SHR大鼠肥厚左室心肌microRNA表达与室性心律失常关系的研究
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摘要
microRNA(miRNA)是内源性非编码蛋白质的单链小RNA,它通过调控基因表达来参与生命过程中的一系列重要进程。有研究表明miRNA可能与疾病发生有密切的关系,在不同类型疾病的发生过程中,miRNA的表达水平及其调节作用是不同的。已有研究发现miRNA与心肌肥厚、心律失常等心血管疾病的发生发展密切相关。动物实验研究表明,大鼠心肌梗死后miRNA-1的表达上调抑制心肌连接蛋白43(connexin 43,Cx43)和内向整流钾通道2.1(Kir2.1)的表达而引发室性心律失常(VA)。
高血压病是常见的心血管疾病,长期的血压增高可导致左室肥厚(LVH)。已有临床研究表明,高血压病伴LVH患者VA的发生率可数倍增高,甚至可发生猝死。目前有关高血压心肌肥厚发生VA的机制尚未完全阐明。因此,为了进一步阐明高血压LVH者发生VA的分子机制,寻找新的VA治疗靶点,本研究以SHR大鼠为研究对象,进行了以下三方面的系列研究。
第一部分:SHR大鼠LVH与VA诱发的实验研究。
17周龄的雄性SHR大鼠12只为LVH组,8周龄的雄性SHR大鼠12只为非LVH组,17周龄的雄性WKY大鼠12只为对照组。通过Langendorff灌注大鼠的离体心脏,观察低钾液灌注诱发VA的情况;组织病理学染色观察离体心脏LVH情况。结果发现:1、HE染色结果显示LVH组大鼠左心室心肌细胞明显肥大,心肌间质有不同程度增多。2、LVH组灌注低钾液后出现VA时间明显早于非LVH组和对照组(P<0.01,P<0.05)。3、LVH组的心律失常评分、VT发生率明显高于非LVH组和对照组(P<0.05)。
第二部分:SHR大鼠肥厚左室心肌组织中miRNA表达的研究。本部分包括二个方面的研究:1、SHR大鼠肥厚左室心肌组织中miRNA差异表达研究。2、实时荧光定量PCR(RT-PCR)检测SHR大鼠肥厚左室心肌组织miRNA-1表达。
在SHR大鼠肥厚左室心肌组织中miRNA差异表达的研究中, 10只16周龄雄性SHR大鼠为LVH组,10只8周龄雄性SHR大鼠为对照组。左室质量指数(LVMI)、组织病理学染色和测量心肌细胞横径(TDM)方法观察LVH状况;取左心室心肌组织常规抽提总RNA,应用含有924条探针的miRNA芯片检测系统进行miRNA表达谱差异分析。结果发现: 1、与对照组比较,LVH组和非LVH组的收缩压和舒张压明显增高(P<0.05, P<0.01)。2、HE染色结果显示LVH组大鼠左室心肌呈肥大表现,心肌间质有不同程度增多;与对照组比较,LVH组大鼠LVMI和CVF明显增高(P<0.05)。3、LVH组大鼠左室心肌组织miRNA表达谱中差异表达的miRNA共有13个,其中有4个miRNA上调,9个miRNA下调。
在RT-PCR检测SHR大鼠肥厚左室心肌组织miRNA-1表达的研究中,通过RT-PCR法定量检测第二部分(研究一)大鼠心肌组织miRNA-1表达水平。结果发现:1、与对照组比较,LVH组左室心肌组织miRNA-1表达水平明显升高为对照组的2.08±0.21倍。2、SHR大鼠左室心肌组织miRNA-1表达水平与LVMI、CVF均呈正相关(分别r=0.712和0.674,均P<0.05)。
第三部分:miRNA-1对心肌组织Cx43、Kir2.1的调控作用研究。分为两方面的研究:1、SHR大鼠肥厚左室心肌组织中Cx43、Kir2.1表达及其与miRNA-1相关性研究。2、干预SHR大鼠肥厚左室心肌组织中miRNA-1的表达对Cx43、Kir2.1蛋白表达水平的影响。
在SHR大鼠肥厚左室心肌组织中Cx43、Kir2.1表达及其与miRNA-1相关性研究中,取第二部分两组大鼠心肌组织,用免疫组织化学和Western Blot检测心肌组织中Kir2.1和Cx43蛋白表达。结果发现:LVH组左室心肌组织Cx43、Kir2.1相对含量均明显低于对照组(均P<0.05),并且与心肌组织miRNA-1表达水平均呈显著负相关(均P<0.05)。
在干预SHR大鼠肥厚左室心肌组织中miRNA-1的表达对Cx43、Kir2.1水平的影响研究中,选择16只高血压持续发展期并有LVH的17周龄SHR大鼠作为研究对象,随机分为干预组6只,空白对照组和阴性对照组各5只。采用尾静脉高压注射脂质体瞬时转染miRNA-1抑制剂(Anti-miRNA-1)技术基因沉默miRNA-1。结果发现:1、与两个对照组比较,干预组转染Anti-miRNA-1后大鼠左室心肌组织miRNA-1表达水平明显减少了(40±8%,P<0.05)。2、转染Anti-miRNA-1基因沉默miRNA-1使干预组大鼠左室心肌组织Cx43、Kir2.1蛋白表达水平明显高于阴性对照组和空白对照组(均P<0.05)。
本课题研究结果表明:1、伴有LVH的SHR大鼠心脏低钾灌注液诱发VA几率明显增高,提示LVH与VA的发生密切相关。2、SHR大鼠肥厚的左室心肌组织miRNA的表达有明显改变,提示miRNA可能参与SHR大鼠高血压左室肥厚的发生发展。3、SHR肥厚左室大鼠心肌组织中miRNA-1表达明显上调,并与LVMI、CVF呈正相关。4、SHR大鼠肥厚左室心肌组织Cx43、Kir2.1表达下调,并与心肌组织miRNA-1表达水平呈显著负相关。5、抑制miRNA-1表达能使SHR肥厚左室心肌组织Cx43和Kir2.1蛋白表达水平升高,提示miRNA-1可能通过抑制Cx43和Kir2.1蛋白表达而参与SHR大鼠左室肥厚时VA的发生。
