血管外膜成纤维细胞microRNA-21在血管重构中的作用及其机制的研究
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摘要
研究背景
血管重构是多种心血管疾病(如冠心病、血管移植后狭窄、动脉粥样硬化和高血压等)的最终病理结局。既往研究者对于血管重构的研究重点主要放在是血管的内皮细胞和中膜的平滑肌细胞(VSMC)上,然而外膜细胞在血管重构过程中的作用没有受到重视。近年来,大量研究表明血管外膜在调控血管生理和病理方面同样发挥着重要作用。研究显示,在损伤和细胞因子等病理刺激下,作为血管外膜最主要的细胞成分,外膜成纤维细胞(adventitial fibroblast, AF)可通过激活、表型分化和腔内迁移参与并促进血管重构。被激活的AF,增殖活性增强,并发生表型变化而分化为肌成纤维细胞(myofibroblast, MF),发生表型变化的特征是获得α-SMA(α-平滑肌肌动蛋白)的表达。相对于AF, MF具有更强的增殖活性和分泌细胞外基质(extracellular matrix, ECM)的能力。MF的出现是组织器官对损伤进行修复过程中的一个特征。血管外膜细胞的增殖和凋亡之间的平衡对于维持血管正常生理功能起着至关重要的作用。当AF和MF的正常的细胞周期出现紊乱时,就会导致凋亡减少和过多的ECM沉积,最终导致病理性血管重构。在这一病理过程中,会伴随着一部分MF向血管腔内迁移参与并促进血管新生内膜形成。血管新生内膜形成是病理性血管重构的一个重要病理特征。因此,血管外膜细胞(包括AF和MF)增殖和凋亡之间的平衡失控而导致的细胞过度增殖和凋亡抑制是血管重构过程中的重要细胞事件。血管的AF和MF已成为抗血管重构治疗的两个重要靶细胞。
microRNA (miRNA)代表了一种新的基因表达调控机制。研究表明,miRNA参与了诸多重要的细胞生命活动过程,包括细胞的增殖、凋亡和分化等。而异常的细胞增殖和凋亡在肿瘤发生和发展过程中的扮演着重要角色。正因为如此,目前关于miRNA的众多研究集中在肿瘤方面。
在近些年来已发现的诸多miRNA分子中,microRNA-21(miR-21)受到研究者更多的关注。研究发现,miR-21在许多肿瘤细胞中过表达,并且是多种肿瘤组织中表达升高最明显的miRNA分子之一,被称作为"oncomir",意思是具有致癌特性的miRNA分子,具有促增殖和抗凋亡作用。许多研究者认为,对miR-21或其靶基因的表达进行干预或许会成为将来抗癌治疗的有效措施。
相对于肿瘤,miRNA在心血管方面的研究起步较晚。近年来,miR-21在心血管领域的功能也逐渐受到关注。研究发现,仅次于肿瘤,在心血管疾病中miR-21的表达也明显失调。在许多心血管疾病,如血管球囊损伤、心脏肥厚和缺血性心脏病等,miR-21的表达异常。有研究显示,大鼠颈动脉球囊损伤的血管壁中miR-21表达明显升高；miR-21在VSMC中表达,并且能调控细胞的增殖和凋亡。有报道称,miR-21在血管内皮中也有表达,miR-21可以通过调控内皮细胞的凋亡而影响其生理功能。但是目前国内外关于miRNA在血管外膜领域的研究报道很少,miR-21在血管AF和MF中是否表达以及是否对增殖和凋亡发挥调控功能尚不清楚。
研究显示,血管增殖性疾病(如冠心病、动脉粥样硬化等)和肿瘤在多种细胞事件和分子通路方面有着许多相似的特征。增殖和凋亡同是血管重构过程和肿瘤的两个重要的细胞生命活动,既然miRNA在肿瘤中发挥着重要作用,因此我们完全有理由相信,miRNA在血管重构的发生和发展过程中同样会发挥重要作用。
近些年一些研究表明,以血管外膜细胞为靶点进行干预可以改善血管重构。有研究显示,17β-雌二醇可以通过抑制外膜细胞的增殖而减少球囊损伤后血管新生内膜的形成。也有研究报道,通过在血管外膜转染smad7腺病毒使外膜细胞过表达Smad7,可以抑制TGF-β1通路,从而明显减弱球囊损伤后成纤维细胞的促新生内膜形成和血管重构的作用。因此,以血管外膜细胞为靶点的血管外膜基因转染代表了一种新的治疗心血管疾病的方法。
综上所述,我们提出如下假说：miR-21在血管AF和MF中内源性表达,并且miR-21在调控AF和MF的增殖和凋亡方面发挥着重要作用；通过干预miR-21的表达能有效调控血管AF和MF的增殖和凋亡,并能进一步改善由球囊损伤导致的血管重构。
目的
1.建立体外肌成纤维细胞模型,检测并比较miR-21在成纤维细胞和肌成纤维细胞的表达水平。
2.通过调节成纤维细胞和肌成纤维细胞中miR-21的表达水平,研究miR-21对增殖和凋亡的影响。
3.检测能否通过干预miR-21的表达影响大鼠髂动脉球囊损伤所致的血管重构。
方法
1.成纤维细胞体外培养
取4-6周(120-180克)雄性Wistar大鼠胸主动脉外膜,采用组织贴壁法原代培养外膜成纤维细胞。实验用第3-6代细胞。
2.细胞试验分组
(1)为确定TGF-β1诱导AF分化为MF的合适剂量和时间,行RT-PCR反应,用TGF-(31(10ng/ml)刺激不同的时间:0、6、12、24、36、48小时,分为6组；不同浓度的TGF-β1刺激24小时：0、1、5、10、20、30ng/ml,分为6组。行Western blot检测,按照TGF-β1(10ng/ml)刺激不同的时间:0、12、24、36小时,分为4组。
(2)为检测细胞内miR-21的表达水平能否被调节,分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂(即miR-21inhibitor)组、前体阴性对照组和pre-miR-21(即miR-21前体)组。
(3)为检测细胞的增殖和凋亡,根据干预措施不同分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂组、前体阴性对照组和pre-miR-21组。
3.建立肌成纤维细胞模型
肌成纤维细胞由TGF-β1诱导成纤维细胞而成,并经RT-PCR、Western blot和细胞免疫荧光实验方法鉴定。
4.细胞免疫荧光实验
将细胞玻片上生长的用(或未用)TGF-β1刺激的成纤维细胞,经固定、0.5%Triton-X100处理、BSA封闭后,α-SMA和vimentin一抗过夜,用含有FITC和TRITC的二抗液孵育玻片,然后用DAPI染核,最后于荧光显微镜下观察α-SMA和vimentin的表达情况.
5. miRNA转染
体外培养的细胞用转染试剂Lipofectamine2000转染pre-miR-21或miR-21inhibitor分别造成miR-21的过表达或表达抑制,同时分别设阴性对照组。48小时后检测细胞样本。实验中用含FAM荧光标签的寡核苷酸转染细胞以确定转染效率。最终寡核苷酸的终浓度为50nM。
6.实时定量RT-PCR使用miRNA提取试剂盒提取用于检测miR-21表达的细胞总RNA,用含有特异茎环结构的引物进行逆转录,然后TaqMan探针法在ABI7500PCR仪上检测miR-21的表达。用于检测其他指标(如α-SMA)的细胞总RNA的提取使用Trizol法,逆转录成cDNA后,采用荧光实时定量的方法在Bio-Rad PCR仪上检测。miR-21的相对表达用U6做内参,α-SMA mRNA等的相对表达以β-actin为内参,并用△ACT的方法计算。
7.蛋白印迹分析
利用组织裂解液提取细胞总蛋白。采用BCA法进行蛋白浓度测定。等量的蛋白样品在SDS-PAGE凝胶上电泳,然后转膜至PVDF膜上,膜封闭后,一抗过夜,用洗膜液清洗,用HRP结合的二抗孵育。ECL法发光,以β-actin作为内参,计算目的蛋白的相对表达量。
8.大鼠髂动脉球囊损伤模型的建立
按照Gabeler报道的方法建立。Wistar大鼠(320-350g)戊巴比妥麻醉,2F的导管引入到右髂总动脉,球囊在3-4atm压力下来回拉动3次造成内膜损伤。左侧未损伤血管作为对照。
9.血管外膜局部孵育寡核苷酸和动物实验分组
手术后在损伤血管的周围均匀孵育20%(w/v)的F-127缓释凝胶200μ1,凝胶中含有0.24mg miR-21antagomir(用于体内实验的miR-21的特异抑制剂)或等体积的PBS。实验动物分为3组：miR-21antagomir组(凝胶中含有miR-21antagomir,n=15)、损伤组(凝胶中仅含有PBS,n=20)和未损伤组(左侧未损伤血管,n=20)。损伤组于手术后1天、3天和21天处死,miR-21antagomir组于手术后3天和21天处死,分别用于进行增殖检测(术后3天,n=5)、凋亡检测(术后1天和3天,n=5)和HE染色(术后21天,n=5)。
10.细胞增殖检测
细胞转染pre-miR-21或miR-21inhibitor后48小时,采用EdU的方法检测细胞的增殖率。操作按照EdU细胞增殖检测试剂盒的说明书进行,增殖的细胞EdU染核阳性,用Hoechst33342染细胞核,荧光镜下观察高倍视野下细胞EdU阳性数占细胞总数的比例,计算细胞增殖率。
动物实验中,采用BrdU方法检测细胞增殖。分别在动物处死前12和24小时各注射BrdU一次。按照说明书操作,使用特异的BrdU抗体检测组织细胞的增殖率。细胞增殖率按高倍视野中BrdU阳性数占总细胞数的百分比计算。
11.细胞凋亡检测
细胞转染后48小时分别采用Annexin-V/7-AAD双染的方法和Caspase-3活性检测的方法检测细胞的凋亡情况。Annexin-V/7-AAD双染利用流式细胞仪(FACS)进行检测。Caspase-3活性检测采用比色法检测Caspase-3相对活性。
在动物实验中,按照试剂盒说明,采用TUNEL方法检测细胞凋亡。细胞的凋亡率按高倍视野中TUNEL阳性数占总细胞数的百分比计算。
12.免疫组织化学
为检测血管外膜成纤维细胞的分化,取石蜡切片,按照SABC免疫组织试剂盒的操作说明,行免疫组织化学检测α-SMA的表达。
13.形态测定分析
通过测定血管组织形态学的改变来分析血管重构的情况。用HE染色法,对新生内膜／中膜面积(Neointimal/Medial area, N/M)和管腔大小(Lumen Size)进行形态学分析,用IPP软件进行测量。
14.统计学分析
所有数据以均数士标准误计算,来源于3次以上独立实验。两组间的差异用t检验,多组间的差异用单因素方差分析。数据用GraphPad Prism5.0软件分析,P<0.05代表有显著性差异。
结果
1.体外用TGF-β1诱导成纤维细胞分化为肌成纤维细胞
RT-PCR和Western blot结果显示,在蛋白和mRNA水平上,TGF-β1都能显著促进α-SMA的表达,并呈现一定的时间和浓度依赖性；TGF-β1(10ng/ml,24小时)能明显促进α-SMA的表达。细胞免疫荧光实验研究显示,经TGF-β1(10ng/ml,24小时)处理后,细胞共表达vimentin和α-SMA,同时胞浆内形成大量肌丝,表明AF分化为MF,体外建立肌成纤维细胞模型成功。
2.miR-21在AF中内源性表达并且在MF中表达升高
实时定量RT-PCR(探针法)检测发现,AF中内源性表达miR-21。相对于AF,MF中miR-21的表达水平明显升高。
3.调节miR-21在AF和MF中的表达
为检测AF和MF中miR-21的表达水平能否被调节,我们采用了表达抑制和过表达的方法。在AF和MF中,转染pre-miR-21后,miR-21的表达水平明显升高；转染miR-21抑制剂(miR-21inhibitor)后,miR-21的表达明显下降,然而转染对照核苷酸后miR-21的表达量没有明显变化,说明miR-21在AF和MF中的表达水平是可以调节的。
4.miR-21对AF和MF增殖的作用
EdU增殖检测分析结果显示,在AF和MF中,抑制miR-21的表达后,两种细胞的EdU掺入率减少；转染pre-miR-21使细胞过表达miR-21后,AF和MF的EdU掺入率减少增加；上述结果表明,miR-21具有促进AF和MF的增殖活性的作用。研究还发现,无论在基础水平还是经pre-miR-21干预之后,MF较AF都显示出更高的增殖活性。
5.miR-21对AF和MF凋亡的作用
Annexin-V/7AAD流式细胞术和Caspase-3活性检测结果显示,AF和MF转染miR-21inhibitor抑制miR-21表达后,annexin Ⅴ阳性细胞数和Caspase-3的活性都明显增加；而过表达miR-21后则出现相反的结果,说明在AF和MF中,miR-21发挥了为抗凋亡的作用。
6.miR-21抑制剂对球囊损伤的血管的外膜成纤维细胞的增殖和凋亡的影响
免疫组化结果显示,在球囊损伤术后3天,血管外膜中可见α-SMA阳性细胞,这一结果提示已有部分AF分化为MF。
术后3天,损伤组的血管外膜细胞增殖活性较未损伤组明显升高；而相对于损伤组,miR-21antagomir组的血管外膜细胞增殖率明显下降。
TUNEL结果显示,未损伤的血管外膜凋亡细胞很少；术后1天,损伤组的外膜可见较多凋亡细胞；术后3天,损伤组外膜中凋亡细胞数明显下降,与未损伤组无明显差别；然而,miR-21antagomir (miR-21抑制剂)组相较于未拉伤组和损伤组(术后3天),凋亡率明显升高。
7.miR-21抑制剂对球囊所致的血管重构的影响
为了检测miR-21抑制剂对球囊损伤所致的血管重构的影响,我们对球囊损伤后21天的血管进行了形态学的分析。相对于未损伤组,损伤组的血管呈现出明显的新生内膜形成和管腔的损失；相对于损伤组,miR-21antagomir组的内膜／中膜面积比(N/M)明显降低,并且管腔损失程度得到改善。
结论
1.miR-21在血管成纤维细胞内源性表达,并且在TGF-β1诱导而成的肌成纤维细胞中miR-21表达明显升高。
2.miR-21是血管调控成纤维细胞和肌成纤维细胞增殖和凋亡的重要分子。
3.miR-21抑制剂可以通过影响AF和MF的增殖和凋亡从而改善球囊损伤所致的血管重构。
研究背景
研究表明,作为血管外膜的最重要的细胞成分,血管外膜成纤维细胞(adventitial fibroblast, AF)是血管损伤后新生内膜形成和病理性血管重构的重要病理基础。当血管受到来自内外的病理损伤或外界刺激时,AF会被“激活”,表现为增殖活性迅速增强,同时发生表型变化而成为肌成纤维细胞(myofibroblast, MF)。MF较AF表现出更强的增殖、迁移和分泌细胞外基质的能力。同时会有一部分MF迁移至内膜并分泌大量ECM从而积极参与新生内膜.的形成。因此,AF和MF是治疗心血管疾病的两种重要的靶细胞。
血管外膜细胞的增殖和凋亡之间的平衡对于维持血管正常生理功能至关重要。在血管增殖性疾病中,AF和MF过度增殖和凋亡缺陷使得这一平衡打破,从而使胶原分泌过度,细胞外基质过多沉积,最终导致不良性血管重构。因此,阐明调控这两种细胞增殖和凋亡的机制对于预防和改善不良血管重构有着重要意义。
