P4Hα1与动脉粥样硬化斑块的关系及miR-124的调节作用
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摘要
1.背景
目前,心脑血管病仍然是全球引起致死致残的主要原因,而且心脑血管病的发病率仍然呈上升趋势。动脉粥样硬化斑块的不稳定性与斑块内胶原含量减少密切相关。增加斑块中胶原含量,对稳定斑块,减少斑块破裂,降低临床血管事件的发生具有重要的意义。
Ⅰ型、Ⅲ型胶原是动脉及粥样硬化斑块中主要的胶原类型,在斑块重塑过程中发挥着重要作用。成熟的Ⅰ型和Ⅲ型胶原,尤其是纤维帽中的胶原大分子可形成保护层,对抗斑块外的拉力及剪切力的应变,是防止斑块破裂的有效阻抗。除此之外,Ⅰ型和Ⅲ型胶原能抑制动脉平滑肌细胞的增生和迁移,减少基质金属蛋白酶与炎性因子释放,与斑块的稳定性密切相关。
P4Ha1胶原合成的关键酶。能通过胶原的翻译后修饰作用促进胶原的成熟与分泌。理论上,在前胶原表达丰富的前提下,过表达胶原合成的关键酶P4Ha1能够促进脯氨酸的羟化,从而增加成熟胶原的表达。
动脉粥样硬化斑块是脂质和胶原等成份在大中型动脉内中膜的异常聚集。它是一个渐进的病理过程。不同时期的动脉粥样硬化斑块有不同的组织学特征。斑块内的各种成分所占的比例也随斑块的不同阶段而有所增减。同样,动脉粥样硬化斑块发展过程中P4Ha1的表达也会有所不同,明确P4Hα1在动脉粥样硬化斑块发展过程中的表达规律,对理解动脉粥样硬化斑块病理过程及正确选择治疗干预的时间窗具有一定的意义。
研究表明,动脉粥样硬化斑块中胶原成分的减少与急性血管事件的发生密切相关。目前公认的易损斑块的组织学特征是纤维帽薄,胶原含量少,脂质含量多(>40%),炎性因子浸润,伴有血管的正性重构。文献表明,P4Hα1能有效的促进胶原的合成,动脉粥样硬化斑块中的Ⅰ型、Ⅲ型胶原能有效的稳定斑块,降低斑块的的易损指数。我们以往的许多研究措施可以间接地通过提高斑块中P4Ha1的表达而增加斑块内胶原的含量,提高斑块的稳定性。但是直接在动脉粥样硬化斑块中过表达P4Hαl观察斑块稳定性的研究,到目前为止尚无相关的报道。
此外,PTEN是近年来被发现的一个抑癌基因,具有抑癌作用,同时,还参与细胞的增殖、迁移和凋亡,与细胞体积大小及细胞稳态有密切关系。在血管病中,被称为平滑肌细胞增生的负性调节因子,能抑制早期斑块新生内膜的增生,并且胶原与PTEN的关系密切。但是,斑块中过表达P4Hα1,对PTEN有何影响,到目前为止,未见相关报道。
基于此,提出本研究的科学假说。在动脉粥样硬化斑块发展的早、晚不同期,即套管4周的斑块和套管12周的斑块中过表达P4Hα1,能增加动脉粥样硬化斑块中P4Hα1蛋白的表达,增加动脉粥样硬化斑块中Ⅰ型和Ⅲ型胶原含量,降低斑块的易损指数,发挥稳定斑块的作用。不同阶段斑块的组织学特征及形态学特征可能与过表达P4Hα1影响PTEN的表达有关。
研究目的
(1)探讨ApoE-/-小鼠动脉粥样硬化斑块发展过程中P4Hα1的表达变化规律。
(2)在ApoE-/-小鼠早、晚不同时期的动脉粥样硬化斑块中转染P4Hα1慢病毒,观察P4Ha1对斑块的组织学、形态学及易损性的影响。
(3)在分子生物学、组织学水平探讨过表达P4Hα1作用的可能机制。
2.材料和方法
2.1构建含目的基因的病毒载体
由上海inventrogen公司用化学合成的方法合成目的基因P4Ha1并携带增强型绿色荧光蛋白报告基因的慢病毒载体(Lenti-P4Hal),及仅携带增强型绿色荧光蛋白报告基因但无目标基因P4Hα1的阴性对照空病毒载体(Lenti-EGFP).