microRNAs (miRNA) are endogenous non-coding single-chain small RNA,which participate in a series of important life processes through regulate gene expression. Researchs had shown that miRNA may be closely related with diseases occurred. miRNA expression and those regulation effects are different in the different types of diseases process. It has been demonstrated that miRNA are closely related to the development of cardiac hypertrophy, arrhythmia and other cardiovascular diseases. An animal study had shown that overexpression of miRNA-1 in the ischemic myocardial tissue of the myocardial infarction rat model could inhibit cardiac connexin 43 (Cx43) and inward rectifier potassium channel 2.1 (Kir2.1) expression and triggered ventricular arrhythmia (VA).
Hypertension is a common cardiovascular disease, long-term high blood pressure will lead to left ventricular hypertrophy (LVH). Clinical studies have shown that the rate of VA in hypertensive patients with LVH was several times higher than the normaltensive ones. Currently the mechanisms of VA due to LVH are not yet fully clarified. Therefore, in order to further clarify the molecular mechanism of the occurrence of VA due to hypertensive LVH, and to find new therapeutic targets , we choosed SHR rats as research subjects to practice the following three aspects studies in this study.
Part I: The Experimental Research Of Provocative VA In SHRs With LVH
The research had three groups. 17-week-old male SHRs were assigned into LVH group (n=12), 8-week-old SHRs were assigned into non-LVH group(n=12), 17-week-old male WKYs rats were assigned into the control group(n=12). The three groups were used to observe the situation of VA induced by Langendorff perfusion of isolated rat hearts perfused with K+-deficient solution. Results:1. Pathologic results showed that LVH group had left ventricular myocardial cellular hypertrophy and increased myocardial interstitial in varying degrees; 2. After perfusioning K+-deficient solution, the time of VA occurred in LVH group significantly earlier than non-LVH group and the control group (P<0.01, P<0.05). 3. Arrhythmia score and the VA incidences of LVH group were significantly higher than non-LVH group and the control group (P <0.05). Part II: Analysis Of MicroRNAs Differential Expression Patterns In The Hypertrophic Left Ventricular Tissue Of SHRs . There were two researches in this part: 1.The research of miRNAs differential expression patterns in the hypertrophic left ventricular tissue of SHRs; 2. The research of miRNA-1 expression in SHRs hypertrophic left ventricle by fluorescent quantitative real time polymerase chain reation (RT-PCR) .
