基于新一代测序筛选急性心肌梗死患者血清外泌体miRNA的差异表达谱

Screening the differential expression profile of serum exocrine miRNA associated with acute myocardial infarction based on the next generation sequencing technology

目的 分析基于新一代测序筛选急性心肌梗死患者血清外泌体miRNA的差异表达谱,阐明该病与血清外泌体miRNA的相关性。方法收集郴州市第一人民医院心内科2020年1月~2021年12月急性心肌梗死患者和正常体检者的血液样本各200例,提取并鉴定外泌体、总RNA,测序分析miRNA表达谱,运用qRT-PCR对测序结果进行验证。对比急性心肌梗死患者和正常体检者的外泌体标记CD63和CD81表达、总RNA鉴定结果、miRNA的差异表达谱、qRT-PCR检测结果。结果 外泌体标记CD63和CD81的测定结果显示,急性心肌梗死患者和正常体检者均能检测到CD63和CD81,提示CD63和CD81均为外泌体标记蛋白;急性心肌梗死患者和正常体检者的OD260/OD280比值均处于1.8~2.1之间,提示总RNA的质量较高,可进行下一步实验检测;经过新一代测序筛选,检测出急性心肌梗死患者和正常体检者血清外泌体miRNA的差异表达谱共89个,其中包括上调63个,下调26个;对三个增加倍数较大的miRNA(miR-1、miR-133a、miR-208a)进行qRT-PCR检测,结果显示急性心肌梗死患者miR-1、miR-133a、miR-208a的Ct值均高于正常体检者(P <0.05)。结论 新一代测序筛选在急性心肌梗死患者中的应用价值好,能检出血清外泌体miRNA的差异表达谱,可作为该病早期预测、诊断的生物标志物。

Objective To analyze the differential expression profile of serum exosomal miRNA in patients with acute myocardial infarction based on new generation sequencing, and to clarify the correlation between the disease and serum exosomal miRNA. Methods Blood samples from 200 patients with acute myocardial infarction and 200 normal subjects in the department of cardiology of Chenzhou first people’s hospital from January 2020 to December 2021 were collected, exosomes and total RNA were extracted and identified, miRNA expression profiles were sequenced and analyzed, and the sequencing results were verified by qRT-PCR. The expression of exosome markers CD63 and CD81, the identification results of total RNA, the differential expression profile of miRNA, and the detection results of qRT-PCR were compared between patients with acute myocardial infarction and normal subjects. Results The determination results of exosome markers CD63 and CD81 showed that CD63 and CD81 could be detected in patients with acute myocardial infarction and normal physical examination, suggesting that both CD63 and CD81 were exosome marker proteins;The OD260/OD280 ratio of patients with acute myocardial infarction and normal physical examination was between 1.8 and 2.1, suggesting that the quality of total RNA was high, which could be tested in the next step;After a new generation of sequencing screening, a total of 89 differential expression profiles of serum exosomal miRNAs in patients with acute myocardial infarction and normal physical examination were detected, including 63 up-regulated and 26 down-regulated;qRT-PCR was performed on three miRNAs(miR-1, mir-133a, and mir-208a) with large increase times. The results showed that the Ct values of miR-1, mir-133a, and mir-208a in patients with acute myocardial infarction were higher than those in normal physical examination(P < 0.05). Conclusion The new generation sequencing screening has good application value in patients with acute myocardial infarction. It can detect the differential expression profile of serum exosome miRNA, and can be used as a biomarker for early prediction and diagnosis of the disease.