Carmona-Luque Maria Dolores, Gonzalez-Alvarez Laura, Guerrero Orriach José Luis
Maimonides Institute of Biomedical Research in Cordoba (IMIBIC), 14004 Cordoba, Spain.
Cellular Therapy Unit, Reina Sofia University Hospital, 14004 Cordoba, Spain.
Life (Basel). 2022 Dec 25;13(1):64. doi: 10.3390/life13010064.
Many clinical studies have identified some circulating micro-RNAs (miRNAs) as potential biomarkers with regard to the cardioprotective effects of halogenated agents administered perioperatively during myocardial conditioning procedures. However, there is a major methodological difficulty in identifying these potential miRNA targets in cardiac cells.
We developed an in vitro protocol to analyze the differential expression of target miRNAs at the intracellular level in non-genetically modified primary human cardiomyocytes (HCMs) through their exposure to different hypnotic compounds (i.e., halogenated versus non-halogenated). For this purpose, we performed a validated in vitro model of "ischemia and reperfusion" with the transfection of specific miRNA mimics (MIMICs) designed to simulate naturally occurring mature miRNAs as a functional study. Afterwards, next-generation sequencing (NGS) was used to identify and quantify miRNAs and elucidate their function. The differences in miRNA expression between HCMs exposed to different hypnotic drugs, along with the prediction of functional miRNA targets, were assessed using a meticulous in-house bioinformatics pipeline in order to derive diagnostic biomarkers and possible therapeutic targets.
In brief, this methodological procedure was designed to investigate whether the cardioprotective effects of halogenated agents are a phenomenon mediated by either the activation or the suppression of miRNAs targeted by halogenated anesthetics.
许多临床研究已确定一些循环微RNA(miRNA)可作为围手术期在心肌预处理过程中给予卤化剂的心脏保护作用的潜在生物标志物。然而,在心脏细胞中鉴定这些潜在的miRNA靶标存在一个主要的方法学难题。
我们开发了一种体外实验方案,通过将未经过基因修饰的原代人心肌细胞(HCM)暴露于不同的催眠化合物(即卤化与非卤化)来分析细胞内水平上靶标miRNA的差异表达。为此,我们进行了一个经过验证的“缺血再灌注”体外模型,转染设计用于模拟天然存在的成熟miRNA的特异性miRNA模拟物(MIMICs)作为功能研究。之后,使用下一代测序(NGS)来鉴定和定量miRNA并阐明其功能。使用精心设计的内部生物信息学流程评估暴露于不同催眠药物的HCM之间miRNA表达的差异,以及对功能性miRNA靶标的预测,以得出诊断生物标志物和可能的治疗靶点。
简而言之,该方法学程序旨在研究卤化剂的心脏保护作用是否是由卤化麻醉剂靶向的miRNA的激活或抑制介导的现象。