mi RNAs结果进行验证,通过mirbase、miranda、targetscan数据库进行靶基因分析,并对重要mi RNAs进行双荧光素酶结合实验,分析验证靶基因。结果房颤患者术前冠状窦血与自身外周血比较,共有142种mi RNAs表达存在显著差异,其中6种表达上调,8种表达下调(P<0.05)。射频消融术后外周血再与自身术前外周血比较,术前上调的6种mi RNAs中,分别有3种表达上调和下调,其中mi R-1266下调–204.17倍,术前下调的8种mi RNAs中,术后7种继续下调,其中mi R-3664-5p下调–44.66倍,仅mi R-574-3p上调5.25倍。荧光素酶结合实验证实SCN5A是mi R-1266的直接靶基因,而CACNA1C是mi R-4279的直接靶基因。结论冠状窦血mi RNAs的表达差异能直接反映房颤发作时心肌mi RNAs的调控状况。房颤射频消融手术逆转或改变了术前冠状窦血显著改变的mi RNAs表达异常。手术前后表达差异幅度较大和同时调控多个离子通道蛋白的mi RNAs如mi R-1266有可能成为未来房颤干预的靶点。

Objective To investigate the effect of radiofrequency ablation（RFA） on mi RNA expression in coronar y sinus blood（CSB） of patients with atrial fibrillation（AF）, especially after the termination of AF by RFA. And try to identif y the possible mi RNAs which can play real regulatory role in AF, so that to find the pathogenesis of AF and possible mi RNA targets for intervention. Methods Thirty AF patients（paroxysmal, persistent and permanent AF, 10 each） were enrolled, alongside of 10 healthy subjects as controls. Peripheral blood（PB） samples were obtained before and 3 months after RFA, respectively. The total RNA was extracted and hybridized with the micro RNA chips（micro RNA v18.0）, and the differential expressions of mi RNA and clustering analysis in whole genome were performed with Volcano Plot and t MEV software respectively, and validated by Real-time PCR. The target gene of mi RNAs was analyzed by retrieving the mirbase, miranda and targetscan databases. The important mi RNAs were undergone with dual luciferase binding experiment to analyze and validate target genes. Results Comparing the CSB with PB of patient-self before RFA, 142 mi RNAs expressed differently, of which 6 up-regulated and 8 down-regulated significantly（P〈0.05）. W hen comparing the PB of patients-self after and before RFA, 3 mi RNAs up-regulated and another 3 down-regulated in above mentioned up-regulated 6 mi RNAs, and mi R-1266 was down-regulated 204.17-fold. Of 8 down-regulated mi RNAs mentioned above, 7 were down-regulated again after RFA, and mi R-3664-5p down-regulated 44.66-fold, while mi R-574-3p up-regulated 5.25-fold. SCN5 A was the target gene of mi R-1266, and CACNA1 C was the target gene of mi R-4279 confirmed by luciferase binding experiments. Conclusions The differential expression of mi RNAs in CSB may directly reflect the regulatory status of mi RNAs in cardiomyocytes in AF attack. RFA can reverse or change the abnormal expressions of mi RNAs in CSB of AF patients in pro-RFA. The mi RNAs which altered significantly before and after RFA, or regulate multi-ion channel proteins simultaneously, like mi R-1266, may become a future target for AF intervention.