微小核糖核酸对血管平滑肌细胞表型转换影响的研究进展

吴 昊 周 甜 邓巧莉综述 胡家才审校



微小核糖核酸对血管平滑肌细胞表型转换影响的研究进展

吴 昊 周 甜 邓巧莉综述 胡家才*审校

血管平滑肌细胞(VSMC)作为血管中膜的主要构成细胞，其功能作用与其表型变化密切相关。合成表型具有增生及迁移能力，是各种心血管及周围血管病发生发展的主要病理基础，而收缩表型对维持血管弹性和收缩功能至关重要。较多微小核糖核酸(microRNAS, miRNAS)可通过相关途径和方式调控VSMC表型转换，从而在机体病理生理变化中发挥重要作用。本文综述涉及上述调节的miRNA研究的有关进展。

微小核糖核酸；血管平滑肌细胞；表型

血管平滑肌细胞(Vascular Smooth Muscle Cell, VSMC)是动脉中膜的组成部分之一，其收缩/舒张可调节血管张力，控制血压。某些病理刺激，如血管炎性损伤、血流切应力异常变化、内皮生长因子增加等能引起VSMC表型转换，使其由具有维持血管弹性、收缩功能的收缩表型(分化表型)向具有强大增殖及迁移能力的合成表型(未分化表型)变化[1]。VSMC表型转化是机体生理功能维护和较多动脉病变如血管成型术后、肺动脉高压及动脉粥样硬化等过程的关键步骤。已有研究[1-10]表明多种微小核糖核酸(MicroRNAs，miRNAs)具有调控VSMC表型转换作用，该作用通过抑制靶向物的信使核糖核酸(Messenger RNA，mRNA)和/或蛋白转录，精确调节心血管系统的相关通路来实现。研究miRNA对VSMC表型转化对认识血管疾病的发生机制具有重要意义。

1 miRNAs的产生和作用

miRNAs是一类19-25个碱基的内源性非编码RNA单链，成熟miRNA的产生，首先是在细胞核中由DNA转录生成较长的初级miRNA(Pri-miRNA)；Pri-miRNA在核内经Drosha酶剪切为70-100个核苷酸且具有发卡样结构的miRNA前体(Pre-miRNA)[11]；Pre-miRNA从胞核转运到细胞质中，再被Dicer酶剪切成19-25个核苷酸长度的miRNA双链；最后双链miRNA被组装进RNA

诱导的沉默复合体(RNA-induced Silencing Complex,RISC)，通过碱基互补配对方式识别靶mRNA，并根据互补程度的不同降解靶mRNA或者阻遏靶mRNA的翻译[12]。近10余年来的研究发现，miRNAs在RNA介导的转录后基因调控中起重要作用。 其通过与靶mRNA的 3’非转录端(Untranslated Region，UTR )的完全或不完全结合来降解mRNA或抑制其转录，负向调控基因表达。目前证实的人类miRNA有1 000余种，各种miRNA功能和作用不同，人类约60%的蛋白受miRNA调控[13, 14]。 对于VSMC，部分miRNA主要参与调节其合成和收缩。

2 VSMC的表型特点及其调节

VSMC可依据其功能及形态分为分化型和未分化型两种表型。分化型是其成熟表型，分化程度较高，细胞多呈纺锤形，直径一般2-5μm，长度通常在8-800μm。分化型VSMC胞浆内的高尔基复合体和线粒体等具有合成功能的细胞器较少，但含有大量肌丝，表达如平滑肌肌动蛋白(α-Smooth Muscle Actin，α-SMA)、平滑肌肌球蛋白重链(Smooth Muscle Myosin Heavy Chain，SM-MHC)和钙调节蛋白(Calponin)等，主要维持血管舒张与收缩能力，而增殖和迁移能力较差。未分化型VSMC分化程度较低，甚至未分化，多呈纤维母细胞样，短小，胞浆内肌丝、致密体和致密板极少，但含有大量高尔基复合体、线粒体和粗面内质网，因而具有良好的合成和分泌功能；主要合成和分泌胶原蛋白和细胞基质金属蛋白酶等细胞外基质，参与血管壁的形成、修复、增殖和迁移[15]。