In the research of the differential expression of miRNA in SHR hypertrophic left ventricular tissuce , 10 seventeen-week-old male SHRs were assigned into LVH group, 10 eight-week-old male SHRs were served as control group. The left ventricular mass index (LVMI), histopathological examination and measurement of myocardial cell diameter (TDM) were used to observe the LVH situation.The total RNA of the two groups were extracted from left ventricle tissue and the differential expression of miRNAs was detected using miRNA microarray chip which containd 924 miRNA microarray probe system.Results: 1. Compared with the control group, systolic and diastolic blood pressure were significantly higher in LVH group and non-LVH group (P<0.05, P<0.01).2.Pathologic results showed that LVH group had left ventricular myocardial cellular hypertrophy and increased myocardial interstitial in varying degrees; Compared with the control group, LVMI and TDM of the rats in LVH group were significantly bigger (all P<0.05). 3. 13 miRNAs were showed differential expression in LVH rats, 4 were up-regulated and 9 were down-regulated.
In the research of miRNA-1 expression in SHR hypertrophic left ventricle by qRT-PCR, we used qRT-PCR to detect miRNA-1 expression in SHR hypertrophied left ventricular tissue. Results: 1. the level of miRNA-1 significantly increased (to 2.08±0.21-fold) higher in the hypertrophic left ventricular tissue of SHRs. 2. miRNA-1 expression in SHR left ventricular myocardium and were positively correlated with LVMI, CVF (r= 0.712 and 0.674, respectively, both P <0.05).
Part III: The Regulative Role Of miRNA-1 Working On Myocardium Cx43 And Kir2.1 In SHRs. There were two researches in this part:1. The correlation between Cx43, Kir2.1 expression and miRNA-1 level in SHR hypertrophied left ventricular myocardium. 2.The intervention on miRNA-1 expression in SHR hypertrophied left ventricle affecting on Cx43, Kir2.1 expression levels.
In the research of the correlation between Cx43, Kir2.1 expression and miRNA-1 level in SHR hypertrophied left ventricular myocardium ,we used immunohistochemistry and Western Blot to detect Kir2.1 and Cx43 protein expression in myocardial tissue. The results showed that: Cx43, Kir2.1 relative levels in LVH left ventricular myocardium were significantly lower than the control group (all P<0.05), and significant negatively correlated with the myocardial expression of miRNA-1 level (P <0.05).
In the research of the intervention on miRNA-1 expression in SHR hypertrophic left ventricle affecting on Cx43, Kir2.1 expression levels, 17-week-old SHR with the sustainable development of hypertension and LVH as research subjects were randomly divided into the intervention group(n=6) ,blank control group (n=5)and negative control group(n=5). The technology of high-pressure tail vein injection with liposome transiently transfected miRNA-1 inhibitor (Anti-miRNA-1) was use to silence miRNA-1. The results showed: 1.Compared with two control groups, miRNA-1 expression in the left ventricular myocardium of intervention group by being transfected Anti-miRNA-1 was significantly reduced (40±8%, P <0.05). 2. Compared with negative control group and blank control group ,gene silencing by transfection Anti-miRNA-1 can increase Cx43, Kir2.1 protein expression in rat left ventricular myocardium significantly (all P <0.05).
The results of this research project show that: 1. The occurred earlier with provocative VA incidences of ventricular arrhythmia increased significantly, which implies that LVH is associated with VA. 2. The expression profile of miRNAs changes in hypertrophic myocardium, suggesting those changed miRNAs may involve in the formation of LVH.3. The level of miRNA-1 increased obviously in the left ventricular hypertrophic tissue of SHRs, its expression was positively correlated with LVMI and CVF in LVH rats. 4. The protein level of Cx43 and Kir2.1 decreased remarkably in the left ventricular hypertrophic tissue of SHRs, and negative correlation with the myocardial expression of miRNA-1. 5. After miRNA-1 was silenced, the level of miRNA-1 significantly decreased, accompanying with the protein level of Cx43 and Kir2.1 increased, suggesting that miRNA-1 might play an important role on the formation of VA due to LVH by inhibiting the expression of Cx43 and Kir2.1.