MicroRNA (miRNA)是一类具有重要基因调控功能的内源性小RNA分子。近年来研究表明,miRNA广泛参与了增殖、凋亡和分化等机体多种细胞生物学过程的调控。在目前已发现和鉴定的miRNA分子中,microRNA-2(miR-21)备受关注。研究显示,miR-21在多种肿瘤中表达明显上调,参与了细胞增殖、凋亡、分化和迁移等诸多细胞生物学过程,与肿瘤的发生和进展密切相关。我们的研究发现,miR-21在血管外膜细胞中也有表达,并且miR-21可以有效调控AF和MF的增殖和凋亡；在大鼠髂动脉球囊损伤的血管重构模型中,通过在外膜孵育miR-21抑制剂可以抑制新生内膜形成。但是目前miR-21对AF和MF的增殖和凋亡发挥调控作用的具体机制尚不清楚。
miRNA分子发挥其功能主要是通过调控其靶基因的表达而实现的。PDCD4是近年来发现的一个肿瘤抑制基因。研究发现,PDCD4在调控细胞的增殖和凋亡方面发挥着重要作用,与肿瘤的发生发展有密切关系。但是目前尚未有PDCD4在血管外膜AF和MF的研究报道。在多种肿瘤细胞中PDCD4被证实为miR-21的靶基因,可以调控细胞的增殖和凋亡。miRNA与其靶基因在表达和基因调控方面呈现出组织特异性,所以,在AF和MF中,PDCD4是否表达以及是否为miRNA的靶基因,还有待于研究确定。JNK/c-Jun信号通路是与细胞生长密切相关的通路。目前关于PDCD4和JNK通路两者关系的研究较少。有研究报道,PDCD4可以干扰JNK介导的c-Jun的磷酸化,并且可通过抑制c-Jun的激活及随后的AP-1依赖性的转录而抑制肿瘤的转化。但是,miR-21能否调控JNK通路以及PDCD4是否参与此调控过程目前还没有报道。
针对以上问题,我们提出如下假说：在血管AF中,PDCD4是miR-21的靶基因；PDCD4能有效调控血管AF和MF的增殖和凋亡,并且直接参与了miR-21介导的细胞事件；JNK/c-Jun是PDCD4参与miR-21介导的细胞效应的下游信号通路。
目的
1.在AF和MF中,验证PDCD4是否是miR-21的一个直接靶基因。
2.检测PDCD4对AF和MF的增殖和凋亡的作用。
3.检测PDCD4是否直接参与了miR-21对AF和MF的增殖和凋亡的调控作用。
4.检测调控miR-21和PDCD4对JNK活性的影响,并确定JNK/c-Jun信号通路在miR-21介导的AF和MF细胞事件中的作用。
方法
1.成纤维细胞体外培养及建立肌成纤维细胞模型
取4-6周(120-180克)雄性Wistar大鼠胸主动脉外膜,采取组织贴壁法原代培养外膜成纤维细胞。实验用3-6代细胞。肌成纤维细胞由TGF-β1诱导成纤维细胞而成,并经RT-PCR. Western blot和细胞免疫荧光实验方法鉴定。
2.实验分组
(1)为验证miR-21能否直接与PDCD4的3'-UTR的靶位点结合而行双荧光素酶报告基因检测,分为2组：荧光素酶载体组(pMIR-REPORT荧光素酶质粒组)和载体对照组；每组又分3个亚组：空白对照组、前体阴性对照组和pre-miR-21(即miR-21前体)组。
(2)为验证miR-21能否调控PDCD4蛋白的表达行Western blot检测,分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂(即miR-21inhibitor)组、前体阴性对照组和pre-miR-21组。
(3)为检测细胞的增殖和凋亡,分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂组、PDCD4siRNA组和miR-21inhibitor+PDCD4siRNA组。
(4)为检测过表达miR-21后JNK活性的改变,分为3组：空白对照组、前体阴性对照组和pre-miR-21组。
(5)为检测抑制miR-21的表达后JNK活性的改变以及PDCD4是否参与了其中过程,分为4组：空白对照组、抑制剂阴性对照组、miR-21抑制剂组和miR-21inhibitor+PDCD4siRNA组。
3. miRNA and siRNA转染
寡核苷酸的转染是用转染试剂Lipofectamine2000按照试剂说明要求进行操作。体外培养的细胞用转染试剂Lipofectamine2000转染pre-miR-21或miR-21inhibitor分别造成miR-21的过表达或表达抑制,最终寡核苷酸浓度的终浓度为50nM。对于PDCD4siRNA, PDCD4siRNA的终浓度80nM。
4.细胞增殖检测
细胞转染pre-miR-21或：niR-21inhibitor后48小时,细胞的增殖检测采用EdU的方法检测。按照EdU细胞增殖检测试剂盒中的说明书进行操作,增殖的细胞EdU染核阳性,用Hoechst33342染细胞核,荧光镜下观察高倍视野下细胞EdU阳性数占细胞总数的比例,计算细胞增殖率。
5.细胞凋亡检测
细胞转染48小时后,分别采用Caspase-3活性检测的方法进行检测。Caspase-3活性检测是在多功能酶标仪上检测细胞样本的OD值。
6.蛋白印迹分析
利用组织裂解液提取细胞总蛋白。采用BCA法测定蛋白浓度。等量的蛋白样品在SDS-PAGE凝胶上电泳,然后转膜在PVDF膜上,膜封闭后,一抗过夜,洗膜液清洗,用HRP结合的二抗孵育。ECL法发光,以β-actin作为内参,计算各目的蛋白的相对表达量。
7.双荧光素酶报告基因检测
为检测miR-21能否与PDCD4的3'-UTR mRNA特异结合,我们采用了双荧光素酶报告基因检测的方法。将含有PDCD4的3'-UTR mRNA部分序列(含有与miR-21潜在结合的位点的mRNA序列)的荧光素酶载体(简称：荧光素酶载体)或不含有PDCD43'-UTR mRNA部分序列的荧光素酶载体(空白载体对照)和pre-miR-21共转染293T细胞。同时转染表达海参素报告基因的pRL-TK作为内参照。转染24小时后,用双荧光素酶检测试剂盒在酶标仪上分别检测萤火虫和海参素的信号强度。
8.统计学分析
所有数据以均数±标准误计算,来源于3次以上独立实验。两组间的差异用t检验,多组间的差异用单因素方差分析。数据用GraphPad Prism5.0软件分析,P<0.05代表有显著性差异。
结果
1.在AF和MF中,PDCD4是miR-21的一个直接靶基因
(1)AF和MF中PDCD4蛋白表达水平的比较
Western blot检测分析显示,PDCD4在MF中的表达水平较AF中明显下降。而我们前期研究显示,在MF中miR-21的表达水平较AF明显增高。PDCD4和miR-21在两种细胞中的表达呈现负相关,这一结果提示：在AF中,PDCD4可能是miR-21的一个靶基因。
(2)双荧光素酶报告基因系统分析结果
双荧光素酶报告基因检测显示,相对于对照组,pre-miR-21能够明显降低荧光素酶信号的强度,说明miR-21能直接与PDCD4mRNA序列中的3′-UTR的靶位点结合。
(3)miR-21过表达和表达抑制后PDCD4的表达
将miR-21过表达或表达抑制后,行Western blot检测PDCD4蛋白的表达。研究发现,在AF和MF中,转染pre-miR-21后,PDCD4蛋白的表达水平明显下降；转染miR-21inhibitor后,PDCD4蛋白的表达水平明显升高,这一结果说明在AF和MF中,miR-21能直接调控PDCD4蛋白的表达。
以上结果充分说明,在AF和MF中,miR-21可以直接靶作用于PDCD4, PDCD4是miR-21的一个直接靶基因。
2.抑制PDCD4表达对miR-21介导的对AF和MF增殖作用的影响
相对于对照组,PDCD4siRNA组的AF和MF的EdU阳性率明显增多,即增殖率明显增加。在AF和MF中,miR-21抑制剂组增殖率明显降低,然而,相对于miR-21抑制剂组,miR-21抑制剂+PDCD4siRNA组的增殖率明显升高。以上结果说明,抑制PDCD4的表达不仅可以促进AF和MF的增殖,而且可以明显缓解miR-21抑制剂对AF和MF增殖的抑制作用,说明PDCD4不仅可以调控AF和MF的增殖,而且直接参与了miR-21对AF和MF增殖的调控。
3.抑制PDCD4表达对miR-21介导的对AF和MF凋亡作用的影响
用PDCD4siRNA沉默PDCD4的表达后,相对于对照组, AF和MF的Caspase-3活性明显降低。在AF和MF中,miR-21抑制剂组的Caspase-3活性明显增强,然而,相对于miR-21抑制剂组,miR-21抑制剂+PDCD4siRNA组的Caspase-3活性明显降低。以上结果表明,PDCD4siRNA不仅可以降低AF和MF的Caspase-3活性,而且还可以消弱miR-21抑制剂增强AF和MF的Cspase-3活性的作用,说明PDCD4直接参与了miR-21对AF和MF凋亡的调控。
4.抑制PDCD4表达对JNK活性的影响
Western blot结果显示,PDCD4siRNA可以明显增强JNK活性,表现为p-AKT和p-c-Jun水平明显升高。
5.调节miR-21表达对JNK活性的影响
在AF和MF中,转染pre-miR-21后,PDCD4蛋白表达减少,JNK活性增强；然而转染miR-21抑制剂后,PDCD4蛋白表达增多,而JNK活性降低。这
研究结果说明,过表达miR-21可以增强JNK活性,抑制miR-21表达后可以降低JNK活性。
6.抑制PDCD4表达对miR-21调控是JNK活性的作用的影响
在AF和MF中,相对于对照组,miR-21抑制剂组的JNK活性活性明显减弱；然而,相对于miR-21抑制剂组,miR-21抑制剂+PDCD4siRNA组的JNK活性明显增强。研究结果说明,PDCD4不仅能影响JNK活性,而且还可以影响miR-21对JNK活性的调控,同时也说明JNK/c-Jun是PDCD4参与miR-21介导的细胞事件的下游信号通路。
结论
1.在血管成纤维细胞和肌成纤维细胞中,PDCD4是miR-21的一个直接靶基因。
2. PDCD4能调控血管成纤维细胞和肌成纤维细胞的增殖和凋亡,并且直接参与了miR-21介导的对细胞增殖和凋亡的调控作用。
3. JNK/c-Jun是PDCD4参与miR-21介导的细胞事件的重要下游信号通路。
Background
Vascular remodeling is the pathological endpoint of diverse cardiovascular diseases such as coronary heart diseases, post-angioplasty restenosis, atherosclerosis, and hypertension. The previous studies on vascular remodeling focused on endothelial cells (ECs) and medial vascular smooth muscle cells (VSMCs), however,the role of adventitial cells in vascular remodeling did not receive attention. Increasing evidence suggests that the adventitia is a critical regulator of vessel wall function in health and disease. Many Studies show that vascular adventitial fibroblasts (AFs) contribute to vascular remodeling through activation, differentiation and intraluminal migration. Under pathological stimuli such as injury and cytokines, AFs, the principal cell type in the adventitia, are activated and undergo phenotypic changes, including proliferation and differentiation to myofibroblasts (MFs). The characteristic of differentiation is that AFs acquire α-smooth muscle actin (α-SMA) expression. Increased proliferative capacity and increased secretion of the extracellular matrix (ECM) proteins are key hallmarks of MFs. Transient appearance of MFs is a feature of normal wound repair. The balance in adventitial cells (including AFs and MFs) proliferation and apoptosis is decisive for the physiology of the vasculature. When the MF life cycle is not regulated properly, absence of apoptosis leads to overproduction of ECM proteins deposition and pathological vascular remodeling. Moreover, these processes are accompanied by adventitial cells migration toward the lumen, thereby contribute to neointimal formation. Neointima formation is an important feature of pathological vascular remodeling. Thus, uncontrolled cell proliferation and suppression of apoptosis are critical cellular events in vascular remodeling. Vascular AFs and MFs have become2potential target cells for anti-vascular remodeling therapeutics.
