miR-199a/mTOR在自发性高血压大鼠心肌纤维化和左心室肥厚中的作用机制研究
|
摘要
目的研究miR-199a和哺乳动物雷帕霉素靶蛋白(mTOR)对自发性高血压(SHR)大鼠心肌纤维化和左心室肥厚的影响,并探讨其可能的作用机制。方法正常雄性WKY大鼠作为正常组(n=15),SHR大鼠随机分为SHR组(n=15)与抑制剂组(n=15)。正常组与SHR组大鼠尾静脉注射生理盐水,抑制剂组大鼠尾静脉注射miR-199a抑制剂。采用尾动脉容积法测定三组大鼠血压;超声心动图检测左心室舒张/收缩末期内径(LVEDD/LVESD)、左心室后壁厚度(LVPWT)、室间隔厚度(IVST)、心肌缩短分数(FS)、左心室射血分数(LVEF);右颈总动脉插管记录心功能指标左心室压力最大升高或降低速率(+dp/dt,-dp/dt)、左心室舒张末期内压(LVEDP),测定左心室重量(LVM),并计算左心室质量指数(LVMI);HE染色、Masson染色观察大鼠心肌纤维化程度;实时定量PCR(qRT-PCR)、蛋白免疫印迹(WB)分析心肌组织中miR-199a和mTOR的mRNA及蛋白表达情况;Pearson评估miR-199a和mTOR相关性。体外培养H9c2细胞,转染miR-199a mimic使其过表达,qRT-PCR、WB检测细胞中miR-199a和mTOR的mRNA及蛋白表达情况;荧光素酶活性检测mTOR是否为miR-199a的靶基因。结果与正常组相比,SHR组LVEDD、LVESD、IVST、LVPWT均升高,差异均有统计学意义(P均<0.05);与SHR组相比,抑制剂组LVEDD、LVESD、IVST、LVPWT均降低,差异均有统计学意义(P均<0.05)。与正常组相比,SHR组收缩压、LVM、LVMI、LVEDP均升高,+dp/dt、-dp/dt降低,差异均有统计学意义(P均<0.05);与SHR组相比,抑制剂组收缩压、LVM、LVMI、LVEDP均降低,+dp/dt、-dp/dt升高,差异均有统计学意义(P均<0.05)。与正常组相比,SHR组病理学评分、心肌胶原纤维比例升高,差异均有统计学意义(P均<0.05);与SHR组相比,抑制剂组病理学评分、心肌胶原纤维比例降低,差异均有统计学意义(P均<0.05)。与正常组相比,SHR组心肌组织miR-199amRNA表达升高,mTOR的mRNA及蛋白表达水平降低,差异均有统计学意义(P均<0.05);与SHR组相比,抑制剂组心肌组织miR-199a mRNA表达降低,mTOR的mRNA及蛋白表达水平升高,差异均有统计学意义(P均<0.05)。Pearson相关性分析显示,miR-199a与mTOR的mRNA和蛋白表达均呈显著性负相关(r=-4.873,P=0.000;r=-5.126,P=0.000)。与空白对照组、阴性转染组相比,miR-199a过表达组miR-199a mRNA表达升高,mTOR的mRNA及蛋白表达降低,差异均有统计学意义(P均<0.05)。与阴性转染组相比,在转染3’UTR-Wt的细胞中,miR-199a过表达组组荧光素酶活性显著降低(P<0.05);而在转染3’UTR-Mut的细胞中,miR-199a过表达组与阴性转染组荧光素酶活性无显著差异(P>0.05)。结论 miR-199a表达上调可能与自发性高血压大鼠心肌纤维化和左心室肥厚有关,其机制可能是通过靶向调控mTOR实现。
Objective To study the influence of miR-199 a and mammalian target of rapamycin(mTOR) on myocardial fibrosis and left ventricular hypertrophy(LVH) in spontaneous hypertensive rats(SHR), and discuss possible mechanism. Methods Normal male WKY rats were chosen into normal group(n=15), and SHR were randomly divided into SHR group and inhibitor group(each n=15). The normal group and SHR group were given caudal vein injection of normal saline, and inhibitor group was given caudal vein injection of miR-199 a inhibitor.The blood pressure was determined by using caudal artery volume method in 3 groups. LVEDD/LVESD, LVPWT,IVST, FS and LVEF were detected by using echocardiogram. The heart function indexes(+dp/dt,-dp/dt, LVEDP,LVM and LVMI) were recorded and calculated by using right common carotid artery intubation. The severity of myocardial fibrosis was observed after HE and Masson staining. The mRNA and protein expressions of myocardial miR-199 a and mTOR were detected by using qRT-PCR and Western blotting assay. The correlation between miR-199 a and mTOR was reviewed by using Pearson correlation analysis. H9 c2 cells were cultivated in vitro and transfected with miR-199 a mimic leading to over-expression. The mRNA and protein expressions of miR-199 a and mTOR were detected by using qRT-PCR and Western blotting assay. Whether or not mTOR was the target gene of miR-199 a was detected by using luciferase activity. Results LVEDD, LVESD, IVST and LVPWT increased in SHR group compared with normal group(all P<0.05), and decreased in inhibitor group compared with SHR group(all P<0.05). LVM, LVMI and LVEDP increased and +dp/dt and-dp/dt decreased in SHR group compared with normal group(all P<0.05), and LVM, LVMI and LVEDP decreased and +dp/dt and-dp/dt increased in inhibitor group compared with SHR group(all P<0.05). The pathologic scores and myocardial collagen fiber ratio increased in SHR group compared with normal group(all P<0.05), and decreased in inhibitor group compared with SHR group(all P<0.05). The expression of miR-199 a mRNA increased and mTOR mRNA expression decreased in SHR group compared with normal group(all P<0.05). The expression of miR-199 a mRNA decreased and mTOR mRNA expression increased in inhibitor group compared with SHR group(all P<0.05). The results of Pearson correlation analysis showed that miR-199 a was significantly and negatively correlated to mTOR mRNA