tsRNA在急性心肌梗死早期诊断中的价值

Diagnostic value of tRNA-derived small RNA in early stage of acute myocardial infarction

目的 探讨转运RNA衍生的小RNA(transfer RNA-derived small RNA,tsRNA)在急性心肌梗死(AMI)早期诊断中的价值及其作为AMI诊断标志物的潜能。方法 将年龄及体质量匹配的雄性C57小鼠(8~10周龄)随机分为MI组及对照组(Sham组),每组4只。两组均于建模24 h后提取左室心肌组织RNA,经去修饰预处理,连接人工化接头后扩增,筛选包含15~40个核苷酸的小RNA扩增产物,构建文库并测序,将测序结果与GtRNAdb数据库成熟的t RNA和tRNA前体序列比对,获得在AMI前后心肌中差异表达的tsRNA谱。采用生物信息学分析同一密码子对应的tRNA在AMI前后剪切方式的改变。依据AMI前后tsRNA表达谱差异,获得在AMI后特异性高丰度表达的tsRNA,并在AMI小鼠心肌和血浆中验证,探讨tsRNA作为AMI诊断标志物的潜能。结果 tsRNA表达谱在组内有较好的重复性,组间差异较大。发生AMI后,Asn-GTT、Glu-TTC、Gly-ACC、Gly-GCC、His-GTG、Ile-AAT、Ile-GAT、Pro-TGG、Ser-AGA和Trp-CCA在心肌中剪切方式发生改变,生成有明显差异的tsRNA表达谱。与Sham组相比,MI组有268个tsRNA表达上调,1 228个tsRNA表达下调,且有64个特异性tsRNA。AMI建模第1天,小鼠tRF-Gly-CCC-2-31、tiRNA-Val-CAC-1-32、tiRNA-ValAAC-2-32、tiRNA-Glu-TTC-2-32和tiRNA-Lys-TTT-1-34在心肌中特异性表达,其中tiRNA-Val-AAC-2-32和tiRNA-LysTTT-1-34在AMI小鼠血浆中特异性高丰度表达,并随AMI持续时间动态变化。结论 tsRNA表达谱在AMI前后差异显著,其中在小鼠心肌和血浆中特异性表达的tiRNA-Val-AAC-2-32和tiRNA-Lys-TTT-1-34有AMI诊断潜能,可能在AMI修复过程中发挥重要作用。

Objective To explore the difference of transfer RNA-derived small RNA(tsRNA)expression profile before and after acute myocardial infarction(AMI)and the diagnostic value of tsRNA for AMI.Methods Age-and weight-matched male C57 mice(8-10 weeks)were randomly divided into MI group and Sham group,with 4 in each group.AMI model was surgically induced in mice in MI group.After 24 h of modeling,RNA was extracted from left ventricular myocardial tissue.After removing modifications,total RNA of each sample was sequentially ligated to 3'and 5'small RNA adapters.Subsequently,reverse transcription PCR was performed.cDNA was then synthesized and amplified.The amplified products corresponding to the size of 15-40 nt RNA were screened to construct a library for sequencing. The sequencing results were compared with the mature tRNAand tRNA precursor sequences in GtRNAdb database. Differentially expressed tsRNA profiles before and after AMI were obtained.The alterations of the cleaved patterns of tRNA corresponding to the same codon before and after AMI were analyzed. Accordingto the profile of differentially expressed tsRNA before and after AMI, tsRNA only abundantly expressed in MI group were selectedand verified in myocardial tissue and plasma of mice to explore the potential of these tsRNAs as diagnostic markers of AMI.Results tsRNA profile showed good repeatability within the same group and great distinctiveness between the different groups.After AMI occurred, the cleaved patterns of a variety of tRNAs changed, including tRNA Asn-GTT, Glu-TTC, Gly-ACC, Gly-GCC,His-GTG, Ile-AAT, Ile-GAT, Pro-TGG, Ser-AGA, and Trp-CCA. Compared with the Sham group, 268 tsRNAs significantly upregulatedand 1 228 tsRNAs down-regulated in MI group, and 64 tsRNAs were uniquely expressed in MI group. tRF-Gly-CCC-2-31,tiRNA-Val-CAC-1-32, tiRNA-Val-AAC-2-32, tiRNA-Glu-TTC-2-32, and tiRNA-Lys-TTT-1-34 were specifically expressed incardiac tissue on the 1st day post AMI. Among them, tiRNA-Val-AAC-2-32 and tiRNA-Lys-TTT-1-34 showed specifically abundantlevels in plasma from MI group and dynamically changed with AMI duration. Conclusions The expression profile of tsRNA issignificantly different before and after AMI. tiRNA-Val-AAC-2-32 and tiRNA-Lys-TTT-1-34 are uniquely highly expressed inmyocardial tissue and plasma from AMI mice, and might have the potential as diagnostic markers of AMI.