As a new layer of gene regulation mechanism, microRNAs (miRNAs) have been implicated in a multitude of important cellular processes, including regulation of cellular proliferation, apoptosis and differentiation. The abnormal proliferation and apoptosis play an important role during tumor occurrence and development process. Therefore, most previous research into the role of miRNAs has focused on tumor.
MiR-21has attracted more attention than any other miRNA. Multiple studies have identified miR-21overexpressed in a wide variety of cancer cell lines and causally linked to cancer-related processes such as proliferation and apoptosis. miR-21was reported to be strongly overexpressed in a wide variety of cancer cell lines. On the basis of these findings, recently, miR-21has been referred to as an "oncomir"(i.e., a miRNA with oncogenic properties) with pro-proliferative and anti-apoptotic functions. Interfere with mir-21expression or with its interaction with their target genes has been suggested as a potential anticancer therapeutic approach.
Related to tumor, the research of miRNA in cardiovascular started later. In recent years, the function of miR-21in cardiovascular also attracts much attention gradually. Next to tumor, miR-21appears to be strongly deregulated in cardiovascular disease. Under some cardiovascular disease conditions such as proliferative vascular disease, vascular walls balloon injury, cardiac hypertrophy and ischemic heart disease, the mir-21expression is deregulated. It was reported that miR-21was strongly overexpressed in vascular walls after balloon angioplasty of the rat carotid artery. MiR-21is expressed in VSMCs and regulates proliferation and apoptosis. MiR-21is also expressed in vascular ECs. It was reported that shear stress forces regulate the expression of miR-21in ECs, which influencing endothelial biology by decreasing apoptosis. However, the research of miRNA in vascular adventitia area is little at present. It is unclear whether miR-21is expressed in AFs and MFs and whether play a role in the proliferation and apoptosis of the2kinds cells.
Numerous studies have demonstrated some characteristics of proliferative vascular disease are similar to those in tumor in cellular events and molecular pathways. Because proliferation and apoptosis are2important cellular events in vascular remodeling and tumor, miRNAs may play important roles in vascular remodeling.
In recent years, many studies suggest that the intervention measures which treat vascular adventitial cells as therapeutic targets can improve vascular remodeling. A study showed that estrogen may attenuate the injury response in balloon injury model by inhibiting proliferation of adventitial cells and their contribution to neointima formation. A study showed that smad7overexpression through gene transfer of smad7in the adventitial surface antagonized the TGF-β1signaling pathway and attenuated vascular remodeling and contribution of AFs to neointima formation after balloon angioplasty. Therefore, perivascular gene transfer which targets vascular adventitial cells represents a novel therapeutic strategy of vascular disease.
Taken together, We put forward the hypothesis:miR-21was endogenously expressed in AFs and MFs; miR-21play an important role in regulating proliferation and apoptosis of AFs and MFs; moudulting miR-21expression could regulation proliferation and apoptosis of vascular AFs and MFs and further improve balloon injury-induced rat iliac artery remodeling.
Objectives
1. To established the cell model of MFs and determine the miR-21expression in AFs and MFs.
2. To investigate the effects of miR-21modulation on proliferation and apoptosis of AFs and MFs.
3. To investigate the effects of miR-21inhibition on neointima formation after iliac artery balloon injury.
Materials
1. Cell culture
AFs were isolated from thoracic aortas of4-to6-week-old male Wistar-Kyoto rats weighing120to180g and cultured by the tissue adherence method. Cells from passage3to6were used.
2. Grouping in vitro experiment
2.1RT-PCR and Western blot were used to inspect the effection of TGF-β1on AF differentiation. In RT-PCR experiment, cell were grouped into6groups depending different time (0、6、12、24、36、48hours) or6groups at different concentrations (0、1、5、10、20、30ng/ml). In Western blot experiment, cell were grouped into4groups according to different time (0、12、24、36hours).
2.2To determine whether the expression of miR-21in AFs and MFs was regulated, we used both gain-and loss-of-function approaches. Cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.
2.3To determine the proliferation and apoptosis, cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.
3. Myofibroblast model
Subconfluent AFs were incubated with different concentrations of TGF-β1in serum-free medium. The MF phenotype in TGF-β1-treated fibroblasts was identified by determining the expression of a-SMA by RT-PCR, Western blot analysis and fluorescent immunocytochemistry.
4. Fluorescent immunocytochemistry
Fibroblasts with or without TGF-β1treatment were grown on glass coverslips, rinsed with PBS, fixed with3%paraformaldehyde, permeabilized with0.5%Triton-X100and blocked with2%BSA. Cells were incubated with the primary antibodies mouse anti-a-SMA and rabbit anti-vimentin. Immunofluorescent staining for a-SMA and vimentin involved incubation with FITC-conjugated anti-mouse IgG and TRITC-conjugated anti-rabbit IgG, respectively. Cellular nuclei were stained with DAPI. Images were examined under confocal microscope.
5. miRNA transfection
The oligonucleotide was transfected into cells by use of Lipofectamine2000reagentas per the manufacturer's instructions. Cells were transfected with pre-miR-21and miR-21inhibitor for miR-21overexpression and knockdown, respectively. The samples were assayed after48h. The transfection efficiency was determined by use of the negative control oligonucleotide with6-FAM at the5'end. After optimization, transfection complexes were added to cells at a final oligonucleotide concentration of50nM.
6. Real-time quantitative RT-PCR
Total RNA was isolated for miR-21and a-SMA from cultured cells48h post-transfection with use of the mirVanaTM miRNA Isolation Kit and Trizol according to the manufacturer's protocol. cDNA synthesis involved carried out in a thermal cycler. PCR reactions for miRNA and mRNA involved the ABI Prism7500Sequence Detection System and iQ5Real-Time PCR Detection System, respectively. Real-time RT-PCR analyses of miRNA expression involved use of TaqMan miRNA assays. RT-PCR analyses of the mRNA expression of a-SMA and (3-actin involved PrimeScript(?) RT-PCR kits. Analysis of relative miRNA and mRNA expression involved the△△CT method, with U6and β-actin used as endogenous controls, respectively.
7. Western blot analysis
Cells were harvested with lysis buffer. Protein concentration was determined using the BCA method. Protein samples were separated on SDS-PAGE gels, transferred to PVDF membrane, and blocked with5%nonfat milk/TBST and then incubated with primary antibodies. Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit IgG. Immunoreactive bands were visualized by ECL. The relative intensities of the bands were analyzed using P-actin as an internal control.
8. Establishment of rat iliac artery balloon injury model
Iliac artery balloon injury was performed according to Gabeler et al. Briefly, Wistar rats (320-350g) were generally anesthetized with sodium pentobarbital. A2F Fogarty catheter was inserted to the right iliac artery. To produce iliac artery injury, the balloon was inflated to3-4atm and withdrawn three times. The left iliac arteries were not injured and served as controls.
9. Adventitial oligo transfer and animal grouping
200βl of20%(w/v) F-127pluronic gel (Sigma) containing0.24mg antagomir against miR-21(miR-21antagomir) or PBS was applied to the adventitial around injured artery segments. Animals were divided into three groups:miR-21antagomir group (treated with miR-21antagomir dissolved in F-127pluronic gel, n=15), vehicle group (treated with PBS dissolved in F-127pluronic gel, n=20), and uninjured group (n=20). Animals of vehicle group were sacrificed at1days,3days, and21days after injury. Animals of miR-21antagomir group were sacrificed at3days and21days after injury. The vasclular sample were used to performe proliferation assay (3days after injury, n=5), apoptosis assay (1and3days after injury, n=5) and HE staining (21days after injury, n=5)
10. Proliferation assay
Cell proliferation in vitro was determined by EdU. After48h of transfection, cell proliferation was quantified by the uptake of EdU into DNA by use of a Cell-LightTM EdU DNA Cell Proliferation Kit. All procedures for EdU incorporation experiments followed the kit's protocol. Cell proliferation rate was calculated as the percentage of EdU-positive nuclei to total nuclei in high-power fields.
To detect AF and MF proliferation in vivo, BrdU dissolved in PBS, was injected12and24hours before euthanasia. BrdU-incorporating nuclei were detected with a specific anti-BrdU antibody according to the manufacturer's instructions. Cell proliferation rate was calculated as the percentage of BrdU-positive nuclei to total nuclei in high-power fields.
11.Apoptosis assay
The level of apoptosis in cultured cells was evaluated by FACS of annexin-V-stained cells with use of the Annexin V Apoptosis Detection Kit and measurement of Caspase-3activity by use of the Caspase-3Colorimetric Assay Kit.
Detection of apoptotic cells in vivo was performed by the TUNEL procedure with the In Situ Cell Death Detection Kit. Cell apoptotic rate was calculated as the percentage of TUNEL-positive nuclei to total nuclei in high-power field.
12. Immunocytochemistry
To detect AF differentiation in vivo, immunohistochemistry was performed in paraffin-embedded vessel sections. The expression of a-SMA was detected by immunohistochemistry SABC method.
13. Morphometric analysis
Vascular remodeling was evaluated using morphometric analysis. Neointimal formation is expressed as the neointimal to medial area ratio (N/M). Morphometric analysis of N/M and lumen size was performed in sections stained with hematoxylin-eosin (H-E) using the Image Pro Plus computer software.
14. Statistical analysis
All data are presented as mean±SEM from at least3independent experiments. Differences between2groups were analyzed by Student's t test and between multiple groups by one-way ANOVA. Data analysis involved use of GraphPad Prism5.0. Differences were considered statistically significant at P<0.05.
Results
1. Differentiation of AFs to MFs
RT-PCR and Western blot revealed that TGF-β1induced a time-and dose-dependent steady increase in a-SMA mRNA and protein expression expression in cultured AFs. Immunoblotting revealed a-SMA protein expression in AFs significantly increased with TGF-β1treatment (10ng/ml,24h). Double immunofluorescent staining revealed that AFs with TGF-β1(10ng/ml,24h) treatment co-expressed vimentin and a-SMA and formed numerous actin stress fibers, characteristic of MFs.
2. MiR-21was endogenously expressed in AFs and markedly increased in MFs
We used real-time RT-PCR (TaqMan probes) to detect and quantify miRNAs expression of miR-21. MiR-21was found endogenously expressed in AFs and markedly increased in MFs.
3. Modulating miR-21expression in AFs and MFs
To determine whether the expression of miR-21in AFs and MFs was regulated, we used both gain-and loss-of-function approaches. The ectopic expression of miR-21was confirmed by real-time quantitative RT-PCR. Pre-miR-21significantly increased but miR-21inhibition decreased miR-21expression. Control oligos had no effect on miR-21expression. The results suggest that miR-21could be modulated both in AFs and MFs
4. The effects of miR-21on proliferation of AFs and MFs
To determine the role of miR-21in the proliferation of AFs and MFs, EdU incorporation assay was used. MiR-21inhibitor decreased EdU incorporation while pre-miR-21significantly increased EdU incorporation in AFs and MFs, which indicated that miR-21promoted the proliferation of AFs and MFs. Moreover, MFs showed higher proliferation compared with AFs both at basal condition and with pre-miR-21treatment.
5. The effects of miR-21on apoptosis of AFs and MFs
We further evaluated the effects of miR-21on the apoptosis of AFs and MFs by Annexin-V-staining and Caspase-3activity. Transfection with the miR-21inhibitor increased the percentage of Annexin V positive cells and Caspase-3enzymatic activities. However, pre-miR-21decreased and Caspase-3enzymatic activities. These results suggest that miR-21plays an anti-apoptosis role in AFs and MFs.