and protein expressions(r=-4.873, P=0.000; r=-5.126, P=0.000). The expression of miR-199 a mRNA increased and mTOR mRNA and protein expressions decreased in miR-199 a over-expression group compared with blank control group and negative transfection group(all P<0.05). The luciferase activity decreased in 3'UTR-Wt transfected cells in miR-199 a over-expression group compared with negative transfection group(P<0.05), and had no significant difference in 3'UTR-Mut transfected cells between miR-199 a over-expression group and negative transfection group(P<0.05). Conclusion The up-regulated expression of miR-199 a may be related to myocardial fibrosis and LVH, and targeted-regulation of mTOR is possibly the mechanism.
Objective To study the influence of miR-199 a and mammalian target of rapamycin(mTOR) on myocardial fibrosis and left ventricular hypertrophy(LVH) in spontaneous hypertensive rats(SHR), and discuss possible mechanism. Methods Normal male WKY rats were chosen into normal group(n=15), and SHR were randomly divided into SHR group and inhibitor group(each n=15). The normal group and SHR group were given caudal vein injection of normal saline, and inhibitor group was given caudal vein injection of miR-199 a inhibitor.The blood pressure was determined by using caudal artery volume method in 3 groups. LVEDD/LVESD, LVPWT,IVST, FS and LVEF were detected by using echocardiogram. The heart function indexes(+dp/dt,-dp/dt, LVEDP,LVM and LVMI) were recorded and calculated by using right common carotid artery intubation. The severity of myocardial fibrosis was observed after HE and Masson staining. The mRNA and protein expressions of myocardial miR-199 a and mTOR were detected by using qRT-PCR and Western blotting assay. The correlation between miR-199 a and mTOR was reviewed by using Pearson correlation analysis. H9 c2 cells were cultivated in vitro and transfected with miR-199 a mimic leading to over-expression. The mRNA and protein expressions of miR-199 a and mTOR were detected by using qRT-PCR and Western blotting assay. Whether or not mTOR was the target gene of miR-199 a was detected by using luciferase activity. Results LVEDD, LVESD, IVST and LVPWT increased in SHR group compared with normal group(all P<0.05), and decreased in inhibitor group compared with SHR group(all P<0.05). LVM, LVMI and LVEDP increased and +dp/dt and-dp/dt decreased in SHR group compared with normal group(all P<0.05), and LVM, LVMI and LVEDP decreased and +dp/dt and-dp/dt increased in inhibitor group compared with SHR group(all P<0.05). The pathologic scores and myocardial collagen fiber ratio increased in SHR group compared with normal group(all P<0.05), and decreased in inhibitor group compared with SHR group(all P<0.05). The expression of miR-199 a mRNA increased and mTOR mRNA expression decreased in SHR group compared with normal group(all P<0.05). The expression of miR-199 a mRNA decreased and mTOR mRNA expression increased in inhibitor group compared with SHR group(all P<0.05). The results of Pearson correlation analysis showed that miR-199 a was significantly and negatively correlated to mTOR mRNA and protein expressions(r=-4.873, P=0.000; r=-5.126, P=0.000). The expression of miR-199 a mRNA increased and mTOR mRNA and protein expressions decreased in miR-199 a over-expression group compared with blank control group and negative transfection group(all P<0.05). The luciferase activity decreased in 3'UTR-Wt transfected cells in miR-199 a over-expression group compared with negative transfection group(P<0.05), and had no significant difference in 3'UTR-Mut transfected cells between miR-199 a over-expression group and negative transfection group(P<0.05). Conclusion The up-regulated expression of miR-199 a may be related to myocardial fibrosis and LVH, and targeted-regulation of mTOR is possibly the mechanism.