2.2构建小鼠动脉粥样硬化斑块模型
购买北京大学维通利华动物技术公司的8周龄ApoE-/-雄性小鼠,全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。
2.2.1构建小鼠主动脉根部自发粥样硬化斑块模型
ApoE-/-雄性小鼠(8周龄,80只),全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。随机分为4组：10周龄组、12周龄组、14周龄组、16周龄组；每组20只。高脂喂养至10周龄、12周龄、14周龄、16周龄,行安乐死处死动物,取材检测。见流程图1.
2.2.2构建小鼠颈总动粥样硬化斑块模型
为诱发颈总动脉斑块形成,8周龄ApoE-/-雄性小鼠行右侧颈总动脉套管术。基因转染干预后并分别于套管术后4周(套管4周的斑块)及12周(套管12周的斑块)取材,进行检测。
颈动脉套管和基因转染流程
(1)颈动脉套管：8周龄ApoE-/-雄性小鼠行右侧颈总动脉套管术,用0.08%戊巴比妥钠(40mg/Kg),术前行腹腔注射麻醉。将小鼠固定于手术台,用脱毛剂给予颈部脱毛处理后,碘酒消毒。沿颈正中线小心地切开皮肤。分离腺体与肌肉,暴露气管,将腺体上翻,清晰看到右侧颈总动脉,小心分离,避免损伤伴行的迷走神经。将预先剖开消毒处理的硅胶套管轻柔小心地套于颈总动脉上,丝线结扎固定套管。恢复组织结构层次后,缝合皮肤切口。
(2)基因转染流程：
①套管4周的斑块干预组：60只8周龄ApoE-/-雄性小鼠,右侧颈动脉套管术后2周,随机分为三个亚组：生理盐水组(Mock组),空病毒组(Lenti-EGFP组)和P4Ha1组(Lenti-P4Hal组),每组20只。分别小鼠尾静脉注射20ml生理盐水,空病毒(Lenti-EGFP,1.75x10.Tfu/ml,20u1),携带增强型绿色荧光蛋白报告基因的慢病毒载体(Lenti-P4Hal,1.75x10.Tfu/m1,20u1)的悬液。转染2周行安乐死后取材。见流程图2。
②套管12周的斑块干预组：60只右侧颈总动脉套管术后8周开始给予精神应激刺激,刺激持续到术后12周。精神应激刺激方法：将实验小鼠封闭于50m1大小、带有通气孔的塑料管中,给与强度为110dB的噪音刺激,噪音刺激间隔为5分钟,每次持续3秒,每天持续6小时,共刺激四周到实验结束。基因转染：套管术后10周,所有实验小鼠随机分为生理盐水组(Mock组),空病毒组(Lenti-EGFP组)和P4Hα1组(Lenti-P4Hal组),每组20只。分别小鼠尾静脉注射20u1生理盐水,空病毒(Lenti-EGFP,1.75x108Tfu,20ul),携带增强型绿色荧光蛋白报告基因的慢病毒载体(Lenti-P4Hal,1.75x108Tfu/ml,20ul)的悬液。转染2周后予以安乐死后取材生理盐水组。见流程图3。
2.3显微超声检测斑块：显微超声影像系统Vevo7700(RMV708探头)分别于套管术后2周、10周转染干预前和4周、12周安乐死前行套管侧颈总动脉动脉粥样硬化斑块的血管超声检测。
2.4体重及血液学指标的检测：分别于套管术后2周、10周转染干预前和4周、12周安乐死前检测小鼠体重；血脂水平；血清中羟脯氨酸水平。
2.5组织学检测：小心分离套管侧颈总动脉,常规冰冻切片后,分别行苏木素一伊红、天狼猩红、油红0染色。使用Image-Pro Plus6.0图像分析软件测量斑块面积、纤维帽面积、纤维帽厚度、纤维帽中胶原含量及脂质核面积并计算易损指数。
2.6免疫组化：
颈动脉组织冰冻切片漂洗后,常规行5%血清封闭,为检测不同指标,分别行相应的一抗二抗孵育：P4Ha1、MOMA-2,α-actin,Ⅰ型,Ⅲ型前胶原,Ⅰ型及Ⅲ型胶原,IL-6、TNFa、MCP-1、MMP9, MMP2, TIMP-1, TIMP-2。使用Image-Pro Plus6.0图像分析软件分别计算以上不同指标在相应斑块中的阳性染色面积(%)。
