MicroRNA-150在房颤及急性心梗中的表达及机制研究

发布时间：2018-07-03 16:45

本文选题：miR-150 + 诊断指标　；参考：《华中科技大学》2014年博士论文

【摘要】：近几年,microRNA (miRNA)在心血管生理及疾病机制中的研究获得了重大的进步,非编码RNA在生物学机理过程中的重要作用目前已经取得共识,而miRNAs就是其中的一类,其大约只有22个碱基的RNA序列,通过部分互补结合信使RNA上的靶位点,调控蛋白的合成。在心血管系统中,miRNAs已经被证实起着重要的作用,它几乎参与所有和心血管系统相关细胞的功能调控,例如内皮细胞、心肌细胞、平滑肌细胞,炎症细胞和成纤维细胞,因此,miRNAs直接参与了很多心血管疾病的病理发展。基于它们在疾病中的作用,研究它们对于疾病的诊断、预防和治疗都成为了目前的热点,然而要使它们进入临床现在还需要克服许多的困难,本课题主要探讨miRNAs在心血管疾病中的诊断以及作用机制。一、血浆中降低的miRNA-150作为潜在诊断房颤的新指标 1、基因筛查并验证miRNA-150在房颤病人血浆中表达降低本课题分别收集5例孤立性阵发性房颤(PAF)病人血浆、5例孤立性永久性房颤(PersAF)病人血浆和5例正常对照组,运用深度测序技术筛查出22个miRNAs表达异常,其中miRNA-146a、miRNA-150、miRNA-19a和miRNA-375符合标准进一步验证。同时收集90例独立血浆标本运用探针PCR技术验证这4个miRNAs,发现只有miRNA-150的表达趋势符合筛查结果,其表达分别在PersAF病人血浆中及PAF病人血浆中相对于正常对照组下降约17倍和20倍。 2、下调的miRNA-150表达与房颤相关联结合病人资料和测得的miRNA-150表达值,逻辑回归分析显示,降低的miRNA-150表达(优势比OR1.96,95%可信区间CI1.5～3.57,P0.001)、年龄(OR1.1,95%CI1.36-2.73, P0.001)和左房直径(LAD)(OR1.5,95%CI1.36～1.8, P0.001)分别为房颤的独立相关因素。数据库分析显示miRNA-150的靶基因中有18个基因与AF相关,其中11个与炎症系统有联系,炎症因子不仅可以作为诊断AF的标准,也参与了AF的发病机理,为此我们探测了病人血浆中C-反应蛋白(CPR)的表达水平,发现其与miRNA-150的表达呈负相关(相关系数0.77)。二、miRNA-150在急性心肌梗死(AMI)中调控单核细胞功能保护心肌功能 1. miRNA-150在AMI后的外周单核细胞中表达下调收集20例AMI病人外周血和10例正常对照组,磁珠分选出外周血CD14+单核细胞,qRT-PCR测试发现miRNA-150表达量下调,而在CD14的外周单核细胞(PBMCs)中表达无显著差异。同时建立小鼠心梗模型,从心梗小鼠和对照组中的PBMCs中磁珠分选出CD115+单核细胞,相对于对照组,心梗后单核细胞中的miRNA-150表达显著下调,而在CD115-PBMCs中表达无差异。 2、miRNA-150调控人类单核细胞(THP-1)的迁移和炎症因子的产生体外实验在THP-1细胞系中转染miRNA-150的拟合物(agomiRNA150)、抑制剂(antagomiRNA-150)和对照物,迁移实验发现高表达组相对对照组阻止了THP-1的迁移(3.7±0.5倍),而抑制miRNA-150组促进了迁移(6.3±0.8倍)。另外,转然后的THP-1给予脂多糖(LPS)刺激,ELLSA测试发现,相对于对照组高表达miRNA-150组减少了促炎因子的产生,增加了白介素-10(IL-10)的表达；抑制miRNA-150组得到了相反的结果。 3、上调单核细胞中miRNA-150的表达保护AMI后的心肌功能动物实验显示在外周细胞高表达miRNA-150的心梗小鼠模型中,相对于对照组小鼠改善了心脏功能,减少了心梗的面积,阻止了心肌细胞的凋亡,并且降低了炎症单核细胞Ly-6Chigh的聚集；而在移植了miRNA-150敲除鼠骨髓的小鼠心梗模型中,这些保护效应则被消除。 4、miRNA-150调控趋化因子受体4(CXCR4)的表达数据库分析得出CXCR4为miRNA-150的靶基因,体外实验在THP-1细胞中分别转染agomiRNA-150和对照拟合物,WESTERN实验发现CXCR4表达相对于对照组下调；在高表达miRNA-150的小鼠中,分离CD115+单核细胞,同样发现CXCR4表达相对于对照组下调。结论 1、血浆中降低的miRNA-150可以作为潜在诊断房颤的新指标； 2、miRNA-150调控单核细胞功能,对AMI造成的心肌损伤起着保护作用,为治疗急性心梗提供了新的研究靶点。
[Abstract]:In recent years , microRNA ( miRNA ) has made great progress in the research of cardiovascular physiology and disease mechanism , and the important role of non - coding RNA in the process of biological mechanism has now reached consensus , and the miRNA is one of them , which has been proved to play an important role in the pathogenesis of cardiovascular diseases , such as endothelial cells , myocardial cells , smooth muscle cells , inflammatory cells and fibroblasts .

