中华急诊医学杂志  2016, Vol. 25 Issue (4): 439-443
大鼠心力衰竭细胞microRNA表达谱及生物信息学分析
朱海娟, 何淑芳, 金世云, 胡军, 张野     
230061 合肥,安徽省妇幼保健院麻醉科(朱海娟);
230601合肥,安徽医科大学第二附属医院麻醉科(何淑芳、金世云、胡军、张野)
摘要目的 观察大鼠心力衰竭细胞中microRNA (miRNA)表达变化,利用生物信息学分析技术探索差异miRNA靶基因的功能。 方法 18只成年雄性SD大鼠,体质量200~220 g,随机数字表法随机分为两组:正常对照组(CON组)和心力衰竭组(HF组)。HF组制备阿霉素致心力衰竭模型,CON组注射等量生理盐水。提取大鼠心脏,Largendorff逆行灌注法分离心室肌细胞,提取总RNA行miRNA表达谱检测,筛选两组差异表达的miRNA,荧光定量RT- PCR 技术验证结果, Targetscan、miRanda软件预测差异表达miRNA的靶基因,并对靶基因行生物信息学分析。 结果 芯片检测结果显示,与CON组相比,HF组共有37个miRNA表达发生显著改变,其中22个miRNA上调,15个miRNA下调(均P<0.01,FDR<0.05)。荧光定量RT- PCR检测miR-133b-5p (t=14.56,P<0.1)、 miR-6216 (t=9.32,P<0.1)、let-7e-5p (t=13.92,P<0.1)表达水平,变化趋势与芯片结果一致。生物信息学分析显示,差异表达miRNA调控的靶基因显著富集于31个基因组(均P<0.01,FDR<0.05)和12条信号通路(均P<0.05,FDR<0.05),其中泛素蛋白酶体系统、MAPK信号通路、Toll样受体信号通路富集程度较高。 结论 阿霉素诱导的心力衰竭模型大鼠心肌细胞中miRNA表达谱发生显著改变,这些差异表达miRNA可能通过调控靶基因功能参与心力衰竭的病理生理过程。
关键词心力衰竭     心肌细胞     microRNA表达谱     生物信息学分析    荧光定量RT-PCR    靶基因    基因组    KEGG通路    
Screening of specific microRNAs and bioinformatics analysis on cardiomyocytes in rat with heart failure
Zhu Haijuan, He Shufang, Jin Shiyun, Hu Jun, Zhang Ye     
Department of Anesthesiology, Anhui Women and Child Health Care Hospital, Hefei 230061, China(Zhu HJ);
Department of Anesthesiology, Second Affiliated Hospital of Anhui Medical University, Hefei 230601, China ( He SF, Jin SY, Hu J, Zhang Y)
Fund Program: National Natural Science Foundation of China Youth Fund (81200171);Key Programs of Anhui Science and Technology Department(1301043031)
Corresponding author: Zhang Ye, Email:zhangye_hassan@sina.co
Abstract: Objective To investigate microRNAs (miRNAs) expression profiling of cardiomyocytes in rats with heart failure, and predict miRNAs-regulated target genes and their functions. Methods Total of 18 male SD rats weighing 200-220gwere randomly divided into 2 groups: the control group (CON) and the heart failure group (HF). The rats in HF group were injected by adriamycin via tail vein to induce heart failure, meanwhile in CON group, rats were received an equal volume of 0.9% sodium chloride intravenously. The cardiomyocytes isolated from the rat hearts in two groups and cultured overnight. After that, total RNA was extracted and then subjected to miRNA microarray to screen differentially expressed miRNAs. The reults of microarray were further verified by quantitative real-time PCR (qRT-PCR). The target genes regulated by differentially expressed miRNAs were predicted by the software of Targetscan and miRanda. Bioinformatics analysis was performed to predict the miRNAs-regulated target genes and analyze the enriched gene ontology (GO) and signaling pathway (KEGG Pathway). Results The results of miRNA microarray showed thatatotal of 37 miRNAs were differentially expressed in HF group as compared to CON group, among which 22 miRNAs were up-regulated and 15 miRNAs were down-regulated (P<0.01, FDR<0.05). The expression of miR-133b-5p (t=14.56, P<0.01), miR-6216 (t=9.32, P<0.01) and let-7e-5p (t=13.92, P<0.01) which were detected by qRT-PCR exhibited the similar tendency of up or down regulation to those shown in microarray results. Bioinformatics analysis indicated that miRNAs-regulated target genes were significantly enriched in 31 GOs (P<0.01, FDR<0.05) and 12 signal pathways (P<0.05,FDR<0.05), among which ubiquitin-proteasome system, MAPK signaling pathway and Toll like siganling pathway exhibitedahigher enrichment. Conclusions MiRNA expression profile on cardiomyocytes in rat with adriamycin-induced heart failure was significantly changed. These differentially expressed miRNAs might participate in the process of heart failing by regulating their target genes in rat cardiomyocytes.
Key words: Heart failure     Cardiomyocytes     MiRNA expressional profiling     Bioinformatics analysis     Quantitative real-time PCR     Target genes     Gene ontology (GO)     KEGG pathway    