2.7蛋白印迹检测：
常规提取动脉斑块的组织蛋白,行凝胶电泳,转膜,孵育一抗二抗,发光。观察目的蛋白P4H α1、Ⅰ型及Ⅲ型胶原、MMP2、MMP9、PTEN的相对表达变化。Quantity-one图像分析软件分析计算三组中各种不同的目的蛋白的相对含量。
2.8实时定量检测：收集主动脉根部或右侧套管近心端颈总动脉各组斑块组织,常规提取总的mRNA,实时荧光定量RT-PCR反应检测各组斑块干预后P4H α1、Ⅰ型及Ⅲ型胶原mRNA的表达水平。
2.9明胶酶谱法：检测MMP2/MMP9的活性。常规方法分别制备10%分离胶、4%浓缩胶；在10%的分离胶中加入明胶储存液,至终浓度为0.1%；然后常规上样、电泳。取下电泳后的凝胶,放入100m1酶谱复性缓冲液(2.5%Triton X-100)中,室温条件下振荡30min;再在酶谱显影缓冲液中平衡30mim;将凝胶置于新鲜的酶谱显影缓冲液中,37℃过夜；凝胶染色、脱色；分析条带大小并测定灰度；计算相对量的变化。
3.结果
3.1实验动物一般情况
实验过程中生理盐水组中1只小鼠死于麻醉,套管12周的斑块P4Hα1组中1只小鼠死于转染后第13天,解剖后发现明显的肝硬化。余实验小鼠未观察到局部和全身性的不良反应,均完成实验。套管4周的斑块组中的3组,与套管12周的斑块组中3组的ApoE-/-小鼠的体重、及血脂指标：TC、TG、LDL-C和HDL-C的血清水平无显著统计学差异。而套管4周的斑块组与套管12周的斑块组中P4Hal组中血清羟脯氨酸水平显著高于生理盐水组与空病毒组。
3.2显微超声检测斑块形成
小鼠显微血管超声检测显示,套管2周套管侧颈动脉内膜增厚,4周见小斑块形成；10周、12周套管侧的颈总动脉有明显斑块形成。而对侧未套管的颈总动脉管腔光滑。
3.3动脉粥样硬化斑块的转染效率的检测
慢病毒转染预实验于慢病毒转染7天、10天、14天、21天、28天、35天后,分别取材,检测粥样硬化斑块内绿色荧光表达。慢病毒转染14天P4Hα1组和空病毒组动脉粥样硬化斑块内GFP表达率最高(70%),而生理盐水组的颈总动脉粥样硬化斑块内无GFP表达。证实体内转染有效。所以取慢病毒转染2周取材作为本实验的慢病毒转染干预时间点。
3.4过表达P4Ha1对套管4周的斑块组织病理学的影响
(1)过表达P4Hα1对套管4周的斑块中P4Hα1表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：三组比较,P4Hα1组P4Hαl的mRNA和蛋白表达显著增加(P<0.05)。
(2)过表达P4Hα1对套管4周的斑块中内胶原表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：三组比较,Ⅰ型和Ⅲ型胶原的mRNA水平无显著性差异(P>0.05)；但P4Hα1组Ⅰ型和Ⅲ型胶原的蛋白表达显著增加(P<0.05)。
(3)过表达P4Hα1对套管4周的斑块组份和易损指数的影响
病理学染色显示：三组比较,P4Hα1组胶原含量、平滑肌细胞含量显著增加(P<0.05)；脂质含量三组间无显著性差异(P>0.05)：P4Hα1组巨噬细胞含量显著降低(P<0.05)；易损指数显著显著降低(P<0.05)。
(4)过表达P4HαI对套管4周的斑块纤维帽的影响
HE及天狼星红染色显示：三组比较,P4Hα1组斑块平均纤维帽厚度显著性增厚(P<0.05)；纤维帽面积显著增大(P<0.05)；纤维帽中胶原的含量显著增多(P<0.05)。
(5)过表达P4Hα1对套管4周的斑块形态学的影响
HE及油红0染色显示：三组比较,P4Hα1组斑块的面积及主动脉表面AS病变范围显著增大(P<0.05)。
(6)过表达P4Hα1对套管4周的斑块中PTEN的表达影响
免疫组化、Western blot显示,P4Hα1组与空病毒组和生理盐水组比较PTEN的阳性染色面积,及PTEN的蛋白相对表达量显著性减少(P<0.05)。
