循环miR-19a/b作为心肌缺血再灌注损伤标志物的研究

Investigation of Circulating miR-19a/b as the Biological Marker for Myocardial Ischemia Reperfusion Injury

目的:探讨miR-19a/b在调节心肌缺血再灌注损伤中的作用及临床意义。方法:实时荧光定量聚合酶链式反应(PCR)检测miR-19a/b在经过10 h缺氧、2 h复氧后的H9c2心肌细胞中的表达,通过荧光素酶实验分析miR-19a/b的潜在靶基因。同时,以40例健康者的血清作为对照,采集40例急性心肌梗死(AMI)患者发病24 h内的外周血标本,分离血清后采用实时荧光定量PCR法检测血清中miR-19a/b表达水平,并采用化学发光免疫分析法检测血心肌肌钙蛋白I(c Tn I)含量;免疫抑制法测定肌酸激酶同工酶(CK-MB)的酶活力。结果:miR-19a和miR-19b在经过10 h缺氧、2 h复氧的H9c2心肌细胞中的表达比在正常H9c2细胞中分别增加了5.876倍和2.761倍(P均<0.01)。转染miR-19a/b类似物和miR-19a/b抑制剂分别上调和下调细胞中miR-19a/b的表达。荧光素酶报告基因实验发现,miR-19a/b与第10号染色体缺失的磷酸酶及张力蛋白同源基因(PTEN)蛋白3'UTR区有靶向作用关系。转染miR-19a/b类似物上调miR-19a/b的表达后,PTEN的表达降低;转染miR-19a/b抑制剂下调miR-19a/b的表达后,PTEN的表达增加。与健康受试者比较,miR-19a/b在AMI患者血清中的表达增加(P均<0.01);AMI患者胸痛发作后0~3 h开始升高,6~12 h达高峰;血清CK-MB酶活力和c Tn I水平在AMI发作后2~6 h开始升高,24 h左右达高峰。结论:miR-19a/b在心肌缺血再灌注过程中高表达,PTEN是miR-19a/b的潜在靶基因;循环miR-19a/b可作为心肌缺血再灌注损伤新的无创性诊断标志物。

Objective： To investigate the role of miR-19a/b in myocardial ischemia reperfusion injury （R/I） with its clinical significance. Methods： Our research included in 2 parts. Part 1： in H9c2 cells. miR-19a/b expression in H9c2 cells with 10 h hypoxia and 2 h re-oxygenation was detected by real-time PCR, miR-19a/b potential target gene was assessed by luciferase reporter activity assay. Part 2： in natural person. Control group,n=40 healthy subjects and AMI （acute myocardial infarction） group,n=40 relevant patients. Peripheral blood levels of miR-19a/b were detected as Part 1, cTnI were measured by chemiluminescence immune analysis and CK-MB were assessed by immune inhibition method. Results： Part 1： Compared with normal H9c2 cells, miR-19a/b expressions were increased 5.876 times and 2.761 times in H9c2 cells with 10 h hypoxia and 2 h re-oxygenation, bothP〈0.01. With respectively transfected miR-19a/b mimic and inhibitor, miR-19a/b expression was up-regulated and down-regulated respectively. miR-19a/b and chromosome-10 deleted phosphatase, tensing homolog gene （PTEN） had the targeting effect. With up-regulated miR-19a/b expression, PTEN level was decreased and with down-regulated miR-19a/b expression, PTEN level was increased. Part 2： Compared with Control group, AMI group had elevated blood level of miR-19a/b,P〈0.01. In AMI patients, miR-19a/b was increasing at 0-3 h of＆nbsp;chest pain and reaching the peak at 6-12 h; CK-MB enzyme activity and cTnI content were elevating at 2-6 h of onset and reaching the peak at 24 h. Conclusion： miR-19a/b expression was up-regulated by myocardial ischemia reperfusion in H9c2 cells, PTEN was the potential target gene of miR-19a/b. Circulating miR-19a/b might be used as a new non-invasive biological marker for myocardial ischemia R/I diagnosis.