心力衰竭是各种心脏疾病发展的终末阶段,随着人口老龄化,心力衰竭的发病率和病死率逐年上升[1]。心力衰竭病理生理机制研究已发展到分子水平,多种基因参与心力衰竭的发生发展[2]。microRNA (miRNA) 是一类单链小分子非编码RNA,特异度识别靶基因3’非翻译区的互补片段(3’UTR),通过在转录后水平负性调控基因的表达[3],在各类心血管疾病的发生发展中发挥重要作用[4],因此,作为重要的基因表达调控因子,miRNA在心力衰竭过程中的作用受到广泛关注。本研究拟观察阿霉素致大鼠心力衰竭模型心肌细胞miRNA表达谱的变化,并应用生物信息学分析软件预测差异表达miRNA的靶基因及其功能,为进一步明确心力衰竭的发生机制提供参考。

1 材料与方法 1.1 主要试剂及仪器

盐酸阿霉素(Pfizer,意大利),M199培养基 (Hyclone,美国),Trizol (Invitrogen,美国),新生牛血清(杭州四季青,中国),ITS、BDM、BSA(Sigma,美国),Vivid 7.0彩色超声仪(GE,美国),缺氧小室(Stem Cell,加拿大),Chip ID Rat Sanger miRBase V19.0 miRNA微阵列芯片(LC Sciences,美国),GenePix 4000B激光扫描仪(Molecular Device,美国),Mx3000P TM Real Time PCR 扩增仪(Agilent,美国),All-in-One TM miRNA qRT-PCR Detection Kit(广州复能基因,中国)。

1.2 动物分组及模型制备

成年雄性SD大鼠,200~220 g,江苏大学实验动物中心提供 [合格证批号:SCXK (苏) 2009-0002],随机(数字法)分为两组:心衰模型组(HF组)和正常对照组(CON组)。HF组参照文献[5]的方法制备大鼠慢性心力衰竭模型,每周尾静脉注射阿霉素2 mg/kg,连续6周;CON组注射等量生理盐水。于8周末采用Vivid 7.0彩色超声仪测量左心室射血分数(LVEF)和左心室短轴缩短率(LVFS),以评价大鼠心功能。参照文献[6]采用Langendorff逆行灌注法分离大鼠心室肌细胞,杆状率>90%的细胞用于进一步实验研究,细胞在含ITS、BDM、BSA的M199培养基中培养过夜。

1.3 心肌细胞miRNA表达检测 1.3.1 miRNA表达谱芯片检测

分离成功的心肌细胞培养过夜后采用Trizol试剂提取总RNA,紫外分光光度计和非变性凝胶电泳鉴定RNA的质量和浓度,每组留取4只大鼠心肌细胞的RNA样本。鉴定合格的RNA送联川生物技术公司行miRNA表达谱芯片检测,芯片采用大鼠miRNA V Chip 19.0版本(包含722个成熟大鼠miRNA)。miRNA的表达水平为5次生物学重复的平均值,采用Student’ s -test法比较CON组与HF组差异表达的miRNA,筛选标准为:荧光信号强度>500,P<0.01,假阳性率(false discovery rate,FDR)<0.05。

1.3.2 荧光定量RT-PCR验证芯片结果

随机选取两组大鼠(n=4)分离心室肌细胞并提取总RNA,运用Mx3000P TM Real Time PCR 扩增仪行荧光定量RT-PCR检测,对芯片结果进行验证。