血小板源性生长因子(Platelet Derived Growth Factor, PDGF)及转化生长因子-β(Transforming Growth Factor-β，TGF-β )均参与VSMC表型的转化，其中PDGF被证实可以刺激VSMC有丝分裂、迁移及其表型从分化型向未分化型转换[8, 16]。而TGF-β能通过刺激VSMC的某些特定基因组如α-SMA (也就是ACTA2)和转凝蛋白(Smooth Muscle α，SM-22α)使得VSMC分化成熟[3, 17-19]。研究[20]表明敲除心肌蛋白或/及心肌蛋白相关转录因子(Myocardin Related Transcription Factors，MRTFs)的小鼠，因α-SMA和SM22α基因表达能力低下无法形成正常血管而不能存活，提示两者与VSMC向分化表型转换有关；体外实验[21]也显示，血清反应因子(Serum Response Factor，SRF)联合心肌蛋白及MRTFs可以促进胚胎干细胞向成熟VSMC分化。

3 miRNAs与VSMC表型

Dicer基因与miRNAs的产生密切相关，敲除Dicer基因小鼠，会出现胚胎发育停滞或无法形成正常新生血管[22]；同时会表现出严重低血压、血管收缩功能减退及Calponin、a-SMA等与收缩相关蛋白的表达缺失[23]。表明miRNA对于VSMC既与合成表型有关，也与收缩表型关联，从而影响VSMC的发生、成熟、分化及收缩功能。

3.1 miRNAs可使VSMC向未分化表型转换

Chan等[18]报道受PDGF刺激小鼠，miR-24表达上调，可促使VSMC向未分化表型转换，可能途径为miR-24在PDGF诱导下靶向作用于内质网应激因子(Tribble)TRB3，而抑制TRB3会使得将TGF-β配体信号从细胞核外传导入核内激活下游基因转录的Smad蛋白(Sma and Dgainst Decapentaplegic Protein)表达下调，进而影响骨形态蛋白(Bone Morphogenetic Protein，BMP)和TGF-β信号通路，促进VSMC增殖并向未分化型转换。表明miR-24是PDGF调节VSMC表型的调控点，上调miR-24会使得VSMC向未分化型转换。

Wang等[24]在体外培养的VSMC中发现了miR-31的表达，而且在增殖性VSMCs和具有新生内膜的血管中miR-31表达更多，故认为miR-31可能使VSMC向未分化型转换。还有研究显示，在大鼠颈动脉球囊损伤时miR-31靶向作用于其下游产物大型肿瘤抑制基因2(Large Tumor Suppressor Homolog 2，LATS2)，而LATS2可抑制增殖细胞核抗原(Proliferating Cell Nuclear Antigen, PCNA)介导的VSMC增殖[25]。另有实验证实，敲除miR-31可抑制PDGF介导的VSMC增殖[26]。且这两种作用均与丝裂原激活蛋白激酶/胞外信号调节激酶(MAPK/ERK)抑制剂有关，因为MAPK/ERK是胞外细胞生长刺激和增殖信号的重要反应路径，所以MAPK/ERK/miR-31/LATS2/PCNA可能是VSMC向未分化型转化的新信号通路。

miR-146a靶向作用于Kruppel样因子4(Kruppel-like Factor 4,KLF4)的30非转录区，促进体外培养的细胞增殖[27]；转染miR-146a反转录基因的球囊损伤大鼠中由KLF4介导的α-SMA和SM22α表达上调， 导致颈动脉内膜及VSMC增生