6. The effects of antagonist of miR-21on proliferation and apoptosis of AFs and MFs in injured artery
Immunocytochemistry revealed that a-SMA positive cells were visible in the adventitia3days after balloon injury, which indicated some AFs differentiation into MFs.
3days after injury, the adventitia cells in the injured arteries treated with vehicle showed higher proliferation rate compared with the uninjured arteries, however, treatment with miR-21antagomir led to a significant reduction in proliferation rate compared with vehicle group.
TUNEL revealed that rare positive cells were detectable in the adventitia from uninjured vessels and many apoptotic cells could be seen in the adventitia from vehicle group at1day after injury. At3days after injury, numbers of apoptotic cells in adventitia from vehicle group were significantly reduced and showed no significant differences compared with uninjured group. However, the apoptosis level were significantly increased in miR-21antagomir group compared with the uninjured group and vehicle group (3days after injury).
7. The effects of antagonist of miR-21on vascular remodeling induced by balloon injury
To determine the effect of miR-21inhibition on vascular remodeling, morphometric analyses were carried out on the21days balloon-injured arteries. The injured arteries treated with vehicle showed extensive neointima formation and dramatic reduction in lumen size compared with the uninjured arteries. N/M (Neointimal/Medial area ratio) of the miR-21antagomir group was significantly lower than that of the vehicle group, while lumen size of the miR-21antagomir group was significantly larger than that of the vehicle group.
Conclusions
1. miR-21expressed in AFs and overexpressed in MFs dedifferentiated from AFs after treatment with TGF-β1.
2. miR-21is an important regulator in proliferation and apoptosis of AFs and MFs.
3. Antagonist of miR-21could improve vascular remodeling induced by balloon injury through regulating the proliferation and apoptosis of AFs and MFs.
Background
Recent studies show that vascular adventitial fibroblasts (AFs), the most main cell components of adventitia, are the the important pathologic basis of neointima formation and pathological vascular remodeling after vascular injury. When blood vessels suffere the pathological injury or stimulation from both inside and outside, AFs will be "activated". The "activated" AFs show higher proliferative activity, and at the same time undergo phenotypic conversion into myofibroblasts (MFs). MFs display stronger ability in proliferation, synthesis of extracellular matrix (ECM) and migration. A part of the MFs will migrate to vascular intima and secrete ECM, which actively involved in neointimal formation. Therefore, AF and MF are two important therapeutic targets for cardiovascular disease.
The balance in proliferation and apoptosis of AFs and MFs is decisive for the physiology of the vasculature. In vascular proliferative diseases, the balance is disturbed, which causes AFs and MFs excessive proliferation and apoptosis defects, excessive collagen secretion, deposition of ECM, finally results in adverse vascular remodeling. Therefore, to clarify the mechanism of regulating the proliferation and apoptosis of the2type cells has important significance for the prevention and improvement of adverse vascular remodeling.
MicroRNA (miRNA) is endogenous small RNA molecule that plays important role in gene regulation. The recent studies show that miRNA is widely involved in multiple cell biological processes, including cell proliferation, apoptosis and differentiation. Among the miRNAs which have been found and identified, microRNA-21(miR-21) has attracted much attention. Much research showed that miR-21expression was up-regulated in a wide variety of tumor, which were closely related with tumor occurrence and development. Our study showed that miR-21was also expressed in vascular adventitia cells and played an important role in regulating proliferation and apoptosis of AFs and MFs. We found that antagonist of miR-21attenuated balloon injury-induced neointimal formation by regulating proliferation and apoptosis of AFs and MFs. However, the mechanism of miR-21regulation proliferation and apoptosis of AFs and MFs remains unclear.
MiRNA exert their functions mainly through regulating target genes. PDCD4is a new kind of tumor suppressor gene found in recent years. Recent research shows that PDCD4plays an important role in the regulation of cell proliferation and apoptosis, which are closely related whith tumor development. Until now, there are no research reports about PDCD4in vascular adventitia. PDCD4is confirmed as miR-21target genes and played an important role in regulating of cell proliferation, apoptosis in a variety of tumor cells. The expression and gene regulation of miRNA and its target genes exhibit tissue specificity, therefore, whether PDCD4is a target gene of miR-21in the AFs and MFs still need to be identificated. The JNK/c-Jun signaling pathway is closely related to the cell growth. There is little study on the relationship between PDCD4and JNK pathway. Studies have reported PDCD4can interfere with JNK mediated phosphorylation of c-Jun and inhibit tumor transformation through inhibition of c-Jun activation and subsequent AP-1dependent transcription. However, at present, whether miR-21can regulat JNK pathway and PDCD4is involved in the regulation process are still not reported.
In view of the above problems, we propose the following hypothesis:in vascular AFs and MFs, PDCD4is a target gene of miR-21; PDCD4can effectively regulate AFs and MFs proliferation and apoptosis, and directly involved in miR-21-mediated cell events; JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.
Objectives
1. To verify whether PDCD4is a direct target of miR-21in AFs and MFs.
2. To investigate the effects of PDCD4on proliferation and apoptosis of AFs and MFs.
3. To investigate whether PDCD4participates in miR-21-mediated proliferation and apoptosis in AFs and MFs.
4. To investigate the effects of miR-21/PDCD4on JNK activity and determine the role of JNK/c-Jun pathway in miR-21-mediated cellular effect in AFs and MFs.
Methods
1. AFs culture and establishment of myofibroblast model AFs were isolated from thoracic aortas of4-to6-week-old male Wistar-Kyoto rats weighing120to180g and cultured by the tissue adherence method. Cells from passage3to6were used.
MF differentiation was induced with TGF-(31and identified by determining the expression of a-SMA by RT-PCR, Western blot analysis and fluorescent immunocytochemistry.
2. Experimental grouping
2.1The dual-luciferase reporter assay was performed to confirm that miR-21can directly bind to PDCD4. Cells were grouped into2groups:luciferase vector group (pMIR-REPORT luciferase plasmids) and vehicle vector group. Each group was further divided into3subgroups:vehicle control group, pre miR control group and pre-miR-21group.
2.2To investigate whether PDCD4is regulated by miR-21, Western blot analysis was performed to detect PDCD4protein expression after miR-21overexpression and miR-21inhibition. Cells were grouped into5groups:vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.
2.3To determine the proliferation and apoptosis, cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, PDCD4siRNA group, miR-21inhibitor+PDCD4siRNA group.
2.4To investigate the effection of miR-21overexpression on JNK activity, cells were grouped into3groups:vehicle control group, pre miR control group and pre-miR-21group.
2.5To investigate the effection of miR-21inhibition on JNK activity and whether PDCD4participates in the regulation of miR-21on JNK activity, cells were grouped into4groups:vehicle control group, miR inhibitor control group, miR-21inhibitor group, miR-21inhibitor+PDCD4siRNA group.
3. miRNA and siRNA transfection
The oligonucleotide was transfected into cells by use of Lipofectamine2000reagentas per the manufacturer's instructions. Cells were transfected with pre-miR-21and miR-21inhibitor for miR-21overexpression and knockdown, respectively. Transfection complexes were added to cells at a final oligonucleotide concentration of50nM. For PDCD4knockdown, PDCD4siRNA was transfected into cells by use of lipofectamine2000reagent. The final PDCD4siRNA concentration was80nM.
4. Proliferation assay
Cell proliferation was determined by EdU proliferation assay. After48h of transfection, cell proliferation was quantified by the uptake of EdU into DNA by use of EdU DNA Cell Proliferation Kit. All procedures for EdU incorporation experiments followed the kit's protocol. Cell proliferation rate was calculated as the percentage of EdU-positive nuclei to total nuclei in high-power fields.
5. Apoptosis assay
The level of apoptosis in cultured cells was evaluated by measurement of Caspase-3activity by use of the Caspase-3Colorimetric Assay Kit. All procedures followed the kit's protocol.
6. Western blot analysis
Cells were harvested with lysis buffer. Protein concentration was determined using the BCA method. Protein samples were separated on SDS-PAGE gels, transferred to PVDF membrane, and blocked with5%nonfat milk/TBST and then incubated with primary antibodies. Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit IgG. Immunoreactive bands were visualized by ECL. The relative intensities of the bands were analyzed using β-actin as an internal control.
7. Dual-luciferase reporter assay
To confirm that miR-21can directly bind to PDCD4, the dual-luciferase reporter assay was performed, we cloned a construct with a fragment of the3'-UTR of PDCD4mRNA with the putative miR-21binding sequence into pMIR-REPORT luciferase plasmids (luciferase reporter vector) for co-transfection with pre-miR-21into293T cells. The vector without the putative miR-21binding sequence (vehicle vector) was the negative control. The samples were co-transfected with the Renilla luciferase-expressing plasmid pRL-TK (internal control plasmid). At24h after transfection, firefly and renilla luciferase activities were measured by use of the dual-luciferase reporter assay system on a luminometer according to the manufacturer's instructions.
8. Statistical analysis
All data are presented as mean±SEM from at least3independent experiments. Differences between2groups were analyzed by Student's t test and between multiple groups by one-way ANOVA. Data analysis involved use of GraphPad Prism5.0. Differences were considered statistically significant at P<0.05.
Results
1. PDCD4is a direct target of miR-21in AFs and MFs
1.1The expression level of PDCD4in AFs and MFs
Western blot analysis revealed PDCD4protein expression level is significantly lower in MFs than in AFs. However, miR-21expression level markedly increased in MFs compared with AFs. The inverse correlation between miR-21and Pdcd4protein suggests that PDCD4may be a target of miR-21in AFs and MFs. 1.2Dual-luciferase reporter assay
To confirm that miR-21can directly bind to PDCD4, the dual-luciferase reporter assay was performed. Our results indicated that pre-miR-21significantly inhibited luciferase activity as compared with control treatments. Therefore, miR-21could directly bind to PDCD4.1.3The miR-21regulation on PDCD4protein expression
To investigate whether PDCD4is regulated by miR-21, western blot analysis was performed after miR-21overexpression and miR-21inhibition. Our results indicated that miR-21inhibitor increased but pre-miR-21decreased PDCD4protein level in AFs and MFs.
Therefore, the above study results proved that PDCD4is a direct target gene of miR-21in AFs and MFs.
2. The effects of PDCD4inhibition on miR-21-mediated proliferation in AFs and MFs
Knockdown of PDCD4by siRNA significantly increased proliferation in AFs and MFs as determined by EDU proliferation assay. miR-21inhibitor decreased the proliferation of AFs and MFs, however, PDCD4siRNA partly rescued the reduced cellular proliferation with miR-21inhibition. Our data showed that miR-21inhibitor+PDCD4siRNA group demonstrated higher proliferation activity compared with miR-21inhibitor group. The results suggest PDCD4not only plays an essential role in regulaing proliferation of AFs and MFs, but also directly participates in miR-21-mediated proliferation in AFs and MFs
3. The effects of PDCD4inhibition on miR-21-mediated apoptosis in AFs and MFs
Knockdown of PDCD4by siRNA decreased apoptosis as determined by caspase-3activity. MiR-21inhibitor increased the caspase-3activity of AFs and MFs, however, PDCD4siRNA partly alleviated the increase in apoptosis with mir-21inhibition. Our data showed that miR-21inhibitor+PDCD4siRNA group demonstrated a significantly lower proliferation activity compared with miR-21inhibitor group. The results suggest PDCD4is a regulator of apoptosis in AFs and MFs and directly participates in miR-21-mediated apoptosis in AFs and MFs.
4. The effects of PDCD4inhibition on JNK activity
Wstern blot analysis demonstrated that knockdown of PDCD4by siRNA significantly increased the JNK/c-Jun activity, the of p-JNK and p-c-Jun protein showed higher expression level compared with control siRNA.
5. The effects of miR-21on JNK activity
MiR-21inhibitor increased PDCD4expression in AFs and MFs, while decreased the level of p-JNK and p-c-Jun. In contrast, pre-miR-21decreased PDCD4expression, while increased the level of p-JNK and p-c-Jun. The results suggested that miR-21could enhance JNK activity while miR-21inhition could decrease JNK activity.
6. The effects of PDCD4inhibition on the regulation of miR-21in JNK activity
MiR-21inhibition decreased JNK activity in AFs and MFs, however, miR-21inhibitor+PDCD4siRNA group demonstrated a significantly higher JNK activity compared with miR-21inhibitor group. The results suggested that PDCD4not only affected JNK activity, but also affected the regulation of miR-21on JNK activity. It also suggests that JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.
Conclusions
1. PDCD4is a direct target of miR-21in AFs and MFs.
2. PDCD4could regulate proliferation and apoptosis of AFs and MFs, and directly participates in miR-21-mediated cellular effect.
3. JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.
血管重构是多种心血管疾病(如冠心病、血管移植后狭窄、动脉粥样硬化和高血压等)的最终病理结局。既往研究者对于血管重构的研究重点主要放在是血管的内皮细胞和中膜的平滑肌细胞(VSMC)上,然而外膜细胞在血管重构过程中的作用没有受到重视。近年来,大量研究表明血管外膜在调控血管生理和病理方面同样发挥着重要作用。研究显示,在损伤和细胞因子等病理刺激下,作为血管外膜最主要的细胞成分,外膜成纤维细胞(adventitial fibroblast, AF)可通过激活、表型分化和腔内迁移参与并促进血管重构。被激活的AF,增殖活性增强,并发生表型变化而分化为肌成纤维细胞(myofibroblast, MF),发生表型变化的特征是获得α-SMA(α-平滑肌肌动蛋白)的表达。相对于AF, MF具有更强的增殖活性和分泌细胞外基质(extracellular matrix, ECM)的能力。MF的出现是组织器官对损伤进行修复过程中的一个特征。血管外膜细胞的增殖和凋亡之间的平衡对于维持血管正常生理功能起着至关重要的作用。当AF和MF的正常的细胞周期出现紊乱时,就会导致凋亡减少和过多的ECM沉积,最终导致病理性血管重构。在这一病理过程中,会伴随着一部分MF向血管腔内迁移参与并促进血管新生内膜形成。血管新生内膜形成是病理性血管重构的一个重要病理特征。因此,血管外膜细胞(包括AF和MF)增殖和凋亡之间的平衡失控而导致的细胞过度增殖和凋亡抑制是血管重构过程中的重要细胞事件。血管的AF和MF已成为抗血管重构治疗的两个重要靶细胞。
microRNA (miRNA)代表了一种新的基因表达调控机制。研究表明,miRNA参与了诸多重要的细胞生命活动过程,包括细胞的增殖、凋亡和分化等。而异常的细胞增殖和凋亡在肿瘤发生和发展过程中的扮演着重要角色。正因为如此,目前关于miRNA的众多研究集中在肿瘤方面。
在近些年来已发现的诸多miRNA分子中,microRNA-21(miR-21)受到研究者更多的关注。研究发现,miR-21在许多肿瘤细胞中过表达,并且是多种肿瘤组织中表达升高最明显的miRNA分子之一,被称作为"oncomir",意思是具有致癌特性的miRNA分子,具有促增殖和抗凋亡作用。许多研究者认为,对miR-21或其靶基因的表达进行干预或许会成为将来抗癌治疗的有效措施。
相对于肿瘤,miRNA在心血管方面的研究起步较晚。近年来,miR-21在心血管领域的功能也逐渐受到关注。研究发现,仅次于肿瘤,在心血管疾病中miR-21的表达也明显失调。在许多心血管疾病,如血管球囊损伤、心脏肥厚和缺血性心脏病等,miR-21的表达异常。有研究显示,大鼠颈动脉球囊损伤的血管壁中miR-21表达明显升高；miR-21在VSMC中表达,并且能调控细胞的增殖和凋亡。有报道称,miR-21在血管内皮中也有表达,miR-21可以通过调控内皮细胞的凋亡而影响其生理功能。但是目前国内外关于miRNA在血管外膜领域的研究报道很少,miR-21在血管AF和MF中是否表达以及是否对增殖和凋亡发挥调控功能尚不清楚。
研究显示,血管增殖性疾病(如冠心病、动脉粥样硬化等)和肿瘤在多种细胞事件和分子通路方面有着许多相似的特征。增殖和凋亡同是血管重构过程和肿瘤的两个重要的细胞生命活动,既然miRNA在肿瘤中发挥着重要作用,因此我们完全有理由相信,miRNA在血管重构的发生和发展过程中同样会发挥重要作用。
近些年一些研究表明,以血管外膜细胞为靶点进行干预可以改善血管重构。有研究显示,17β-雌二醇可以通过抑制外膜细胞的增殖而减少球囊损伤后血管新生内膜的形成。也有研究报道,通过在血管外膜转染smad7腺病毒使外膜细胞过表达Smad7,可以抑制TGF-β1通路,从而明显减弱球囊损伤后成纤维细胞的促新生内膜形成和血管重构的作用。因此,以血管外膜细胞为靶点的血管外膜基因转染代表了一种新的治疗心血管疾病的方法。
综上所述,我们提出如下假说：miR-21在血管AF和MF中内源性表达,并且miR-21在调控AF和MF的增殖和凋亡方面发挥着重要作用；通过干预miR-21的表达能有效调控血管AF和MF的增殖和凋亡,并能进一步改善由球囊损伤导致的血管重构。
目的
1.建立体外肌成纤维细胞模型,检测并比较miR-21在成纤维细胞和肌成纤维细胞的表达水平。
2.通过调节成纤维细胞和肌成纤维细胞中miR-21的表达水平,研究miR-21对增殖和凋亡的影响。
3.检测能否通过干预miR-21的表达影响大鼠髂动脉球囊损伤所致的血管重构。
方法
1.成纤维细胞体外培养
取4-6周(120-180克)雄性Wistar大鼠胸主动脉外膜,采用组织贴壁法原代培养外膜成纤维细胞。实验用第3-6代细胞。
2.细胞试验分组
(1)为确定TGF-β1诱导AF分化为MF的合适剂量和时间,行RT-PCR反应,用TGF-(31(10ng/ml)刺激不同的时间:0、6、12、24、36、48小时,分为6组；不同浓度的TGF-β1刺激24小时：0、1、5、10、20、30ng/ml,分为6组。行Western blot检测,按照TGF-β1(10ng/ml)刺激不同的时间:0、12、24、36小时,分为4组。
(2)为检测细胞内miR-21的表达水平能否被调节,分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂(即miR-21inhibitor)组、前体阴性对照组和pre-miR-21(即miR-21前体)组。
(3)为检测细胞的增殖和凋亡,根据干预措施不同分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂组、前体阴性对照组和pre-miR-21组。
3.建立肌成纤维细胞模型
肌成纤维细胞由TGF-β1诱导成纤维细胞而成,并经RT-PCR、Western blot和细胞免疫荧光实验方法鉴定。
4.细胞免疫荧光实验
将细胞玻片上生长的用(或未用)TGF-β1刺激的成纤维细胞,经固定、0.5%Triton-X100处理、BSA封闭后,α-SMA和vimentin一抗过夜,用含有FITC和TRITC的二抗液孵育玻片,然后用DAPI染核,最后于荧光显微镜下观察α-SMA和vimentin的表达情况.
5. miRNA转染
体外培养的细胞用转染试剂Lipofectamine2000转染pre-miR-21或miR-21inhibitor分别造成miR-21的过表达或表达抑制,同时分别设阴性对照组。48小时后检测细胞样本。实验中用含FAM荧光标签的寡核苷酸转染细胞以确定转染效率。最终寡核苷酸的终浓度为50nM。
6.实时定量RT-PCR使用miRNA提取试剂盒提取用于检测miR-21表达的细胞总RNA,用含有特异茎环结构的引物进行逆转录,然后TaqMan探针法在ABI7500PCR仪上检测miR-21的表达。用于检测其他指标(如α-SMA)的细胞总RNA的提取使用Trizol法,逆转录成cDNA后,采用荧光实时定量的方法在Bio-Rad PCR仪上检测。miR-21的相对表达用U6做内参,α-SMA mRNA等的相对表达以β-actin为内参,并用△ACT的方法计算。
7.蛋白印迹分析
利用组织裂解液提取细胞总蛋白。采用BCA法进行蛋白浓度测定。等量的蛋白样品在SDS-PAGE凝胶上电泳,然后转膜至PVDF膜上,膜封闭后,一抗过夜,用洗膜液清洗,用HRP结合的二抗孵育。ECL法发光,以β-actin作为内参,计算目的蛋白的相对表达量。
8.大鼠髂动脉球囊损伤模型的建立
按照Gabeler报道的方法建立。Wistar大鼠(320-350g)戊巴比妥麻醉,2F的导管引入到右髂总动脉,球囊在3-4atm压力下来回拉动3次造成内膜损伤。左侧未损伤血管作为对照。
9.血管外膜局部孵育寡核苷酸和动物实验分组
手术后在损伤血管的周围均匀孵育20%(w/v)的F-127缓释凝胶200μ1,凝胶中含有0.24mg miR-21antagomir(用于体内实验的miR-21的特异抑制剂)或等体积的PBS。实验动物分为3组：miR-21antagomir组(凝胶中含有miR-21antagomir,n=15)、损伤组(凝胶中仅含有PBS,n=20)和未损伤组(左侧未损伤血管,n=20)。损伤组于手术后1天、3天和21天处死,miR-21antagomir组于手术后3天和21天处死,分别用于进行增殖检测(术后3天,n=5)、凋亡检测(术后1天和3天,n=5)和HE染色(术后21天,n=5)。
10.细胞增殖检测
细胞转染pre-miR-21或miR-21inhibitor后48小时,采用EdU的方法检测细胞的增殖率。操作按照EdU细胞增殖检测试剂盒的说明书进行,增殖的细胞EdU染核阳性,用Hoechst33342染细胞核,荧光镜下观察高倍视野下细胞EdU阳性数占细胞总数的比例,计算细胞增殖率。
动物实验中,采用BrdU方法检测细胞增殖。分别在动物处死前12和24小时各注射BrdU一次。按照说明书操作,使用特异的BrdU抗体检测组织细胞的增殖率。细胞增殖率按高倍视野中BrdU阳性数占总细胞数的百分比计算。
11.细胞凋亡检测
细胞转染后48小时分别采用Annexin-V/7-AAD双染的方法和Caspase-3活性检测的方法检测细胞的凋亡情况。Annexin-V/7-AAD双染利用流式细胞仪(FACS)进行检测。Caspase-3活性检测采用比色法检测Caspase-3相对活性。
在动物实验中,按照试剂盒说明,采用TUNEL方法检测细胞凋亡。细胞的凋亡率按高倍视野中TUNEL阳性数占总细胞数的百分比计算。
12.免疫组织化学
为检测血管外膜成纤维细胞的分化,取石蜡切片,按照SABC免疫组织试剂盒的操作说明,行免疫组织化学检测α-SMA的表达。
13.形态测定分析
通过测定血管组织形态学的改变来分析血管重构的情况。用HE染色法,对新生内膜／中膜面积(Neointimal/Medial area, N/M)和管腔大小(Lumen Size)进行形态学分析,用IPP软件进行测量。
14.统计学分析
所有数据以均数士标准误计算,来源于3次以上独立实验。两组间的差异用t检验,多组间的差异用单因素方差分析。数据用GraphPad Prism5.0软件分析,P<0.05代表有显著性差异。
结果
1.体外用TGF-β1诱导成纤维细胞分化为肌成纤维细胞
RT-PCR和Western blot结果显示,在蛋白和mRNA水平上,TGF-β1都能显著促进α-SMA的表达,并呈现一定的时间和浓度依赖性；TGF-β1(10ng/ml,24小时)能明显促进α-SMA的表达。细胞免疫荧光实验研究显示,经TGF-β1(10ng/ml,24小时)处理后,细胞共表达vimentin和α-SMA,同时胞浆内形成大量肌丝,表明AF分化为MF,体外建立肌成纤维细胞模型成功。
2.miR-21在AF中内源性表达并且在MF中表达升高
实时定量RT-PCR(探针法)检测发现,AF中内源性表达miR-21。相对于AF,MF中miR-21的表达水平明显升高。
3.调节miR-21在AF和MF中的表达
为检测AF和MF中miR-21的表达水平能否被调节,我们采用了表达抑制和过表达的方法。在AF和MF中,转染pre-miR-21后,miR-21的表达水平明显升高；转染miR-21抑制剂(miR-21inhibitor)后,miR-21的表达明显下降,然而转染对照核苷酸后miR-21的表达量没有明显变化,说明miR-21在AF和MF中的表达水平是可以调节的。
4.miR-21对AF和MF增殖的作用
EdU增殖检测分析结果显示,在AF和MF中,抑制miR-21的表达后,两种细胞的EdU掺入率减少；转染pre-miR-21使细胞过表达miR-21后,AF和MF的EdU掺入率减少增加；上述结果表明,miR-21具有促进AF和MF的增殖活性的作用。研究还发现,无论在基础水平还是经pre-miR-21干预之后,MF较AF都显示出更高的增殖活性。
5.miR-21对AF和MF凋亡的作用
Annexin-V/7AAD流式细胞术和Caspase-3活性检测结果显示,AF和MF转染miR-21inhibitor抑制miR-21表达后,annexin Ⅴ阳性细胞数和Caspase-3的活性都明显增加；而过表达miR-21后则出现相反的结果,说明在AF和MF中,miR-21发挥了为抗凋亡的作用。
6.miR-21抑制剂对球囊损伤的血管的外膜成纤维细胞的增殖和凋亡的影响
免疫组化结果显示,在球囊损伤术后3天,血管外膜中可见α-SMA阳性细胞,这一结果提示已有部分AF分化为MF。
术后3天,损伤组的血管外膜细胞增殖活性较未损伤组明显升高；而相对于损伤组,miR-21antagomir组的血管外膜细胞增殖率明显下降。
TUNEL结果显示,未损伤的血管外膜凋亡细胞很少；术后1天,损伤组的外膜可见较多凋亡细胞；术后3天,损伤组外膜中凋亡细胞数明显下降,与未损伤组无明显差别；然而,miR-21antagomir (miR-21抑制剂)组相较于未拉伤组和损伤组(术后3天),凋亡率明显升高。
7.miR-21抑制剂对球囊所致的血管重构的影响
为了检测miR-21抑制剂对球囊损伤所致的血管重构的影响,我们对球囊损伤后21天的血管进行了形态学的分析。相对于未损伤组,损伤组的血管呈现出明显的新生内膜形成和管腔的损失；相对于损伤组,miR-21antagomir组的内膜／中膜面积比(N/M)明显降低,并且管腔损失程度得到改善。
结论
1.miR-21在血管成纤维细胞内源性表达,并且在TGF-β1诱导而成的肌成纤维细胞中miR-21表达明显升高。
2.miR-21是血管调控成纤维细胞和肌成纤维细胞增殖和凋亡的重要分子。
3.miR-21抑制剂可以通过影响AF和MF的增殖和凋亡从而改善球囊损伤所致的血管重构。
研究背景
研究表明,作为血管外膜的最重要的细胞成分,血管外膜成纤维细胞(adventitial fibroblast, AF)是血管损伤后新生内膜形成和病理性血管重构的重要病理基础。当血管受到来自内外的病理损伤或外界刺激时,AF会被“激活”,表现为增殖活性迅速增强,同时发生表型变化而成为肌成纤维细胞(myofibroblast, MF)。MF较AF表现出更强的增殖、迁移和分泌细胞外基质的能力。同时会有一部分MF迁移至内膜并分泌大量ECM从而积极参与新生内膜.的形成。因此,AF和MF是治疗心血管疾病的两种重要的靶细胞。
血管外膜细胞的增殖和凋亡之间的平衡对于维持血管正常生理功能至关重要。在血管增殖性疾病中,AF和MF过度增殖和凋亡缺陷使得这一平衡打破,从而使胶原分泌过度,细胞外基质过多沉积,最终导致不良性血管重构。因此,阐明调控这两种细胞增殖和凋亡的机制对于预防和改善不良血管重构有着重要意义。