3.5过表达P4Hα1对套管12周的斑块组织病理学的影响
(1)过表达P4Hα1对套管12周的斑块中P4Hα1表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：P4Hα1组与空病毒组和生理盐水组比较P4Hα1的mRNA和蛋白表达显著增加(P<0.05)。
(2)P4H α1过表达对套管12周的斑块内胶原表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：P4Hα1组与空病毒组和生理盐水组比较Ⅰ型和Ⅲ型胶原的蛋白表达显著增加(P<0.05)；三组间Ⅰ型和Ⅲ型前胶原(I型和III型胶原)的mRNA的表达,及Ⅰ型和Ⅲ型前胶原的蛋白表达均无显著性差异(P>0.05)。
(3)过表达P4Hα1对套管12周的斑块组份和易损指数的影响
病理学染色显示：
①P4Hα1组与空病毒组和生理盐水组比较胶原含量显著增加(P<0.05)；
②P4Hα1组与空病毒组和生理盐水组比较平滑肌细胞含量显著增加(P<0.05)；
③P4Hα1组与空病毒组和生理盐水组比较脂质含量三组间无显著性差异(P>0.05)。
④P4Hα1组与空病毒组和生理盐水组比较巨噬细胞含量显著降低(P<0.05)；
⑤P4Hα1组与空病毒组和生理盐水组比较易损指数显著显著降低(P<0.05)；
(4)过表达P4HαI对套管12周的斑块内纤维帽的影响
HE及天狼星红染色显示：
①P4Hα1组与空病毒组和生理盐水组比较平均纤维帽厚度显著性增厚(P<0.05)；
②P4Hα1组与空病毒组和生理盐水组比较斑块的纤维帽面积显著增大(P<0.05)；
③P4Hα1组与空病毒组和生理盐水组比较斑块的纤维帽中胶原的含量显著性增大(P<0.05)；
(5)过表达P4Hα1对套管12周的斑块形态学的影响
①P4Hα1组与空病毒组和生理盐水组比较斑块的面积三组之间无显著地差异(P>0.05)；
②P4Hα1组与空病毒组和生理盐水组比较主动脉表面AS病变范围三组之间无显著地差异(P>0.05)。
(6)过表达P4Hα1对套管12周的斑块内炎性因子的影响
斑块内炎性因子IL-6、TNF-a和MCP-I的免疫组化染色显示：
①P4Hα1组与空病毒组和生理盐水组比较斑块内工L-6阳性染色面积显著性减小(P<0.05)；
②P4Hα1组与空病毒组和生理盐水组比较斑块内TNF-a阳性染色面积显著性减小(P<0.05)；
③P4Hα1组与空病毒组和生理盐水组比较斑块内MCP-1阳性染色面积显著性减小(P<0.05)；
(7)过表达P4Hα1对套管12周的斑块中基质金属蛋白酶表达的影响
斑块内MMP9、MMP2、TIMP-1、TIMP-2的免疫组化染色及Western blot显示：
①P4Hα1组与空病毒组和生理盐水组比较斑块内MMP9、MMP2的阳性染色面积显著性减小(P<0.05)；相反,TIMP-1.TIMP-2的阳性染色面积显著性增大(P<0.05)；
②Western blot显示,P4Hα1组与空病毒组和生理盐水组比较MMP9、MMP2的蛋白相对表达量显著性减少(P<0.05)；
③基质金属蛋白酶原位酶谱法检测显示：P4Ha1组与空病毒组和生理盐水组比较MMP9、MMP2活性显著性降低(P<0.05)；
(8)过表达P4Ha1对套管12周的斑块中PTEN的表达影响
①免疫组化显示,三组中PTEN的阳性染色面积无统计学差异(P>0.05)。
②Western blot显示出相同的趋势：三组中PTEN的蛋白表达无统计学差异(P>0.05)。
4.结论
(1)在ApoE-/-小鼠脉粥样硬化斑块发展过程中,P4Hα1的表达逐渐减少。
(2)在ApoE-/-小鼠套管4周的斑块及套管12周的斑块中,过表达P4Hα1均能升高斑块中胶原的含量,降低易损指数,但在套管4周的斑块模型中过表达P4Hα1却促使斑块增大和主动脉表面斑块总负荷的增多.