能力明显减弱[28]，同时发现KLF4和KLF5竞争性结合并调控miR-146a启动子，但其作用相反，KLF4过表达会抑制miR-146a的转录水平[29]。表明miR-146a在调控VSMC向未分化表型转换的同时也与KLF4形成负反馈环，调节这种转换。 Zhang等[30]研究发现胰岛素可促进VSMC增生，并且提高miR-208的表达，过表达miR-208能增加VSMC的基础增生以及胰岛素诱导的VSMC增生。尽管miR-208抑制剂对于VSMC的基础增生没有影响，但可减弱胰岛素诱导的VSMC增生。进一步研究[2, 3]表明，miR-208还可靶向作用于细胞周期蛋白依赖性激酶相互作用蛋白/激酶抑制蛋白(Cyclin-Dependent Kinase Inhibitor, CDKN)家族的p21，加速细胞从G0/G1期向S期的转变速度。说明胰岛素及过表达miR-208都会增加VSMC增殖，同时均能抑制p21的作用，而使VSMC向未分化表型变换；但是在有miR-208抑制剂存在的情况下，胰岛素不能发挥对p21的抑制作用。

Tallquist等[8]研究发现动脉损伤可能激活PDGF信号通路，使新生内膜增生，同时抑制平滑肌特异性基因组如ACTA2和SM22的表达，促进VSMC由分化表型向未分化表型转换。miR-221作为PDGF信号通路的表型调节器[13]，在已被PDGF刺激的原态VSMC中接受指令下调其靶向物酪氨酸激酶受体蛋白的一种c-Kit和CDKN家族的p27Kip1的表达水平。miR-211靶向下调p27Kip1是PDGF诱导VSMC增生的关键，同时降低c-Kit会抑制VSMC表达特异性收缩基因，从而有利于VSMC向未分化表型转换。

3.2 miRNAs可使VSMC向分化表型转换

部分miRNAs可促进VSMC进一步成熟、稳定，转换为收缩表型(分化表型)。

Xie等[31]研究表明，在胚胎干细胞向VSMC分化过程中miR-1表达水平稳定升高，而将miR-1拮抗基因转录入VSMC后VSMC特异性标志物以及成熟VSMC都会随之减少，说明miR-1在胚胎干细胞源性VSMC分化过程中必不可少。Chen等[32]发现梭形VSMC(分化表型)miR-1的表达水平显著高于上皮样VSMC(未分化表型)，并且抑制VSMC增殖，但此作用可以被miR-1抑制剂拮抗。

Torella等[33]报道，miR-133在体内及体外培养的VSMC中皆有丰富表达。当血管受损，VSMC准备增殖时，miR-133表达水平明显降低，应用转染过表达miR-133可降低VSMC在体内及体外的增殖及迁移；进一步研究发现miR-133抑制增殖作用主要通过特异性抑制转录因子Sp-1的表达，从而抑制VSMC增殖的基因表达。另外，miR-133可能下调受PDGF刺激后以及颈动脉球囊损伤后大鼠VSMC增殖。

Kim等[5]的实验，通过激活骨形态发生蛋白4(Bone Morphogenetic Protein4，BMP4)信号通路使R-Smad 蛋白与miR-21的初级转录物pri-miR-21及Drosha结合，促进pri-miR-21向pre-miR-21转化，进而增加miR-21的表达，而miR-21可下调PDGF介导VSMC迁移的重要调节因子细胞质分裂付出蛋白(Dedicator of cytokinesis, DOCK4、5、7)，上调收缩基因如a-SMA的表达，继而促进VSMC收缩并抑制Rac1蛋白活性及迁移能力，使VSMC向分化表型转换。 但Horita等[21]发现在缺乏血清反应因子(Serum Response Factor，SRF)的体外培养VSMC中miR-21的表达增加，细胞的增殖能力及炎性介质的表达也随之增加。还有研究[34]表明miR-21的靶向物磷酸酶张力蛋白同源基因(Phosphatase and Tensin Homolog，PTEN)失活导致VSMC增殖及凋亡减少，而miR-21的主要下游靶向物就是PTEN蛋白，因此在此研究中miR-21的过表达促进了VSMC退分化并增殖。结合以上两个实验，认为miR-21保持于某一水平可促进VSMC分化成熟，并抑制其增殖及迁移，而当某些刺激使其过表达后，则会使VSMC退分化，增加其增殖及迁移能力。