I . miRNA - 150 lowered in plasma as a new indicator for potential diagnosis of atrial fibrillation

1 . Gene screening and verification of reduced expression of miRNA - 150 in patients with atrial fibrillation

In this study , five patients with isolated paroxysmal atrial fibrillation ( PAF ) were collected from plasma and 5 normal controls . Twenty - five normal controls were screened by depth - sequencing .

2 . Down - regulated miRNA - 150 expression is associated with AF

The results showed that 18 of the target genes of miRNA - 150 were associated with AF , 11 of them were associated with AF , 11 of them were associated with the inflammatory system , and inflammatory factors could not only be used as criteria for diagnosis of AF , but also involved in the pathogenesis of AF , and we detected the expression level of C - reactive protein ( CPR ) in plasma of patients , and found that it had negative correlation with the expression of miRNA - 150 ( correlation coefficient 0.77 ) .

II . Function of miRNA - 150 in regulating the function of monocytes in acute myocardial infarction ( AMI )

1 . Down regulation of miRNA - 150 in peripheral blood mononuclear cells after AMI

The expression of miRNA - 150 was downregulated in peripheral blood mononuclear cells ( PBMCs ) of 20 AMI patients and 10 normal controls , and no significant difference was found in peripheral blood mononuclear cells ( PBMCs ) of CD14 + monocytes .

2 . miRNA - 150 regulates the migration of human monocytes ( THP - 1 ) and the production of inflammatory factors

In vitro experiments were carried out in THP - 1 cell line transfected miRNA - 150 ( agomiRNA150 ) , inhibitor ( miRNA - 150 ) and control . The migration of THP - 1 was inhibited ( 3.7 卤 0.5 times ) in high expression group compared with control group . In addition , THP - 1 inhibited migration ( 6.3 卤 0.8 times ) . In addition , THP - 1 inhibited the production of LPS - 150 group and increased the expression of IL - 10 ( IL - 10 ) .
The suppression of miRNA - 150 groups yielded the opposite results .

3 . Up - regulate the expression of miRNA - 150 in monocytes and protect the myocardial function after AMI

Animal experiments show that in the stem mouse model of the high expression miRNA - 150 of the peripheral cells , the heart function is improved relative to the control group , the area of the myocardial infarction is reduced , the apoptosis of the myocardial cells is prevented , and the aggregation of the inflammatory monocytes is reduced ;
These protective effects were eliminated in mice transplanted with miRNA - 150 knockout mice bone marrow .

4 . miRNA - 150 regulates the expression of chemokine receptor 4 ( CXCR4 )

The results showed that CXCR4 was the target gene of miRNA - 150 , and in vitro experiments were carried out in THP - 1 cells transfected with agomiRNA - 150 and control fitting respectively . Western experiments showed that CXCR4 expression was down - regulated with respect to the control group ;
In mice with high expression of miRNA - 150 , CD115 + monocytes were isolated and CXCR4 expression was also found to be down - regulated with respect to the control group .

Conclusion

1 . miRNA - 150 reduced in plasma can be used as a new index for diagnosis of atrial fibrillation .

2 . miRNA - 150 regulates the function of monocytes , plays a protective role in myocardial injury caused by AMI , and provides a new research target for the treatment of acute myocardial infarction .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R541.75;R542.22

【参考文献】