(3)过表达P4Hα1促进套管4周的斑块的增大及主动脉表面斑块总负荷的增多,与过表达P4Hα1抑制了PTEN的表达有关。
1.背景
MicroRNA (miRNA)是近年来发现的一类内源性的非编码单链小RNA分子,长度约为18-24个核苷酸,能够特异地结合于靶基因mRNA的3’端非翻译区(3’-UTR),干扰靶基因的翻译或促进靶基因mRNA的降解。从而抑制靶蛋白在体内的表达。MicroRNA与其靶基因之间构成网络调控关系,每个MicroRNA可以调控多个靶基因,而多个miRNAs也可以调节同一个靶基因。据推测,miRNA调节着人体内约60%以上的基因,在人体细胞的发育、增殖、分化和凋亡等生理及病理过程中发挥着重要作用。
研究表明,胶原的减少与动脉粥样硬化斑块的不稳定性有关。P4Ha1是胶原合成的限速酶,能使前胶原(procollagen)肽链上的脯氨酸羟化成为羟基脯氨酸,促进胶原的成熟与分泌。本实验室以往的研究表明,炎性因子如IL-6通过MAPK和c-Jun分子通路,下调P4Hal的表达,降低动脉粥样硬化斑块的稳定性；TNFa通过影响转录因子Nono的甲基化等表观遗传学的调控而降低P4Hal的表达。相反,脂连素则可以通过MAPK-AP1分子传导通路上调P4Hal的表达,增加动脉粥样硬化斑块的稳定性。但是动脉粥样硬化斑块中MicroRNA分子与调节胶原合成的关键酶P4Ha1的关系如何目前尚无相关的研究报道。
MicroRNA124(miR-124)最早在神经系统肿瘤-神经胶质瘤中发现其参与肿瘤的病理调控过程。以后,又发现其在动脉粥样硬化性腹主动脉瘤中高表达。提示miR-124可能与动脉重构过程中胶原的代谢有关。Micro.org.等多个MicroRNA应用软件均推测miR-124能特异的与P4Hα1的3’端非翻译区(3’-UTR)结合,并且结合能低,P4Ha1是miR-124理论上的靶基因。那么miR-124是否在动脉粥样硬化斑块中发挥作用?是否与斑块的胶原代谢有关?能否调控P4Ha1,能否进一步影响胶原的合成?到目前为止尚无相关的报道。
本研究提出如下假说：
(1)miR-124在应激条件下的动脉粥样硬化斑块与非应激条件下的斑块中的表达不同,在应激条件下的粥样硬化斑块中表达上调。
(2)P4Ha1是miR-124特定的靶基因。在人主动脉平滑肌细胞中上调miR-124能特异的抑制P4Hal蛋白的表达,并进一步降低Ⅰ、Ⅲ型胶原的合成。相反,抑制主动脉平滑肌细胞中miR-124的表达,促进Ⅰ、Ⅲ型胶原的合成。
研究目的
(1)观察ApoE-/-小鼠的动脉粥样硬化斑块应激组与非应激组中MicroRNA124(miR-124)与P4Ha1表达变化关系。
(2)验证miR-124能特异地结合于P4Hα13’端非翻译区,明确P4Ha1是miR-124作用靶基因的分子机制。
(3)探讨miR一124是否能调节P4Hal,进而影响主动脉平滑肌细胞中胶原的成熟和表达。
2.材料和方法
(1)构建动脉粥样硬化斑块模型：动物来源和颈动脉套管术同论文一
60只套管后8周的ApoE-/-小鼠,随机分成两组：
①应激斑块组：颈动脉套管术后给予脂多糖与应激刺激以诱发斑块中的炎症反应,应激刺激的方法同论文一。
②非应激组：不给予脂多糖与应激刺激,仅持续高脂喂养。见流程图1。
(2)设计与合成miR-124特异性引物
miR-124特异性引物的设计与合成由丹麦Exiqon公司提供(本公司不提供引物序列)。
(3)设计与合成miR-24特异性引物模拟物及抑制物
使用化学合成的方法合成miR-124mimics及miR-124inhibitors。由上海吉玛生物技术有限公司提供。
(4)构建含有P4Hal-3'-UTR的荧光素酶报告基因质粒
分别构建含有野生型P4Hα1基因的3’端非编码区序列3'-UTR(P4Hal-3'-UTR-WT)、突变型P4Ha1基因的3’端非编码区序列3'-UTR(P4Hal-3'-UTR-MUT)的荧光素酶报告基因质粒(pGL3)：PGL3-P4H al-3'-UTR-WT和pGL3-P4H a1-3'-UTR-MUT。通过脂质体2000和miR-124mimic共转染人主动脉平滑肌细胞,检测荧光素酶荧光表达强度。验证软件预测结果。明确miR-124与P4Hα1基因的3’端非编码区特异的碱基互补结合序列。探讨miR-124调控的下游靶基因P4Hα1的分子机制。
(5)miRNA提取和实时荧光定量qRT-PCR
提出颈动脉斑块及人主动脉平滑肌细胞中总的MicroRNAs,通过qRT-PCR检测miR-124在两组斑块中及主动脉平滑肌细胞不同干预组中的相对表达量。
(6)人主动脉平滑肌细胞培养