高血压病是常见的心血管疾病,长期的血压增高可导致左室肥厚(LVH)。已有临床研究表明,高血压病伴LVH患者VA的发生率可数倍增高,甚至可发生猝死。目前有关高血压心肌肥厚发生VA的机制尚未完全阐明。因此,为了进一步阐明高血压LVH者发生VA的分子机制,寻找新的VA治疗靶点,本研究以SHR大鼠为研究对象,进行了以下三方面的系列研究。
第一部分:SHR大鼠LVH与VA诱发的实验研究。
17周龄的雄性SHR大鼠12只为LVH组,8周龄的雄性SHR大鼠12只为非LVH组,17周龄的雄性WKY大鼠12只为对照组。通过Langendorff灌注大鼠的离体心脏,观察低钾液灌注诱发VA的情况;组织病理学染色观察离体心脏LVH情况。结果发现:1、HE染色结果显示LVH组大鼠左心室心肌细胞明显肥大,心肌间质有不同程度增多。2、LVH组灌注低钾液后出现VA时间明显早于非LVH组和对照组(P<0.01,P<0.05)。3、LVH组的心律失常评分、VT发生率明显高于非LVH组和对照组(P<0.05)。
第二部分:SHR大鼠肥厚左室心肌组织中miRNA表达的研究。本部分包括二个方面的研究:1、SHR大鼠肥厚左室心肌组织中miRNA差异表达研究。2、实时荧光定量PCR(RT-PCR)检测SHR大鼠肥厚左室心肌组织miRNA-1表达。
在SHR大鼠肥厚左室心肌组织中miRNA差异表达的研究中, 10只16周龄雄性SHR大鼠为LVH组,10只8周龄雄性SHR大鼠为对照组。左室质量指数(LVMI)、组织病理学染色和测量心肌细胞横径(TDM)方法观察LVH状况;取左心室心肌组织常规抽提总RNA,应用含有924条探针的miRNA芯片检测系统进行miRNA表达谱差异分析。结果发现: 1、与对照组比较,LVH组和非LVH组的收缩压和舒张压明显增高(P<0.05, P<0.01)。2、HE染色结果显示LVH组大鼠左室心肌呈肥大表现,心肌间质有不同程度增多;与对照组比较,LVH组大鼠LVMI和CVF明显增高(P<0.05)。3、LVH组大鼠左室心肌组织miRNA表达谱中差异表达的miRNA共有13个,其中有4个miRNA上调,9个miRNA下调。
在RT-PCR检测SHR大鼠肥厚左室心肌组织miRNA-1表达的研究中,通过RT-PCR法定量检测第二部分(研究一)大鼠心肌组织miRNA-1表达水平。结果发现:1、与对照组比较,LVH组左室心肌组织miRNA-1表达水平明显升高为对照组的2.08±0.21倍。2、SHR大鼠左室心肌组织miRNA-1表达水平与LVMI、CVF均呈正相关(分别r=0.712和0.674,均P<0.05)。
第三部分:miRNA-1对心肌组织Cx43、Kir2.1的调控作用研究。分为两方面的研究:1、SHR大鼠肥厚左室心肌组织中Cx43、Kir2.1表达及其与miRNA-1相关性研究。2、干预SHR大鼠肥厚左室心肌组织中miRNA-1的表达对Cx43、Kir2.1蛋白表达水平的影响。
在SHR大鼠肥厚左室心肌组织中Cx43、Kir2.1表达及其与miRNA-1相关性研究中,取第二部分两组大鼠心肌组织,用免疫组织化学和Western Blot检测心肌组织中Kir2.1和Cx43蛋白表达。结果发现:LVH组左室心肌组织Cx43、Kir2.1相对含量均明显低于对照组(均P<0.05),并且与心肌组织miRNA-1表达水平均呈显著负相关(均P<0.05)。
在干预SHR大鼠肥厚左室心肌组织中miRNA-1的表达对Cx43、Kir2.1水平的影响研究中,选择16只高血压持续发展期并有LVH的17周龄SHR大鼠作为研究对象,随机分为干预组6只,空白对照组和阴性对照组各5只。采用尾静脉高压注射脂质体瞬时转染miRNA-1抑制剂(Anti-miRNA-1)技术基因沉默miRNA-1。结果发现:1、与两个对照组比较,干预组转染Anti-miRNA-1后大鼠左室心肌组织miRNA-1表达水平明显减少了(40±8%,P<0.05)。2、转染Anti-miRNA-1基因沉默miRNA-1使干预组大鼠左室心肌组织Cx43、Kir2.1蛋白表达水平明显高于阴性对照组和空白对照组(均P<0.05)。
本课题研究结果表明:1、伴有LVH的SHR大鼠心脏低钾灌注液诱发VA几率明显增高,提示LVH与VA的发生密切相关。2、SHR大鼠肥厚的左室心肌组织miRNA的表达有明显改变,提示miRNA可能参与SHR大鼠高血压左室肥厚的发生发展。3、SHR肥厚左室大鼠心肌组织中miRNA-1表达明显上调,并与LVMI、CVF呈正相关。4、SHR大鼠肥厚左室心肌组织Cx43、Kir2.1表达下调,并与心肌组织miRNA-1表达水平呈显著负相关。5、抑制miRNA-1表达能使SHR肥厚左室心肌组织Cx43和Kir2.1蛋白表达水平升高,提示miRNA-1可能通过抑制Cx43和Kir2.1蛋白表达而参与SHR大鼠左室肥厚时VA的发生。