MicroRNA (miRNA)是一类具有重要基因调控功能的内源性小RNA分子。近年来研究表明,miRNA广泛参与了增殖、凋亡和分化等机体多种细胞生物学过程的调控。在目前已发现和鉴定的miRNA分子中,microRNA-2(miR-21)备受关注。研究显示,miR-21在多种肿瘤中表达明显上调,参与了细胞增殖、凋亡、分化和迁移等诸多细胞生物学过程,与肿瘤的发生和进展密切相关。我们的研究发现,miR-21在血管外膜细胞中也有表达,并且miR-21可以有效调控AF和MF的增殖和凋亡；在大鼠髂动脉球囊损伤的血管重构模型中,通过在外膜孵育miR-21抑制剂可以抑制新生内膜形成。但是目前miR-21对AF和MF的增殖和凋亡发挥调控作用的具体机制尚不清楚。
miRNA分子发挥其功能主要是通过调控其靶基因的表达而实现的。PDCD4是近年来发现的一个肿瘤抑制基因。研究发现,PDCD4在调控细胞的增殖和凋亡方面发挥着重要作用,与肿瘤的发生发展有密切关系。但是目前尚未有PDCD4在血管外膜AF和MF的研究报道。在多种肿瘤细胞中PDCD4被证实为miR-21的靶基因,可以调控细胞的增殖和凋亡。miRNA与其靶基因在表达和基因调控方面呈现出组织特异性,所以,在AF和MF中,PDCD4是否表达以及是否为miRNA的靶基因,还有待于研究确定。JNK/c-Jun信号通路是与细胞生长密切相关的通路。目前关于PDCD4和JNK通路两者关系的研究较少。有研究报道,PDCD4可以干扰JNK介导的c-Jun的磷酸化,并且可通过抑制c-Jun的激活及随后的AP-1依赖性的转录而抑制肿瘤的转化。但是,miR-21能否调控JNK通路以及PDCD4是否参与此调控过程目前还没有报道。
针对以上问题,我们提出如下假说：在血管AF中,PDCD4是miR-21的靶基因；PDCD4能有效调控血管AF和MF的增殖和凋亡,并且直接参与了miR-21介导的细胞事件；JNK/c-Jun是PDCD4参与miR-21介导的细胞效应的下游信号通路。
目的
1.在AF和MF中,验证PDCD4是否是miR-21的一个直接靶基因。
2.检测PDCD4对AF和MF的增殖和凋亡的作用。
3.检测PDCD4是否直接参与了miR-21对AF和MF的增殖和凋亡的调控作用。
4.检测调控miR-21和PDCD4对JNK活性的影响,并确定JNK/c-Jun信号通路在miR-21介导的AF和MF细胞事件中的作用。
方法
1.成纤维细胞体外培养及建立肌成纤维细胞模型
取4-6周(120-180克)雄性Wistar大鼠胸主动脉外膜,采取组织贴壁法原代培养外膜成纤维细胞。实验用3-6代细胞。肌成纤维细胞由TGF-β1诱导成纤维细胞而成,并经RT-PCR. Western blot和细胞免疫荧光实验方法鉴定。
2.实验分组
(1)为验证miR-21能否直接与PDCD4的3'-UTR的靶位点结合而行双荧光素酶报告基因检测,分为2组：荧光素酶载体组(pMIR-REPORT荧光素酶质粒组)和载体对照组；每组又分3个亚组：空白对照组、前体阴性对照组和pre-miR-21(即miR-21前体)组。
(2)为验证miR-21能否调控PDCD4蛋白的表达行Western blot检测,分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂(即miR-21inhibitor)组、前体阴性对照组和pre-miR-21组。
(3)为检测细胞的增殖和凋亡,分为5组：空白对照组、抑制剂阴性对照组、miR-21抑制剂组、PDCD4siRNA组和miR-21inhibitor+PDCD4siRNA组。
(4)为检测过表达miR-21后JNK活性的改变,分为3组：空白对照组、前体阴性对照组和pre-miR-21组。
(5)为检测抑制miR-21的表达后JNK活性的改变以及PDCD4是否参与了其中过程,分为4组：空白对照组、抑制剂阴性对照组、miR-21抑制剂组和miR-21inhibitor+PDCD4siRNA组。
3. miRNA and siRNA转染
寡核苷酸的转染是用转染试剂Lipofectamine2000按照试剂说明要求进行操作。体外培养的细胞用转染试剂Lipofectamine2000转染pre-miR-21或miR-21inhibitor分别造成miR-21的过表达或表达抑制,最终寡核苷酸浓度的终浓度为50nM。对于PDCD4siRNA, PDCD4siRNA的终浓度80nM。
4.细胞增殖检测
细胞转染pre-miR-21或：niR-21inhibitor后48小时,细胞的增殖检测采用EdU的方法检测。按照EdU细胞增殖检测试剂盒中的说明书进行操作,增殖的细胞EdU染核阳性,用Hoechst33342染细胞核,荧光镜下观察高倍视野下细胞EdU阳性数占细胞总数的比例,计算细胞增殖率。
5.细胞凋亡检测
细胞转染48小时后,分别采用Caspase-3活性检测的方法进行检测。Caspase-3活性检测是在多功能酶标仪上检测细胞样本的OD值。
6.蛋白印迹分析
利用组织裂解液提取细胞总蛋白。采用BCA法测定蛋白浓度。等量的蛋白样品在SDS-PAGE凝胶上电泳,然后转膜在PVDF膜上,膜封闭后,一抗过夜,洗膜液清洗,用HRP结合的二抗孵育。ECL法发光,以β-actin作为内参,计算各目的蛋白的相对表达量。
7.双荧光素酶报告基因检测
为检测miR-21能否与PDCD4的3'-UTR mRNA特异结合,我们采用了双荧光素酶报告基因检测的方法。将含有PDCD4的3'-UTR mRNA部分序列(含有与miR-21潜在结合的位点的mRNA序列)的荧光素酶载体(简称：荧光素酶载体)或不含有PDCD43'-UTR mRNA部分序列的荧光素酶载体(空白载体对照)和pre-miR-21共转染293T细胞。同时转染表达海参素报告基因的pRL-TK作为内参照。转染24小时后,用双荧光素酶检测试剂盒在酶标仪上分别检测萤火虫和海参素的信号强度。
8.统计学分析
所有数据以均数±标准误计算,来源于3次以上独立实验。两组间的差异用t检验,多组间的差异用单因素方差分析。数据用GraphPad Prism5.0软件分析,P<0.05代表有显著性差异。
结果
1.在AF和MF中,PDCD4是miR-21的一个直接靶基因
(1)AF和MF中PDCD4蛋白表达水平的比较
Western blot检测分析显示,PDCD4在MF中的表达水平较AF中明显下降。而我们前期研究显示,在MF中miR-21的表达水平较AF明显增高。PDCD4和miR-21在两种细胞中的表达呈现负相关,这一结果提示：在AF中,PDCD4可能是miR-21的一个靶基因。
(2)双荧光素酶报告基因系统分析结果
双荧光素酶报告基因检测显示,相对于对照组,pre-miR-21能够明显降低荧光素酶信号的强度,说明miR-21能直接与PDCD4mRNA序列中的3′-UTR的靶位点结合。
(3)miR-21过表达和表达抑制后PDCD4的表达
将miR-21过表达或表达抑制后,行Western blot检测PDCD4蛋白的表达。研究发现,在AF和MF中,转染pre-miR-21后,PDCD4蛋白的表达水平明显下降；转染miR-21inhibitor后,PDCD4蛋白的表达水平明显升高,这一结果说明在AF和MF中,miR-21能直接调控PDCD4蛋白的表达。
以上结果充分说明,在AF和MF中,miR-21可以直接靶作用于PDCD4, PDCD4是miR-21的一个直接靶基因。
2.抑制PDCD4表达对miR-21介导的对AF和MF增殖作用的影响
相对于对照组,PDCD4siRNA组的AF和MF的EdU阳性率明显增多,即增殖率明显增加。在AF和MF中,miR-21抑制剂组增殖率明显降低,然而,相对于miR-21抑制剂组,miR-21抑制剂+PDCD4siRNA组的增殖率明显升高。以上结果说明,抑制PDCD4的表达不仅可以促进AF和MF的增殖,而且可以明显缓解miR-21抑制剂对AF和MF增殖的抑制作用,说明PDCD4不仅可以调控AF和MF的增殖,而且直接参与了miR-21对AF和MF增殖的调控。
3.抑制PDCD4表达对miR-21介导的对AF和MF凋亡作用的影响
用PDCD4siRNA沉默PDCD4的表达后,相对于对照组, AF和MF的Caspase-3活性明显降低。在AF和MF中,miR-21抑制剂组的Caspase-3活性明显增强,然而,相对于miR-21抑制剂组,miR-21抑制剂+PDCD4siRNA组的Caspase-3活性明显降低。以上结果表明,PDCD4siRNA不仅可以降低AF和MF的Caspase-3活性,而且还可以消弱miR-21抑制剂增强AF和MF的Cspase-3活性的作用,说明PDCD4直接参与了miR-21对AF和MF凋亡的调控。
4.抑制PDCD4表达对JNK活性的影响
Western blot结果显示,PDCD4siRNA可以明显增强JNK活性,表现为p-AKT和p-c-Jun水平明显升高。
5.调节miR-21表达对JNK活性的影响
在AF和MF中,转染pre-miR-21后,PDCD4蛋白表达减少,JNK活性增强；然而转染miR-21抑制剂后,PDCD4蛋白表达增多,而JNK活性降低。这
研究结果说明,过表达miR-21可以增强JNK活性,抑制miR-21表达后可以降低JNK活性。
6.抑制PDCD4表达对miR-21调控是JNK活性的作用的影响
在AF和MF中,相对于对照组,miR-21抑制剂组的JNK活性活性明显减弱；然而,相对于miR-21抑制剂组,miR-21抑制剂+PDCD4siRNA组的JNK活性明显增强。研究结果说明,PDCD4不仅能影响JNK活性,而且还可以影响miR-21对JNK活性的调控,同时也说明JNK/c-Jun是PDCD4参与miR-21介导的细胞事件的下游信号通路。
结论
1.在血管成纤维细胞和肌成纤维细胞中,PDCD4是miR-21的一个直接靶基因。
2. PDCD4能调控血管成纤维细胞和肌成纤维细胞的增殖和凋亡,并且直接参与了miR-21介导的对细胞增殖和凋亡的调控作用。
3. JNK/c-Jun是PDCD4参与miR-21介导的细胞事件的重要下游信号通路。
Background
Vascular remodeling is the pathological endpoint of diverse cardiovascular diseases such as coronary heart diseases, post-angioplasty restenosis, atherosclerosis, and hypertension. The previous studies on vascular remodeling focused on endothelial cells (ECs) and medial vascular smooth muscle cells (VSMCs), however,the role of adventitial cells in vascular remodeling did not receive attention. Increasing evidence suggests that the adventitia is a critical regulator of vessel wall function in health and disease. Many Studies show that vascular adventitial fibroblasts (AFs) contribute to vascular remodeling through activation, differentiation and intraluminal migration. Under pathological stimuli such as injury and cytokines, AFs, the principal cell type in the adventitia, are activated and undergo phenotypic changes, including proliferation and differentiation to myofibroblasts (MFs). The characteristic of differentiation is that AFs acquire α-smooth muscle actin (α-SMA) expression. Increased proliferative capacity and increased secretion of the extracellular matrix (ECM) proteins are key hallmarks of MFs. Transient appearance of MFs is a feature of normal wound repair. The balance in adventitial cells (including AFs and MFs) proliferation and apoptosis is decisive for the physiology of the vasculature. When the MF life cycle is not regulated properly, absence of apoptosis leads to overproduction of ECM proteins deposition and pathological vascular remodeling. Moreover, these processes are accompanied by adventitial cells migration toward the lumen, thereby contribute to neointimal formation. Neointima formation is an important feature of pathological vascular remodeling. Thus, uncontrolled cell proliferation and suppression of apoptosis are critical cellular events in vascular remodeling. Vascular AFs and MFs have become2potential target cells for anti-vascular remodeling therapeutics.