原代人主动脉平滑肌细胞,购自美国Sciencell,应用VSMC培养液常规培养至80-90%融合后,传代再培养。第4-8代平滑肌细胞用于本实验。
(7)人主动脉平滑肌细胞培养的转染与刺激
①miRNAs转染人主动脉平滑肌细胞
应用脂质体2000进行miRNA (miR-con、miR-124mimics或miR-124inhibitor)转染。具体步骤按GenePORTER2的说明书进行转染操作,转染后37℃,5%CO2培养箱中培养6-8h,更换为常规的细胞培养液,继续培养24-48小时,进行下一步检测。
②TNFa刺激人主动脉平滑肌细胞
六孔板中平滑肌细胞的密度达到80%-90%时,按100ng8mlTNFa的剂量浓度,将TNFa加入到细胞培养基刺激平滑肌细胞,37℃,5%CO2温箱中培养24小时后进行下一步实验。
(8)生物信息软件分析
应用miRanda, TargetScan, RNAhybrid and PicTar等著名的MicroRNA分析预测软件,确定P4Hα1是miR-124用的靶基因。并且miR-124是调控P4Ha1的MicroRNA分子之一。进一步确定miR-124与P4Ha13’非转录区(3’-UTR)特异结合的互补结合位点。
(9)免疫印迹(Western Blot)检测
同论文一
(10)细胞免疫荧光
人主动脉平滑肌细胞干预后,4%甲醛固定,PBS漂洗,常规行5%兔血清封闭,分别行抗P4Hal的一抗及带荧光标记的二抗孵育(避光),DAPI染核后,避光拍照,使用Image-Pro Plus6.0图像分析特异荧光的表达含量。
3.结果
3.1P4Ha1在斑块中的表达
P4Hα1在应激组斑块中的蛋白表达显著低于非应激组(P<0.05)。
3.2miR-124在斑块中的表达
miR-124在应激组斑块中的表达显著高于非应激组(P<0.05)。
3.3P4Hα1在人主动脉平滑肌细胞中的表达
P4Hα1在TNFa刺激组中的蛋白表达显著低于无TNFa刺激组(P<0.05)。
3.4mi R-124在人主动脉平滑肌细胞中的表达
qRT-PCR显示,miR-124在TNFa刺激的人主动脉平滑肌细胞中的表达是无TNFa刺激组的2.57倍(U6标准化后)。两组间的差异有显著性(P<0.05)。
3.5生物信息软件分析结果
P4Hal (NM_000917)是miR-124的理论靶基因,并且结合能低。miR-124与P4Ha1的结合位点位于P4Ha1基因的3’端非翻译区(3,-UTR)的第834-841个碱基。此碱基序列位点在生物界的各物种之间有高度保守性。
3.6miR-124对P4Ha1表达的影响
miR-124mimics转染人主动脉平滑肌细胞48小时后,P4Ha1的蛋白表达量较转染前显著降低(P<0.05)。miR-124inhibitors转染人主动脉平滑肌细胞后,TNFa再刺激主动脉平滑肌细胞24小时后的P4Ha1的蛋白表达量较无miR-124inhibitors预转染组上调(P<0.05)。
3.7miR-124通过与P4Ha1的3’端非翻译区(3’-UTR)的结合位点的特异结合作用
miR-124mimics与含有野生型P4Hαl基因的3’端非编码区序列P4Hα13’UTR(P4Hα1-WT)荧光素酶报告质粒瞬时共转染人主动脉平滑肌细胞48小时后,荧光素荧光表达显著地被抑制(P<0.05)。
3.8miR-124对胶原的合成与成熟的影响
miR-124mimics转染人主动脉平滑肌细胞48小时后,胶原Ⅰ和胶原Ⅲ的蛋白表达量较转染前显著减少(P<0.05)。miR-124inhibitors预转染能使TNFa诱导人主动脉平滑肌细胞降低胶原Ⅰ和胶原Ⅲ的作用部分地消失,差异具有显著性(P<0.05)。
4.结论
(1)在应激条件下,动脉粥样硬化斑块中miR-124表达显著上调,P4Hα1的表达显著下调。
(2)miR-124与P4Hα1的3’端非翻译区(3’-UTR)特异的结合位点结合,抑制P4Hα1蛋白的表达,进而影响Ⅰ型与Ⅲ型胶原的合成过程。
1. Backgroud
The rapture of atherosclerotic plaque is responsible for the clinical acute events of vascular, such as stroke and myocardial infarction, which is the most common cause of mortality and morbidity in developed countries. To stabilize atherosclerotic plaques is an essential strategy to reduce the clinical acute events of vascular.
