病毒性心肌炎患儿外周血中微小RNA-98的表达及其对凋亡相关蛋白/凋亡相关蛋白配体表达的影响

Expression of micro RNA-98 in peripheral blood of children with viral myocarditis and its effect on the expression of factor associated suicide/factor associated suicide ligand

目的探讨病毒性心肌炎(VM)患儿外周血中微小RNA-98(miR-98)的水平及其对凋亡相关蛋白(Fas)/凋亡相关蛋白配体(FasL)表达的影响。方法选择2015年9月至2017年7月郑州大学附属儿童医院收治的VM患儿62例作为VM组,根据左心室射血分数(LVEF)及心肌肌钙蛋白(cTnⅠ)水平将VM组患儿分为轻度组(n=38)和重度组(n=24),另选择56例健康儿童作为对照组。采用实时荧光定量聚合酶链反应(qRT-PCR)测定3组受试者外周血中miR-98、Fas、FasL mRNA表达水平,Western blot检测Fas、FasL蛋白表达水平。体外培养人心肌细胞(HCM)、H9C2心肌细胞至占培养皿底面积80%时,将细胞分为空白组(不做任何处理)、阴性转染组(无意义序列转染至细胞内)和miR-98转染组(miR-98 siRNA转染至细胞内)。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法检测3组细胞培养12、24、48、72 h时细胞增殖情况;平板细胞克隆形成实验检测细胞克隆形成情况;流式细胞仪检测细胞凋亡情况。结果 VM组患儿外周血中miR-98相对表达量低于对照组(P<0.05),Fas、FasL mRNA相对表达量高于对照组(P<0.05),重度组患儿外周血中miR-98相对表达量低于轻度组,Fas、FasL mRNA相对表达量高于轻度组(P<0.05)。VM组患儿外周血中Fas、FasL蛋白相对表达量高于对照组(P<0.05),重度组患儿外周血中Fas、FasL蛋白相对表达量高于轻度组(P<0.05)。VM组患儿外周血中miR-98与Fas、FasL呈显著负相关(r=-516、-463,P<0.05)。miR-98转染组HCM、H9C2心肌细胞抑制率在各时间点均高于空白组和阴性转染组(P<0.05)。miR-98转染组HCM、H9C2心肌细胞克隆数量和miR-98相对表达量低于空白组和阴性转染组(P<0.05),HCM、H9C2细胞凋亡率及HCM、H9C2心肌细胞中Fas、FasL蛋白相对表达量高于空白组和阴性转染组(P<0.05)。结论 miR-98在VM患儿外周血中表达水平下降,其可能是通过调节Fas/FasL基因在心肌细胞中的表达来参与心肌细胞损伤和凋亡。

Objective To explore the level of micro RNA-98（miR-98） in peripheral blood of children with viral myocarditis（VM） and its effect on the expression of factor associated suicide（Fas）/factor associated suicide ligand（FasL）.Methods Sixty-two VM children in the Children′s Hospital Affiliated of Zhengzhou University from September 2015 to July 2017 were selected as VM group,the children in VM group were divided into mild group（ n =38） and severe group（ n =24） according to left ventricular ejection fraction（LVEF） and cardiac troponin（cTnI） levels.And 56 healthy children were selected as control group.The expressions of miR-98 and Fas,FasL mRNA in peripheral blood of children in the three groups were detected by quantitative real time polymerase chain reaction（qRT-PCR） and the expressions of Fas,FasL protein were detected by Western blot.The human cardiac myocytes（HCM）,H9C2 cells were cultured in petri dish and proliferated to the bottom area of the dish 80%.Then the cells were divided into blank group （without any treatment）,negative transfection group （control sequence transfected into cells） and miR-98 transfection group （miR-98 transfected into cells）.The cell proliferation was detected by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay at 12,24,48 and 72 h after cultured;the colony formation was detected by plate cell clone formation assay;the cell apoptosis was detected by flow cytometry.Results The expression of miR-98 mRNA in the peripheral blood of children in the VM group was significantly lower than that in the control group（ P 〈0.05）,but the expressions of Fas,FasL mRNA were significantly higher than those in the control group（ P 〈0.05）;the expression of miR-98 in peripheral blood of children of them in the severe group was lower than that in the mild group,and the expression of Fas,FasL mRNA in peripheral blood of children in the severe group was higher than that in the mild group（ P 〈0.05）.The expression of Fas and FasL protein in the peripheral blood of children in the VM group was significantly higher than that in the control group（ P 〈0.05）,and the expression of Fas and FasL protein in the severe group was significantly higher than that in the mild group（ P 〈0.05）.There was a significant negative correlation between miR-98 and Fas,FasL in peripheral blood of children in the VM group（ r =-516,-463; P 〈0.05）.The inhibition rates of HCM and H9C2 cells in miR-98 transfection group were significantly higher than those in blank group and negative transfection group at each time point（ P 〈0.05）.The number of cell colonies and the expression of miR-98 in HCM and H9C2 cells in the miR-98 transfection group were significantly lower than those in the blank group and the negative transfection group（ P 〈0.05）.The apoptotic rate of HCM and H9C2 cells and the expression of Fas,FasL protein in HCM and H9C2 cells in the miR-98 transfection group were significantly higher than those in the blank group and the negative transfection group（ P 〈0.05）.Conclusion The expression level of miR-98 in peripheral blood of children with VM is decreased.It may be involve in the injury and apoptosis of cardiac myocytes by regulating the expression of Fas/FasL gene in cardiac myocytes.