As a new layer of gene regulation mechanism, microRNAs (miRNAs) have been implicated in a multitude of important cellular processes, including regulation of cellular proliferation, apoptosis and differentiation. The abnormal proliferation and apoptosis play an important role during tumor occurrence and development process. Therefore, most previous research into the role of miRNAs has focused on tumor.
MiR-21has attracted more attention than any other miRNA. Multiple studies have identified miR-21overexpressed in a wide variety of cancer cell lines and causally linked to cancer-related processes such as proliferation and apoptosis. miR-21was reported to be strongly overexpressed in a wide variety of cancer cell lines. On the basis of these findings, recently, miR-21has been referred to as an "oncomir"(i.e., a miRNA with oncogenic properties) with pro-proliferative and anti-apoptotic functions. Interfere with mir-21expression or with its interaction with their target genes has been suggested as a potential anticancer therapeutic approach.
Related to tumor, the research of miRNA in cardiovascular started later. In recent years, the function of miR-21in cardiovascular also attracts much attention gradually. Next to tumor, miR-21appears to be strongly deregulated in cardiovascular disease. Under some cardiovascular disease conditions such as proliferative vascular disease, vascular walls balloon injury, cardiac hypertrophy and ischemic heart disease, the mir-21expression is deregulated. It was reported that miR-21was strongly overexpressed in vascular walls after balloon angioplasty of the rat carotid artery. MiR-21is expressed in VSMCs and regulates proliferation and apoptosis. MiR-21is also expressed in vascular ECs. It was reported that shear stress forces regulate the expression of miR-21in ECs, which influencing endothelial biology by decreasing apoptosis. However, the research of miRNA in vascular adventitia area is little at present. It is unclear whether miR-21is expressed in AFs and MFs and whether play a role in the proliferation and apoptosis of the2kinds cells.
Numerous studies have demonstrated some characteristics of proliferative vascular disease are similar to those in tumor in cellular events and molecular pathways. Because proliferation and apoptosis are2important cellular events in vascular remodeling and tumor, miRNAs may play important roles in vascular remodeling.
In recent years, many studies suggest that the intervention measures which treat vascular adventitial cells as therapeutic targets can improve vascular remodeling. A study showed that estrogen may attenuate the injury response in balloon injury model by inhibiting proliferation of adventitial cells and their contribution to neointima formation. A study showed that smad7overexpression through gene transfer of smad7in the adventitial surface antagonized the TGF-β1signaling pathway and attenuated vascular remodeling and contribution of AFs to neointima formation after balloon angioplasty. Therefore, perivascular gene transfer which targets vascular adventitial cells represents a novel therapeutic strategy of vascular disease.
Taken together, We put forward the hypothesis:miR-21was endogenously expressed in AFs and MFs; miR-21play an important role in regulating proliferation and apoptosis of AFs and MFs; moudulting miR-21expression could regulation proliferation and apoptosis of vascular AFs and MFs and further improve balloon injury-induced rat iliac artery remodeling.
Objectives
1. To established the cell model of MFs and determine the miR-21expression in AFs and MFs.
2. To investigate the effects of miR-21modulation on proliferation and apoptosis of AFs and MFs.
3. To investigate the effects of miR-21inhibition on neointima formation after iliac artery balloon injury.
Materials
1. Cell culture
AFs were isolated from thoracic aortas of4-to6-week-old male Wistar-Kyoto rats weighing120to180g and cultured by the tissue adherence method. Cells from passage3to6were used.
2. Grouping in vitro experiment
2.1RT-PCR and Western blot were used to inspect the effection of TGF-β1on AF differentiation. In RT-PCR experiment, cell were grouped into6groups depending different time (0、6、12、24、36、48hours) or6groups at different concentrations (0、1、5、10、20、30ng/ml). In Western blot experiment, cell were grouped into4groups according to different time (0、12、24、36hours).
2.2To determine whether the expression of miR-21in AFs and MFs was regulated, we used both gain-and loss-of-function approaches. Cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.
2.3To determine the proliferation and apoptosis, cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.
3. Myofibroblast model
Subconfluent AFs were incubated with different concentrations of TGF-β1in serum-free medium. The MF phenotype in TGF-β1-treated fibroblasts was identified by determining the expression of a-SMA by RT-PCR, Western blot analysis and fluorescent immunocytochemistry.
4. Fluorescent immunocytochemistry
Fibroblasts with or without TGF-β1treatment were grown on glass coverslips, rinsed with PBS, fixed with3%paraformaldehyde, permeabilized with0.5%Triton-X100and blocked with2%BSA. Cells were incubated with the primary antibodies mouse anti-a-SMA and rabbit anti-vimentin. Immunofluorescent staining for a-SMA and vimentin involved incubation with FITC-conjugated anti-mouse IgG and TRITC-conjugated anti-rabbit IgG, respectively. Cellular nuclei were stained with DAPI. Images were examined under confocal microscope.
5. miRNA transfection
The oligonucleotide was transfected into cells by use of Lipofectamine2000reagentas per the manufacturer's instructions. Cells were transfected with pre-miR-21and miR-21inhibitor for miR-21overexpression and knockdown, respectively. The samples were assayed after48h. The transfection efficiency was determined by use of the negative control oligonucleotide with6-FAM at the5'end. After optimization, transfection complexes were added to cells at a final oligonucleotide concentration of50nM.
6. Real-time quantitative RT-PCR
Total RNA was isolated for miR-21and a-SMA from cultured cells48h post-transfection with use of the mirVanaTM miRNA Isolation Kit and Trizol according to the manufacturer's protocol. cDNA synthesis involved carried out in a thermal cycler. PCR reactions for miRNA and mRNA involved the ABI Prism7500Sequence Detection System and iQ5Real-Time PCR Detection System, respectively. Real-time RT-PCR analyses of miRNA expression involved use of TaqMan miRNA assays. RT-PCR analyses of the mRNA expression of a-SMA and (3-actin involved PrimeScript(?) RT-PCR kits. Analysis of relative miRNA and mRNA expression involved the△△CT method, with U6and β-actin used as endogenous controls, respectively.
7. Western blot analysis
Cells were harvested with lysis buffer. Protein concentration was determined using the BCA method. Protein samples were separated on SDS-PAGE gels, transferred to PVDF membrane, and blocked with5%nonfat milk/TBST and then incubated with primary antibodies. Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit IgG. Immunoreactive bands were visualized by ECL. The relative intensities of the bands were analyzed using P-actin as an internal control.
8. Establishment of rat iliac artery balloon injury model
Iliac artery balloon injury was performed according to Gabeler et al. Briefly, Wistar rats (320-350g) were generally anesthetized with sodium pentobarbital. A2F Fogarty catheter was inserted to the right iliac artery. To produce iliac artery injury, the balloon was inflated to3-4atm and withdrawn three times. The left iliac arteries were not injured and served as controls.
9. Adventitial oligo transfer and animal grouping
200βl of20%(w/v) F-127pluronic gel (Sigma) containing0.24mg antagomir against miR-21(miR-21antagomir) or PBS was applied to the adventitial around injured artery segments. Animals were divided into three groups:miR-21antagomir group (treated with miR-21antagomir dissolved in F-127pluronic gel, n=15), vehicle group (treated with PBS dissolved in F-127pluronic gel, n=20), and uninjured group (n=20). Animals of vehicle group were sacrificed at1days,3days, and21days after injury. Animals of miR-21antagomir group were sacrificed at3days and21days after injury. The vasclular sample were used to performe proliferation assay (3days after injury, n=5), apoptosis assay (1and3days after injury, n=5) and HE staining (21days after injury, n=5)
10. Proliferation assay
Cell proliferation in vitro was determined by EdU. After48h of transfection, cell proliferation was quantified by the uptake of EdU into DNA by use of a Cell-LightTM EdU DNA Cell Proliferation Kit. All procedures for EdU incorporation experiments followed the kit's protocol. Cell proliferation rate was calculated as the percentage of EdU-positive nuclei to total nuclei in high-power fields.
To detect AF and MF proliferation in vivo, BrdU dissolved in PBS, was injected12and24hours before euthanasia. BrdU-incorporating nuclei were detected with a specific anti-BrdU antibody according to the manufacturer's instructions. Cell proliferation rate was calculated as the percentage of BrdU-positive nuclei to total nuclei in high-power fields.
11.Apoptosis assay
The level of apoptosis in cultured cells was evaluated by FACS of annexin-V-stained cells with use of the Annexin V Apoptosis Detection Kit and measurement of Caspase-3activity by use of the Caspase-3Colorimetric Assay Kit.
Detection of apoptotic cells in vivo was performed by the TUNEL procedure with the In Situ Cell Death Detection Kit. Cell apoptotic rate was calculated as the percentage of TUNEL-positive nuclei to total nuclei in high-power field.
12. Immunocytochemistry
To detect AF differentiation in vivo, immunohistochemistry was performed in paraffin-embedded vessel sections. The expression of a-SMA was detected by immunohistochemistry SABC method.
13. Morphometric analysis
Vascular remodeling was evaluated using morphometric analysis. Neointimal formation is expressed as the neointimal to medial area ratio (N/M). Morphometric analysis of N/M and lumen size was performed in sections stained with hematoxylin-eosin (H-E) using the Image Pro Plus computer software.
14. Statistical analysis
All data are presented as mean±SEM from at least3independent experiments. Differences between2groups were analyzed by Student's t test and between multiple groups by one-way ANOVA. Data analysis involved use of GraphPad Prism5.0. Differences were considered statistically significant at P<0.05.
Results
1. Differentiation of AFs to MFs
RT-PCR and Western blot revealed that TGF-β1induced a time-and dose-dependent steady increase in a-SMA mRNA and protein expression expression in cultured AFs. Immunoblotting revealed a-SMA protein expression in AFs significantly increased with TGF-β1treatment (10ng/ml,24h). Double immunofluorescent staining revealed that AFs with TGF-β1(10ng/ml,24h) treatment co-expressed vimentin and a-SMA and formed numerous actin stress fibers, characteristic of MFs.
2. MiR-21was endogenously expressed in AFs and markedly increased in MFs
We used real-time RT-PCR (TaqMan probes) to detect and quantify miRNAs expression of miR-21. MiR-21was found endogenously expressed in AFs and markedly increased in MFs.
3. Modulating miR-21expression in AFs and MFs
To determine whether the expression of miR-21in AFs and MFs was regulated, we used both gain-and loss-of-function approaches. The ectopic expression of miR-21was confirmed by real-time quantitative RT-PCR. Pre-miR-21significantly increased but miR-21inhibition decreased miR-21expression. Control oligos had no effect on miR-21expression. The results suggest that miR-21could be modulated both in AFs and MFs
4. The effects of miR-21on proliferation of AFs and MFs
To determine the role of miR-21in the proliferation of AFs and MFs, EdU incorporation assay was used. MiR-21inhibitor decreased EdU incorporation while pre-miR-21significantly increased EdU incorporation in AFs and MFs, which indicated that miR-21promoted the proliferation of AFs and MFs. Moreover, MFs showed higher proliferation compared with AFs both at basal condition and with pre-miR-21treatment.
5. The effects of miR-21on apoptosis of AFs and MFs
We further evaluated the effects of miR-21on the apoptosis of AFs and MFs by Annexin-V-staining and Caspase-3activity. Transfection with the miR-21inhibitor increased the percentage of Annexin V positive cells and Caspase-3enzymatic activities. However, pre-miR-21decreased and Caspase-3enzymatic activities. These results suggest that miR-21plays an anti-apoptosis role in AFs and MFs.