Collagen is one kind important composition in atherosclerotic plaque. Type I and type III collagen are the predominant collagen in atherosclerotic plaques and arterial wall. The vulnerable plaque is characterized by less collagen and more lipids. Otherwise, the stable one with more collagen and less lipids.
Prolyl-4-hydroxylase (P4H) is a key intracellular enzyme involved in the synthesis of collagens. It catalyzes the proline residues repeating X-Pro-Gly triplets located in the procollagens to format hydroxyproline. It is essential for collagen posttranslational modification, and this modification facilitate folding the polypeptide procollagen chains into stable triple-helical molecules.
P4H is composed of a and P subunits; The a subunit is a rate-limiting enzyme.The active enzyme is a tetramer composed of two pairs of non-identical subunits (a2p2). P4Hal is the most important form of all kinds of P4Hs. It is responsible for the posttranslational modifications of procollagens. Studies demonstrated that less P4Hal in plaques is closely related to destability of plaques. So to increase the expression of P4Hal in plaques is a good strategy to avoid the plaque rupture.
In theory, overexpression of P4Hal can make over production of collagen under the condition of sufficient procollagen. But whether or not overexpression of P4Ha1influences the collagens and other components in atherosclerotic plaques, and whether or not overexpression of P4Hal influences the vulnerable index are unknown.
Collagen metabolism is composed of collagen synthesis and collagen degradation modulated by extracellular matrix metallopeptidase (MMPs), along with their endogenous tissue inhibitors (TIMPs) constituting an important system for regulating extracellular matrix (ECM), especially, collagen turnover. But whether or not overexpression of P4Hal influences the MMPs and TIMPs is also unknown.