microRNAs (miRNA) are endogenous non-coding single-chain small RNA,which participate in a series of important life processes through regulate gene expression. Researchs had shown that miRNA may be closely related with diseases occurred. miRNA expression and those regulation effects are different in the different types of diseases process. It has been demonstrated that miRNA are closely related to the development of cardiac hypertrophy, arrhythmia and other cardiovascular diseases. An animal study had shown that overexpression of miRNA-1 in the ischemic myocardial tissue of the myocardial infarction rat model could inhibit cardiac connexin 43 (Cx43) and inward rectifier potassium channel 2.1 (Kir2.1) expression and triggered ventricular arrhythmia (VA).
Hypertension is a common cardiovascular disease, long-term high blood pressure will lead to left ventricular hypertrophy (LVH). Clinical studies have shown that the rate of VA in hypertensive patients with LVH was several times higher than the normaltensive ones. Currently the mechanisms of VA due to LVH are not yet fully clarified. Therefore, in order to further clarify the molecular mechanism of the occurrence of VA due to hypertensive LVH, and to find new therapeutic targets , we choosed SHR rats as research subjects to practice the following three aspects studies in this study.
Part I: The Experimental Research Of Provocative VA In SHRs With LVH
The research had three groups. 17-week-old male SHRs were assigned into LVH group (n=12), 8-week-old SHRs were assigned into non-LVH group(n=12), 17-week-old male WKYs rats were assigned into the control group(n=12). The three groups were used to observe the situation of VA induced by Langendorff perfusion of isolated rat hearts perfused with K+-deficient solution. Results:1. Pathologic results showed that LVH group had left ventricular myocardial cellular hypertrophy and increased myocardial interstitial in varying degrees; 2. After perfusioning K+-deficient solution, the time of VA occurred in LVH group significantly earlier than non-LVH group and the control group (P<0.01, P<0.05). 3. Arrhythmia score and the VA incidences of LVH group were significantly higher than non-LVH group and the control group (P <0.05). Part II: Analysis Of MicroRNAs Differential Expression Patterns In The Hypertrophic Left Ventricular Tissue Of SHRs . There were two researches in this part: 1.The research of miRNAs differential expression patterns in the hypertrophic left ventricular tissue of SHRs; 2. The research of miRNA-1 expression in SHRs hypertrophic left ventricle by fluorescent quantitative real time polymerase chain reation (RT-PCR) .