Leeper等[35]观察两种腹主动脉瘤形成小鼠时发现，当miR-26a表达下降时，VSMC会更多的由未分化型向分化型转换，即抑制miR-26a会加速VSMC分化，抑制VSMC增殖及迁移；而过表达miR-26a则会减弱这种分化及抑制功能。这是由于miR-26a能靶向作用于信号传导分子-1(SMAD-1)与信号传导分子-4(SMAD-4)，当miR-26a被抑制时，SMAD-1和 SMAD-4基因表达增加，同时改变TGF-β信号，使VSMC向分化型转换。但miR-26a过表达则会抑制SMAD-1，影响VSMC分化。

Grundmann等[36]发现，miR-100可通过抑制雷帕霉素靶蛋白(Mammalian Target of Rapamycin,mTOR)信号通路调控细胞增殖、血管形成、内皮细胞生长活性及VSMC迁移，过表达miR-100能抑制mTOR表达，抑制细胞增生；但拮抗miR-100或过表达mTOR则可以逆转被miR-100抑制的细胞增殖能力。表明过表达miR-100具有抗血管生成及抗动脉粥样硬化作用。

miR-365在多种细胞的增殖、凋亡及分化调控中也具有重要作用。Zhang等[37]通过化学合成方法使原代大鼠主动脉VSMC的miR-365过表达，经过48h培养后，过表达miR-365的VSMC较正常VSMC的细胞数减少了40%，而72h后这一差别达到了60%；同时发现过表达miR-365的VSMC中溴-脱氧尿嘧啶掺入法表现为阳性的细胞减少了58%。说明miR-365可明显抑制VSMC增殖。后续研究表明，这种作用由miR-365靶向作用于细胞周期蛋白D1(Cyclin D1)，使其不能在mRNA及蛋白水平表达，进而将抑制VSMC增殖所致。

4 小结与展望

miRNAs作为广泛存在并具有重要调控作用的内源性微小RNA，通过抑制表达或降解其靶向作用物的mRNA或蛋白质转录而发挥作用。近年来研究表明miRNAs在VSMC表型转化和各类增殖性疾病的发展中扮演着重要角色，miR-24、miR-31、miR-221、miR-146a、miR-208可通过促进VSMC增殖来诱导VSMC向未分化表型转换；miR-1、miR-21、miR-26a、miR-100、miR-133、miR-365则可通过抑制VSMC增殖和迁移，上调收缩基因以及加速VSMC分化来诱导VSMC向分化表型转换。说明体内的miRNAs可能成为不同细胞群之间的分泌物介质及各种生长因子的信号通路的调控点。因此，更进一步探讨并验证miRNAs的直接靶标基因、miRNAs间的相互作用及信号通路，有助于从新的视角认识动脉粥样硬化、肺动脉高压、血管成形术后再狭窄等因VSMC异常增殖导致的血管疾病，为其发病机制的研究找到新的切入点，也为研发新的治疗药物开拓新的思路。

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本文第一作者简介：

吴 昊(1987-)，男，汉族，硕士研究生，主要研究中西医结合周围血管病

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The Roles of Micro-RNAs in the Modulation of the Vascular Smooth Muscle Cell Phenotype

WU Hao, ZHOU Tian, DENG Qiao-li ,HU Jia-cai*

Departent of Traditional Chinese Medicine, Renmin Hospital of Wuhan University, Wuhan 430060，China；*

The functions of vascular smooth muscle cells(VSMC), as the main constituent of media in artery, are closely related to the modulation of the VSMC. The proliferation and migration function from the dedifferentiated VSMC phenotype is the pathological basis of cardiovascular and peripheral vascular disease. The differentiated VSMC phenotype is essential to maintain vessel elasticity and contractile function. Many miRNAs play as important regulators in pathological and physiological changes through pathways which are related to the modulation of the VSMC. This article will summarize recent advances of miRNA relating to phenotypic modulation of VSMC.

MicroRNAS；Vascular smooth muscle cell；Phenotype

武汉大学人民医院中医科，武汉 430060；*

，E-mail:hujiacai@sohu.com

本文2017-02-22收到，2017-03-24修回

R329.2

A

1005-1740(2017)02-0076-05