1.4 差异miRNA靶基因预测及功能显著性分析

运用miRNA TargetScan、miRanda分析软件对芯片 检测差异性表达的miRNA进行靶基因的预测,选择两种软件交集的靶基因。基于GO(Gene ontology)和KEGG(Kyoto Encyclopedia of Gene and Genomes)数据库,运用DAVID软件进行GO和Pathway显著性分析。计算P值,并根据P值排序计算FDR值,数据采用Fisher 精确检验和χ2检验进行统计,以P<0.01且FDR<0.05 认为差异具有统计学意义[7]

1.5 统计学方法

采用SPSS 10.0统计软件进行分析,正态分布的计量资料以均数±标准差(x±s)表示,组间比较采用单因素方差分析,两两比较采用配对设计资料的t检验分析,以P﹤0.05认为差异具有统计学意义。生物信息学数据分析采用 Fisher 精确检验和χ2检验进行统计。

2 结果 2.1 差异表达的miRNA及qRT-PCR验证

芯片结果显示,共检测出37个差异表达的miRNA,其中22个miRNA上调,15个miRNA下调(P<0.01且FDR<0.05)。上调的miRNA中,荧光信号值>500的有:miR-6216、let-7e-5p、miR-352、miR-29b-3p、miR-30d-5p;下调的miRNA中,荧光信号值>500的有: miR-133b-5p、miR-6215、miR-181a-5p、miR-378a-3p、miR-378b、miR-22-3p、rno-miR-24-3p,见表 1

表 1 CON组与HF组比较差异表达的miRNA Table 1 The differentially expressed miRNA in HF group as compared with CON group
(x±s, n=4)
上调miRNAtPFold change(HF/CON)下调miRNAtPFold change(HF/CON)
miR-62169.96<0.015.31miR-133b-5p19.41<0.010.02
let-7e-5p6.800.00053.39miR-621513.56<0.010.67
miR-35211.800.00131.62miR-181a-5p9.070.00010.61
miR-29b-3p5.4330.00161.44miR-378a-3p7.680.00030.69
miR-30d-5p3.9380.00761.15miR-378b6.840.00050.71
miR-22-3p12.56<0.010.71
rno-miR-24-3p12.42<0.010.78
注:miR: miRNA,Fold change: 差异倍数

对芯片结果中变化最为显著的miRNA(荧光信号值>500,P<0.01,Fold change>2)行qRT-PCR验证,结果表明HF组miR-133b-5p [(0.053 6±0.006 8) vs. (0.004 2±0.000 3),t=14.56,P<0.01]表达显著下调,miR-6216 [(0.000 2±0.000 1) vs. (0.001 3±0.000 2),t=9.32,P<0.01]和let-7e-5p [(0.062 5±0.008 6) vs. (0.161 4±0.011 3),t=13.92,P<0.01]表达显著上调,变化趋势与芯片结果一致。

2.2 差异表达miRNA 靶基因显著富集的 GO和Pathway

靶基因 GO 显著性分析显示,HF组差异表达miRNA的靶基因主要富集于31个GO。其中12个GO被上调miRNA的靶基因调控,富集度最高的GO主要涉及泛素-蛋白酶体系统(GO:0006511、GO:0008234、GO:0070936);19个GO被下调miRNA的靶基因调控,主要涉及G蛋白相关激酶激活(GO:0007189)、钠离子转运(GO:0006814)及MAPK激酶激活(GO:0000186)等,P<0.01,见图 1

图表左侧部分为下调miRNA富集的GO;右侧部分为上调miRNA富集的GO;X轴代表富集度,-lgPP值的负对数;Y轴代表GO terms 图 1 差异表达miRNA靶基因的GO分析 Fig 1 GO analysis on targets of differentially expressed miRNAs

Pathway 显著性分析显示,靶基因显著富集于12条信号通路,主要包括丝裂原活化蛋白激酶通路(MAPK signaling pathway)、Toll样受体信号通路(Toll-like receptor signaling pathway)、细胞代谢(glycine serine and threonine metabolism)等,见图 2

图表左侧部分为下调miRNA富集的Pathway;右侧部分为上调miRNA富集的Pathway;X轴代表富集度,-lgPP值的负对数;Y轴代表Pathway terms 图 2 差异表达miRNA靶基因的Pathway分析 Fig 2 KEGG Pathway analysis on targets of differentially expressed miRNAs
3 讨论