引文
[1]王超,张萍.高血压左心室肥厚形成机制的研究进展[J].重庆医学,2015(22):3143-6.
[2]杨晋静,王敬萍,张月安,等.miRNA在心室重塑中的调控作用[J].中华医学杂志,2015,95(29):2408-10.
[3]张振辉,黎佼,熊旭明,等.微小RNA-199a-5p调控大鼠心肌细胞肥大的研究[J].中国病理生理杂志,2014,30(3):408-13.
[4]李奕,曾凡星,吴迎,等.不同强度运动诱导Akt/mTOR信号对心肌肥大的作用研究[J].北京体育大学学报,2013(6):49-54.
[5]李燕,黄凌波,付兵,等.心脏磁共振T1 Mapping技术和ECV评估扩张型心肌病心肌纤维化应用分析[J].中国循证心血管医学杂志,2018,10(2):185-9.
[6]尚冬升,边云飞,杨慧宇,等.脂联素在高血压致炎症和心肌纤维化中的作用[J].中国动脉硬化杂志,2015,23(5):464-8.
[7]蒋征奎,李晓,田锋奇,等.黄芪多糖对自发性高血压大鼠心功能和心肌纤维化的影响及其机制研究[J].中国药房,2016,27(25):3505-8.
[8]陈裴.自发性高血压大鼠的脑白质损伤及其相关机制[D].山东大学,2013.
[9]黄帧桧,柏松,张年宝,等.葛根素对自发性高血压大鼠心肌纤维化的影响及其机制(英文)[J].中国病理生理杂志,2014,30(3):518-23.
[10]张志敏,赵连友,卢凡,等.高同型半胱氨酸对高血压大鼠心肌细胞GRP78和CHOP表达及左室肥厚的影响[J].中国循证心血管医学杂志,2014(2):223-6.
[11]马东宇,王邦宁,胡泽平,等.肝细胞生长因子联合缬沙坦对自发性高血压大鼠心肌纤维化的影响[J].安徽医科大学学报,2013,48(10):1159-63.
[12]曲巍,于波.依普利酮对自发性高血压大鼠心肌纤维化的影响及其机制探讨[J].山东医药,2015(4):26-7.
[13]王洁.miRNA在心脏疾病中的研究进展[J].同济大学学报(医学版),2014,35(6):124-8.
[14]Huang ZP,Chen J,Seok HY,et al.MicroRNA-22 regulates cardiac hypertrophy and remodeling in response to stress[J].Circ Res,2013,112(9):1234-43.
[15]刘红梅.miR-135a-5p与miR-199a-5p在心肌肥大中的作用及初步机制研究[D].第三军医大学,2014.
[16]张灼,朱杰宁,肖珍,等.微小RNA-199a-5p通过靶向SIRT1促进心肌纤维化相关基因表达[J].中国病理生理杂志,2017,33(10):1781-7.
[17]Azzouzi H,Leptidis S,Dirkx E,et al.The hypoxia-inducible microRNAcluster miR-199a~214 targets myocardial PPARδand impairs mitochondrial fatty acid oxidation[J].Cell Metab,2013,18(3):341-54.