In the research of the differential expression of miRNA in SHR hypertrophic left ventricular tissuce , 10 seventeen-week-old male SHRs were assigned into LVH group, 10 eight-week-old male SHRs were served as control group. The left ventricular mass index (LVMI), histopathological examination and measurement of myocardial cell diameter (TDM) were used to observe the LVH situation.The total RNA of the two groups were extracted from left ventricle tissue and the differential expression of miRNAs was detected using miRNA microarray chip which containd 924 miRNA microarray probe system.Results: 1. Compared with the control group, systolic and diastolic blood pressure were significantly higher in LVH group and non-LVH group (P<0.05, P<0.01).2.Pathologic results showed that LVH group had left ventricular myocardial cellular hypertrophy and increased myocardial interstitial in varying degrees; Compared with the control group, LVMI and TDM of the rats in LVH group were significantly bigger (all P<0.05). 3. 13 miRNAs were showed differential expression in LVH rats, 4 were up-regulated and 9 were down-regulated.
In the research of miRNA-1 expression in SHR hypertrophic left ventricle by qRT-PCR, we used qRT-PCR to detect miRNA-1 expression in SHR hypertrophied left ventricular tissue. Results: 1. the level of miRNA-1 significantly increased (to 2.08±0.21-fold) higher in the hypertrophic left ventricular tissue of SHRs. 2. miRNA-1 expression in SHR left ventricular myocardium and were positively correlated with LVMI, CVF (r= 0.712 and 0.674, respectively, both P <0.05).
Part III: The Regulative Role Of miRNA-1 Working On Myocardium Cx43 And Kir2.1 In SHRs. There were two researches in this part:1. The correlation between Cx43, Kir2.1 expression and miRNA-1 level in SHR hypertrophied left ventricular myocardium. 2.The intervention on miRNA-1 expression in SHR hypertrophied left ventricle affecting on Cx43, Kir2.1 expression levels.
In the research of the correlation between Cx43, Kir2.1 expression and miRNA-1 level in SHR hypertrophied left ventricular myocardium ,we used immunohistochemistry and Western Blot to detect Kir2.1 and Cx43 protein expression in myocardial tissue. The results showed that: Cx43, Kir2.1 relative levels in LVH left ventricular myocardium were significantly lower than the control group (all P<0.05), and significant negatively correlated with the myocardial expression of miRNA-1 level (P <0.05).
In the research of the intervention on miRNA-1 expression in SHR hypertrophic left ventricle affecting on Cx43, Kir2.1 expression levels, 17-week-old SHR with the sustainable development of hypertension and LVH as research subjects were randomly divided into the intervention group(n=6) ,blank control group (n=5)and negative control group(n=5). The technology of high-pressure tail vein injection with liposome transiently transfected miRNA-1 inhibitor (Anti-miRNA-1) was use to silence miRNA-1. The results showed: 1.Compared with two control groups, miRNA-1 expression in the left ventricular myocardium of intervention group by being transfected Anti-miRNA-1 was significantly reduced (40±8%, P <0.05). 2. Compared with negative control group and blank control group ,gene silencing by transfection Anti-miRNA-1 can increase Cx43, Kir2.1 protein expression in rat left ventricular myocardium significantly (all P <0.05).
The results of this research project show that: 1. The occurred earlier with provocative VA incidences of ventricular arrhythmia increased significantly, which implies that LVH is associated with VA. 2. The expression profile of miRNAs changes in hypertrophic myocardium, suggesting those changed miRNAs may involve in the formation of LVH.3. The level of miRNA-1 increased obviously in the left ventricular hypertrophic tissue of SHRs, its expression was positively correlated with LVMI and CVF in LVH rats. 4. The protein level of Cx43 and Kir2.1 decreased remarkably in the left ventricular hypertrophic tissue of SHRs, and negative correlation with the myocardial expression of miRNA-1. 5. After miRNA-1 was silenced, the level of miRNA-1 significantly decreased, accompanying with the protein level of Cx43 and Kir2.1 increased, suggesting that miRNA-1 might play an important role on the formation of VA due to LVH by inhibiting the expression of Cx43 and Kir2.1.
引文
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