心力衰竭是一种心功能进行性下降和循环衰竭的临床综合征,高血压、冠心病、风湿性心脏病等病理信号使心肌细胞发生肥厚、纤维化、凋亡,同时标志性基因表达亦发生改变[8],导致心肌收缩力下降、心脏扩大,最终出现心力衰竭。研究表明阿霉素使心肌细胞膜脂质过氧化、破坏线粒体结构、诱导细胞凋亡,最终导致心力衰竭[9],阿霉素尾静脉注射制作大鼠心力衰竭模型操作方便,贴近临床心力衰竭病理生理[10]。本实验参考文献[5]方法制备大鼠心力衰竭模型,结果表明,与CON组比较,HF组大鼠心功能、心质量/体质量、心质量/肺质量指数均显著减低,与文献报道基本一致,提示模型制备成功。参照文献[6]方法分离大鼠心室肌细胞,心衰细胞获得率约为正常心肌细胞的60%。

miRNA是一类小分子单链非编码RNA,通过互补结合靶基因3’UTR片段,抑制其翻译,在转录后水平调控靶基因的表达[3]。研究显示miRNA控制人类近30%的基因表达,调控各种心血管疾病过程,如心肌缺血再灌注损伤、凋亡、纤维化[4]。miRNA芯片分析技术是一种新型、高通量、高效率的miRNA表达谱检测方法,具有较高的特异度和敏感度,本实验所用V Chip19.0版本大鼠miRNA表达谱芯片共包含722个成熟大鼠miRNA,细胞及RNA提取、芯片杂交技术及生物学重复等过程均严格质控,以确保芯片检测结果的稳定性和重复性。芯片筛选出心衰细胞中差异表达的miRNA 37个,经荧光定量RT-PCR验证显示,HF组与CON组比较,miR-133b-5p表达显著下调,miR-6216和let-7e-5p表达显著上调,其变化趋势与芯片结果一致,提示芯片结果可信。研究表明,miR-133b在人扩张性及缺血性心衰心肌组织中表达下调,过表达miR-133b抑制心肌细胞肥厚相关基因的表达,提示miR-133b有调控肥厚性心肌病的作用[11]。心肌细胞凋亡是心力衰竭发生机制之一[8],研究人员发现miR-133b在胚胎心肌组织中表达下调,通过靶向抑制caspase cleaved-CK18发挥抗凋亡作用[12]。本实验结果显示miR-133b-5p在心衰细胞中显著下调,提示其在心衰发展过程中可能发挥重要作用,但其靶基因及功能尚需进一步深入研究。HF组表达上调的miRNA中,let-7e在梗死后心肌组织中表达下调,过表达let-7e可靶向抑制β1-肾上腺素受体表达发挥抗梗死后心律失常的作用[13]。miR-6216为本研究中发现的新的miRNA,其功能目前尚未见文献报道。

为进一步探索差异表达miRNA的功能,本研究利用Targetscan、miRanda软件预测差异表达miRNA 可能调控的靶基因,并对这些靶基因运用DAVID软件行生物信息学分析。我们同时选择两种软件预测,选取交集的靶基因,以避免单一软件预测的假阳性,提高了分析的准确度。结果显示,这些靶基因功能及信号通路主要涉及泛素蛋白酶体系统、MAPK信号通路、Toll样受体通路。泛素蛋白酶体系统是蛋白质降解的重要途径之一,通过调控凋亡抑制蛋白(IAPs)[14]、核因子κB(NF-κB)[15]介导细胞凋亡及炎症反应。阿霉素通过诱导心肌细胞凋亡致心衰,研究显示热休克蛋白HSP10和HSP60可通过抑制BCL-xl的泛素化降解、增加BCL-xl表达,从而抵抗阿霉素诱导凋亡的作用[16]。P38MAPK活化可诱导心肌细胞凋亡[17]及心肌肥厚[18],在心力衰竭进展中发挥重要作用。Toll样受体信号通路(Toll-like receptor sigaling,TLRS)是哺乳动物固有免疫的重要组成部分,最近研究显示,TLRs活化介导的内在免疫反应激活在梗死后心肌重塑中发挥重要作用。

综上所述,本研究利用miRNA芯片技术发现miRNA在阿霉素性心力衰竭细胞中表达谱发生显著改变,其中miR-133b-5p、miR-6216、let-7e-5p值得关注。通过生物信息学分析预测分析miRNA调控的靶基因及其功能,为进一步探索心力衰竭发生机制提供新的切入点,但对差异miRNA靶基因及其功能的验证尚需进一步探索。

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