[18]黄文秋,黄宏,徐祥,等.mTOR及其下游信号通路在骨髓间充质干细胞氧化应激损伤中的变化及作用[J].第三军医大学学报,2013,35(2):114-8.
[19]王宇霆,周海燕,滕辉.Akt/mTOR信号通路在乌司他丁减轻大鼠心肌细胞氧化应激损伤中的作用[J].中华麻醉学杂志,2017,37(5):637-40.
[20]胡浩然,宣佳利,杨解人,等.芝麻素改善自发性高血压大鼠肾损伤的作用及与PI3K/AKT/mTOR信号通路的关系[J].中国病理生理杂志,2016,32(4):719-25.
[2]杨晋静,王敬萍,张月安,等.miRNA在心室重塑中的调控作用[J].中华医学杂志,2015,95(29):2408-10.
[3]张振辉,黎佼,熊旭明,等.微小RNA-199a-5p调控大鼠心肌细胞肥大的研究[J].中国病理生理杂志,2014,30(3):408-13.
[4]李奕,曾凡星,吴迎,等.不同强度运动诱导Akt/mTOR信号对心肌肥大的作用研究[J].北京体育大学学报,2013(6):49-54.
[5]李燕,黄凌波,付兵,等.心脏磁共振T1 Mapping技术和ECV评估扩张型心肌病心肌纤维化应用分析[J].中国循证心血管医学杂志,2018,10(2):185-9.
[6]尚冬升,边云飞,杨慧宇,等.脂联素在高血压致炎症和心肌纤维化中的作用[J].中国动脉硬化杂志,2015,23(5):464-8.
[7]蒋征奎,李晓,田锋奇,等.黄芪多糖对自发性高血压大鼠心功能和心肌纤维化的影响及其机制研究[J].中国药房,2016,27(25):3505-8.
[8]陈裴.自发性高血压大鼠的脑白质损伤及其相关机制[D].山东大学,2013.
[9]黄帧桧,柏松,张年宝,等.葛根素对自发性高血压大鼠心肌纤维化的影响及其机制(英文)[J].中国病理生理杂志,2014,30(3):518-23.
[10]张志敏,赵连友,卢凡,等.高同型半胱氨酸对高血压大鼠心肌细胞GRP78和CHOP表达及左室肥厚的影响[J].中国循证心血管医学杂志,2014(2):223-6.
[11]马东宇,王邦宁,胡泽平,等.肝细胞生长因子联合缬沙坦对自发性高血压大鼠心肌纤维化的影响[J].安徽医科大学学报,2013,48(10):1159-63.
[12]曲巍,于波.依普利酮对自发性高血压大鼠心肌纤维化的影响及其机制探讨[J].山东医药,2015(4):26-7.
[13]王洁.miRNA在心脏疾病中的研究进展[J].同济大学学报(医学版),2014,35(6):124-8.
[14]Huang ZP,Chen J,Seok HY,et al.MicroRNA-22 regulates cardiac hypertrophy and remodeling in response to stress[J].Circ Res,2013,112(9):1234-43.
[15]刘红梅.miR-135a-5p与miR-199a-5p在心肌肥大中的作用及初步机制研究[D].第三军医大学,2014.
[16]张灼,朱杰宁,肖珍,等.微小RNA-199a-5p通过靶向SIRT1促进心肌纤维化相关基因表达[J].中国病理生理杂志,2017,33(10):1781-7.
[17]Azzouzi H,Leptidis S,Dirkx E,et al.The hypoxia-inducible microRNAcluster miR-199a~214 targets myocardial PPARδand impairs mitochondrial fatty acid oxidation[J].Cell Metab,2013,18(3):341-54.
[18]黄文秋,黄宏,徐祥,等.mTOR及其下游信号通路在骨髓间充质干细胞氧化应激损伤中的变化及作用[J].第三军医大学学报,2013,35(2):114-8.
[19]王宇霆,周海燕,滕辉.Akt/mTOR信号通路在乌司他丁减轻大鼠心肌细胞氧化应激损伤中的作用[J].中华麻醉学杂志,2017,37(5):637-40.
[20]胡浩然,宣佳利,杨解人,等.芝麻素改善自发性高血压大鼠肾损伤的作用及与PI3K/AKT/mTOR信号通路的关系[J].中国病理生理杂志,2016,32(4):719-25.