6. The effects of antagonist of miR-21on proliferation and apoptosis of AFs and MFs in injured artery
Immunocytochemistry revealed that a-SMA positive cells were visible in the adventitia3days after balloon injury, which indicated some AFs differentiation into MFs.
3days after injury, the adventitia cells in the injured arteries treated with vehicle showed higher proliferation rate compared with the uninjured arteries, however, treatment with miR-21antagomir led to a significant reduction in proliferation rate compared with vehicle group.
TUNEL revealed that rare positive cells were detectable in the adventitia from uninjured vessels and many apoptotic cells could be seen in the adventitia from vehicle group at1day after injury. At3days after injury, numbers of apoptotic cells in adventitia from vehicle group were significantly reduced and showed no significant differences compared with uninjured group. However, the apoptosis level were significantly increased in miR-21antagomir group compared with the uninjured group and vehicle group (3days after injury).
7. The effects of antagonist of miR-21on vascular remodeling induced by balloon injury
To determine the effect of miR-21inhibition on vascular remodeling, morphometric analyses were carried out on the21days balloon-injured arteries. The injured arteries treated with vehicle showed extensive neointima formation and dramatic reduction in lumen size compared with the uninjured arteries. N/M (Neointimal/Medial area ratio) of the miR-21antagomir group was significantly lower than that of the vehicle group, while lumen size of the miR-21antagomir group was significantly larger than that of the vehicle group.
Conclusions
1. miR-21expressed in AFs and overexpressed in MFs dedifferentiated from AFs after treatment with TGF-β1.
2. miR-21is an important regulator in proliferation and apoptosis of AFs and MFs.
3. Antagonist of miR-21could improve vascular remodeling induced by balloon injury through regulating the proliferation and apoptosis of AFs and MFs.
Background
Recent studies show that vascular adventitial fibroblasts (AFs), the most main cell components of adventitia, are the the important pathologic basis of neointima formation and pathological vascular remodeling after vascular injury. When blood vessels suffere the pathological injury or stimulation from both inside and outside, AFs will be "activated". The "activated" AFs show higher proliferative activity, and at the same time undergo phenotypic conversion into myofibroblasts (MFs). MFs display stronger ability in proliferation, synthesis of extracellular matrix (ECM) and migration. A part of the MFs will migrate to vascular intima and secrete ECM, which actively involved in neointimal formation. Therefore, AF and MF are two important therapeutic targets for cardiovascular disease.
The balance in proliferation and apoptosis of AFs and MFs is decisive for the physiology of the vasculature. In vascular proliferative diseases, the balance is disturbed, which causes AFs and MFs excessive proliferation and apoptosis defects, excessive collagen secretion, deposition of ECM, finally results in adverse vascular remodeling. Therefore, to clarify the mechanism of regulating the proliferation and apoptosis of the2type cells has important significance for the prevention and improvement of adverse vascular remodeling.
MicroRNA (miRNA) is endogenous small RNA molecule that plays important role in gene regulation. The recent studies show that miRNA is widely involved in multiple cell biological processes, including cell proliferation, apoptosis and differentiation. Among the miRNAs which have been found and identified, microRNA-21(miR-21) has attracted much attention. Much research showed that miR-21expression was up-regulated in a wide variety of tumor, which were closely related with tumor occurrence and development. Our study showed that miR-21was also expressed in vascular adventitia cells and played an important role in regulating proliferation and apoptosis of AFs and MFs. We found that antagonist of miR-21attenuated balloon injury-induced neointimal formation by regulating proliferation and apoptosis of AFs and MFs. However, the mechanism of miR-21regulation proliferation and apoptosis of AFs and MFs remains unclear.
MiRNA exert their functions mainly through regulating target genes. PDCD4is a new kind of tumor suppressor gene found in recent years. Recent research shows that PDCD4plays an important role in the regulation of cell proliferation and apoptosis, which are closely related whith tumor development. Until now, there are no research reports about PDCD4in vascular adventitia. PDCD4is confirmed as miR-21target genes and played an important role in regulating of cell proliferation, apoptosis in a variety of tumor cells. The expression and gene regulation of miRNA and its target genes exhibit tissue specificity, therefore, whether PDCD4is a target gene of miR-21in the AFs and MFs still need to be identificated. The JNK/c-Jun signaling pathway is closely related to the cell growth. There is little study on the relationship between PDCD4and JNK pathway. Studies have reported PDCD4can interfere with JNK mediated phosphorylation of c-Jun and inhibit tumor transformation through inhibition of c-Jun activation and subsequent AP-1dependent transcription. However, at present, whether miR-21can regulat JNK pathway and PDCD4is involved in the regulation process are still not reported.
In view of the above problems, we propose the following hypothesis:in vascular AFs and MFs, PDCD4is a target gene of miR-21; PDCD4can effectively regulate AFs and MFs proliferation and apoptosis, and directly involved in miR-21-mediated cell events; JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.
Objectives
1. To verify whether PDCD4is a direct target of miR-21in AFs and MFs.
2. To investigate the effects of PDCD4on proliferation and apoptosis of AFs and MFs.
3. To investigate whether PDCD4participates in miR-21-mediated proliferation and apoptosis in AFs and MFs.
4. To investigate the effects of miR-21/PDCD4on JNK activity and determine the role of JNK/c-Jun pathway in miR-21-mediated cellular effect in AFs and MFs.
Methods
1. AFs culture and establishment of myofibroblast model AFs were isolated from thoracic aortas of4-to6-week-old male Wistar-Kyoto rats weighing120to180g and cultured by the tissue adherence method. Cells from passage3to6were used.
MF differentiation was induced with TGF-(31and identified by determining the expression of a-SMA by RT-PCR, Western blot analysis and fluorescent immunocytochemistry.
2. Experimental grouping
2.1The dual-luciferase reporter assay was performed to confirm that miR-21can directly bind to PDCD4. Cells were grouped into2groups:luciferase vector group (pMIR-REPORT luciferase plasmids) and vehicle vector group. Each group was further divided into3subgroups:vehicle control group, pre miR control group and pre-miR-21group.
2.2To investigate whether PDCD4is regulated by miR-21, Western blot analysis was performed to detect PDCD4protein expression after miR-21overexpression and miR-21inhibition. Cells were grouped into5groups:vehicle control group, miR inhibitor control group, miR-21inhibitor group, pre miR control group and pre-miR-21group.
2.3To determine the proliferation and apoptosis, cells were grouped into5groups: vehicle control group, miR inhibitor control group, miR-21inhibitor group, PDCD4siRNA group, miR-21inhibitor+PDCD4siRNA group.
2.4To investigate the effection of miR-21overexpression on JNK activity, cells were grouped into3groups:vehicle control group, pre miR control group and pre-miR-21group.
2.5To investigate the effection of miR-21inhibition on JNK activity and whether PDCD4participates in the regulation of miR-21on JNK activity, cells were grouped into4groups:vehicle control group, miR inhibitor control group, miR-21inhibitor group, miR-21inhibitor+PDCD4siRNA group.
3. miRNA and siRNA transfection
The oligonucleotide was transfected into cells by use of Lipofectamine2000reagentas per the manufacturer's instructions. Cells were transfected with pre-miR-21and miR-21inhibitor for miR-21overexpression and knockdown, respectively. Transfection complexes were added to cells at a final oligonucleotide concentration of50nM. For PDCD4knockdown, PDCD4siRNA was transfected into cells by use of lipofectamine2000reagent. The final PDCD4siRNA concentration was80nM.
4. Proliferation assay
Cell proliferation was determined by EdU proliferation assay. After48h of transfection, cell proliferation was quantified by the uptake of EdU into DNA by use of EdU DNA Cell Proliferation Kit. All procedures for EdU incorporation experiments followed the kit's protocol. Cell proliferation rate was calculated as the percentage of EdU-positive nuclei to total nuclei in high-power fields.
5. Apoptosis assay
The level of apoptosis in cultured cells was evaluated by measurement of Caspase-3activity by use of the Caspase-3Colorimetric Assay Kit. All procedures followed the kit's protocol.
6. Western blot analysis
Cells were harvested with lysis buffer. Protein concentration was determined using the BCA method. Protein samples were separated on SDS-PAGE gels, transferred to PVDF membrane, and blocked with5%nonfat milk/TBST and then incubated with primary antibodies. Secondary antibodies were HRP-conjugated anti-mouse or anti-rabbit IgG. Immunoreactive bands were visualized by ECL. The relative intensities of the bands were analyzed using β-actin as an internal control.
7. Dual-luciferase reporter assay
To confirm that miR-21can directly bind to PDCD4, the dual-luciferase reporter assay was performed, we cloned a construct with a fragment of the3'-UTR of PDCD4mRNA with the putative miR-21binding sequence into pMIR-REPORT luciferase plasmids (luciferase reporter vector) for co-transfection with pre-miR-21into293T cells. The vector without the putative miR-21binding sequence (vehicle vector) was the negative control. The samples were co-transfected with the Renilla luciferase-expressing plasmid pRL-TK (internal control plasmid). At24h after transfection, firefly and renilla luciferase activities were measured by use of the dual-luciferase reporter assay system on a luminometer according to the manufacturer's instructions.
8. Statistical analysis
All data are presented as mean±SEM from at least3independent experiments. Differences between2groups were analyzed by Student's t test and between multiple groups by one-way ANOVA. Data analysis involved use of GraphPad Prism5.0. Differences were considered statistically significant at P<0.05.
Results
1. PDCD4is a direct target of miR-21in AFs and MFs
1.1The expression level of PDCD4in AFs and MFs
Western blot analysis revealed PDCD4protein expression level is significantly lower in MFs than in AFs. However, miR-21expression level markedly increased in MFs compared with AFs. The inverse correlation between miR-21and Pdcd4protein suggests that PDCD4may be a target of miR-21in AFs and MFs. 1.2Dual-luciferase reporter assay
To confirm that miR-21can directly bind to PDCD4, the dual-luciferase reporter assay was performed. Our results indicated that pre-miR-21significantly inhibited luciferase activity as compared with control treatments. Therefore, miR-21could directly bind to PDCD4.1.3The miR-21regulation on PDCD4protein expression
To investigate whether PDCD4is regulated by miR-21, western blot analysis was performed after miR-21overexpression and miR-21inhibition. Our results indicated that miR-21inhibitor increased but pre-miR-21decreased PDCD4protein level in AFs and MFs.
Therefore, the above study results proved that PDCD4is a direct target gene of miR-21in AFs and MFs.
2. The effects of PDCD4inhibition on miR-21-mediated proliferation in AFs and MFs
Knockdown of PDCD4by siRNA significantly increased proliferation in AFs and MFs as determined by EDU proliferation assay. miR-21inhibitor decreased the proliferation of AFs and MFs, however, PDCD4siRNA partly rescued the reduced cellular proliferation with miR-21inhibition. Our data showed that miR-21inhibitor+PDCD4siRNA group demonstrated higher proliferation activity compared with miR-21inhibitor group. The results suggest PDCD4not only plays an essential role in regulaing proliferation of AFs and MFs, but also directly participates in miR-21-mediated proliferation in AFs and MFs
3. The effects of PDCD4inhibition on miR-21-mediated apoptosis in AFs and MFs
Knockdown of PDCD4by siRNA decreased apoptosis as determined by caspase-3activity. MiR-21inhibitor increased the caspase-3activity of AFs and MFs, however, PDCD4siRNA partly alleviated the increase in apoptosis with mir-21inhibition. Our data showed that miR-21inhibitor+PDCD4siRNA group demonstrated a significantly lower proliferation activity compared with miR-21inhibitor group. The results suggest PDCD4is a regulator of apoptosis in AFs and MFs and directly participates in miR-21-mediated apoptosis in AFs and MFs.
4. The effects of PDCD4inhibition on JNK activity
Wstern blot analysis demonstrated that knockdown of PDCD4by siRNA significantly increased the JNK/c-Jun activity, the of p-JNK and p-c-Jun protein showed higher expression level compared with control siRNA.
5. The effects of miR-21on JNK activity
MiR-21inhibitor increased PDCD4expression in AFs and MFs, while decreased the level of p-JNK and p-c-Jun. In contrast, pre-miR-21decreased PDCD4expression, while increased the level of p-JNK and p-c-Jun. The results suggested that miR-21could enhance JNK activity while miR-21inhition could decrease JNK activity.
6. The effects of PDCD4inhibition on the regulation of miR-21in JNK activity
MiR-21inhibition decreased JNK activity in AFs and MFs, however, miR-21inhibitor+PDCD4siRNA group demonstrated a significantly higher JNK activity compared with miR-21inhibitor group. The results suggested that PDCD4not only affected JNK activity, but also affected the regulation of miR-21on JNK activity. It also suggests that JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.
Conclusions
1. PDCD4is a direct target of miR-21in AFs and MFs.
2. PDCD4could regulate proliferation and apoptosis of AFs and MFs, and directly participates in miR-21-mediated cellular effect.
3. JNK/c-Jun is a downstream signaling pathway of PDCD4in the miR-21-mediated cellular effect in AFs and MFs.
引文
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