microRNA-1抑制心肌特异性Dicer基因缺失小鼠心脏病理性重构

目的 探讨microRNA-1(miR-1)对他莫昔芬(tamoxifen,TMX)诱导的心肌特异性Dicer 基因缺失小鼠心脏结构重构及心功能的影响.方法 采用Myh6-Cre/Loxp重组系统,通过Dicerloxp/1oxp小鼠和Myh6-creERT小鼠杂交,获得Myh6-creERT/Dicerloxp/loxp小鼠.将18只8周龄雄性Myh6-creERT/Dicerloxp/loxp小鼠按随机数字表法分为对照组、模型组、miR-1治疗组.采用腹腔注射20 mg/mL TMX溶液(0.1 mL),连续注射5d,建立心肌特异性Dicer基因缺失心力衰竭小鼠模型,对照组腹腔注射玉米油;模型建立后,miR-1治疗组通过尾静脉注射miR-1类似物(miR-1 mimic) 10 nmol/只,对照组给予同等剂量生理盐水,模型组给予miRNA阴性对照试剂,2次/周.1周后,经胸二维超声心动图检测各组小鼠舒张末期左室内径(LVIDd)、收缩末期左室内径(LVIDs)、舒张末期左室容积(EDV)、收缩末期左室容积(ESV)、射血分数(EF)及左室短轴缩短率(FS),然后收集心肌标本,通过HE染色、Masson三色染色及天狼猩红染色观察小鼠心肌结构重构程度;利用原位缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling,TUNEL)免疫荧光染色检测心肌组织细胞凋亡情况;通过细胞增殖指标Ki67免疫组化染色评估细胞增殖水平.结果 建模后1周,模型组小鼠心功能明显下降,LVIDs及ESV均高于对照组(P＜0.05),EF及FS则显著低于对照组(P＜0.05),miR-1治疗后上述指标均较模型组明显改善(P＜0.05).组织病理学检测结果显示,与对照组相比,模型组小鼠心肌细胞排列紊乱,细胞肥大,炎症细胞浸润明显,心肌纤维化显著,而治疗组小鼠心肌细胞形态正常,无明显肥大和炎性浸润,未见明显的间质纤维化;TUNEL免疫荧光染色及Ki67免疫组化染色结果显示,miR-1治疗组小鼠较模型组心肌组织凋亡比例增加(P＜0.05),且存在较明显的细胞增殖(P＜0.05).结论 miR-1可抑制TMX诱导的心肌特异性Dicer基因缺失心力衰竭模型中心肌细胞的固有改变和间质的改变,抑制心脏结构重构,维持心功能.

MicroRNA-1 prevents cardiac remodeling in mice with heart failure induced by Dicer ablation

Objective To investigate the effects of microRNA-1 (miR-1) on cardiac remodeling and cardiac function following tamoxifen (TMX)-induced heart failure in mice with myocardium-specific Dicer gene deletion.Methods Myh6-Cre/Loxp mice were crossed with Dicer1oxp/loxp mice to obtain Myh6-creERT/Dicerloxp/loxp mice.Eighteen adult male Myh6-creERT/Dicerloxp/loxp mice were randomly divided into control group (corn oil + normal saline),model group (TMX + negative control miRNA) and miR-1 treatment group (TMX + miR mimic).For each mouse,TMX(20 mg/mL,0.1 mL) or corn oil (0.1 mL) were administered by intraperitoneal injection for 5 consecutive days.Normal saline,negative control miRNA or miR-1 mimic was injected via the caudal vein twice a week.After 1 week,all the mice underwent echocardiography and the end diastolic left ventricular diameter (LVIDd),end systolic diameter (LVIDs),left ventricular end diastolic volume (EDV),end systolic volume (ESV),ejection fraction (EF) and left ventricular fractional shortening (FS) were measured.The cardiac tissues were then collected for assessment of cardiac remodeling by histological analysis using HE staining,Masson staining and Sirius Red staining.Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay and Ki67 immunohistochemistry were used to determine the apoptosis and proliferation of the cardiac myocytes.Results At 1 week after the modeling,the cardiac function were significantly decreased in the mice with TMX-induced heart failure,which showed significantly increased LVIDs and ESV (P ＜ 0.05) and lowered EF and FS (P ＜ 0.05) as compared with the control mice.In miR-1 treatment group,the cardiac function parameters were all significantly improved (P ＜0.05).Histopathological examination of the cardiac tissues of the mice with heart failure revealed irregular cardiomyocyte arrangement,cardiomyocyte hypertrophy,inflammatory cell infiltration,and significant myocardial interstitial fibrosis as compared with the tissues from the control mice.In miR-1 treatment group,the cardiomyocytes showed normal morphology without obvious hypertrophy,and no inflammatory cell infiltration or interstitial fibrosis was observed.TUNEL assay and Ki67 immunohistochemistry showed a significantly higher apoptotic rate of myocardocyte (P ＜ 0.05) and more active cardiomyocyte proliferation (P ＜ 0.05) in miR-1 treatment group than in the model group.Conclusion miR-1 plays a vital role in the development of heart failure in mice with myocardium-specific Dicer knockout.MiR-1 inhibits cardiac remodeling by regulating the changes of the myocardial and interstitial cells to maintain the cardiac function and prevent the occurrence of heart failure.