Guided by above theories, We put forward such scientific hypothesis as:
(1) The expession of P4Hal is developmental decrease in the pathological course of atherosclerotic plaques in ApoE-/-mice.
(2) Overexpression of P4Ha1can increase the product of Type I and type III collagen both in evolving plaques and in established ones.
(3) Overexpression of P4Hal can increase the stability of evolving plaques and established ones. But there may be some different influence in evolving plaques from established ones.
Objectives
(1) To investigate the developmental expession of P4Hal in the pathological course of atherosclerotic plaques of ApoE-/-mice.
(2) To investigate effects of overexpression of P4H a1on the collagen metabolism both in evolving plaques and in established ones of ApoE-/-mice.
(3) To elucidate the effect of overexpression of P4Hal on the stability of evolving plaques and established ones of ApoE-/-mice.
(4) Clarify the different influence of overexpression of P4Hal on evolving plaques from established ones and explore the mechanism under the appearance.
2. Material and Methods
(1) Plasmids and Lentivirus-P4Ha1Construction The Lentivirus-P4Haland empty Lentivirus (control) were constructed by Inventrogen, Shang hai.
(2) Animal Model
Ethics Statement
The animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (document No55,2001) and was approved by Animal Care Committee of Shandong University.
Spontaneous plaque of aortic root
Male apolipoprotein E-deficient mice (n=80,8weeks old, weighing25-30g) were obtained from Peking University (Beijing, China), and were divided into randomly4groups:10weeks old group;12weeks old group;14weeks old group and16weeks old group (n=20). All of them had high-fat diet. At the end of10,12,14,16week, they were killed kindly. Histopathology-Immunohistochemistry and Western, Blot, were carried on for detecting the expression of P4Hal in Spontaneous plaque of aortic root.
Carotid Collar Placement and Transgenic therapy
Male apolipoprotein E-deficient mice (n=120,8weeks old, weighing25-30g) m were obtained from Peking University (Beijing, China). Atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of mice. The Lenti-EGFP groups were used as controls.120mice were divided randomly into two groups:established plaques and early ones.
①Evolving Plaques:60mice (male,8weeks old) were induced carotid atherosclerotic lesions by placement of a restraint perivascular silica collar as above. After surgery2weeks,60were divided into randomly3groups:saline group; Lenti-EGFP group and Lenti-P4H a1group. Three group mice were given intravenously saline, Lentivirus-P4Ha1and empty Lentivirus transgenic therapy.2weeks later, all mice were given euthanasia for research.
②Established plaques:60mice (male,8weeks old) were induced carotid atherosclerotic lesions by placement of a restraint perivascular silica collar, and fed with high-fat diet combined stimulation of mental to create a vulnerable plaque model. After surgery10weeks,60were divided into randomly3groups:saline group; Lenti-EGFP group and Lenti-P4Hal group. Three group mice were also given intravenously saline, Lentivirus-P4Hal and empty Lentivirus transgenic therapy.2weeks later, all mice were given euthanasia for research.
(3) Micro-ultrasonography
Vevo770system was used to measure the baseline ultrasonography parameters of plaques
(4) Blood samples and Biological Measurements
Blood samples were taken to detect:total cholesterol? high-density lipoprotein cholesterol,triglycerides,low-density lipoprotein cholesterol, and hydroxyproline.Weight was also detected before surgery and euthanasia.
(5) Histopathology and Immunohistochemistry
Cryosections were stained with hematoxylin and eosin (H&E), oil O and picosirius red as previous.For immunohistochemistry, Sections were incubated with different primary antibodies and second antibodies for immunohistochemistry.
(6) Western Blot
Proteins were extracted from carotid artery specimens, separated on14%SDS-PAGE and transferred to nitrocellulose membranes. After incubation with primary antibodies and second antibodies and exposure, protein expression was determined.
(7) RT-PCR
Carotid artery specimens were pooled for RNA isolation with TriZol to detect the mRNA of P4Ha1, collagen I (procollagen I) and collagen III (procollagen III).
(8) Gelatin zymography assay
MMP-9and MMP-2activity was detected using the gelatin zymography assay. The tissue proteins were extracted using lysis buffer. The protein concentration was determined. The sample was electrophoresed on SDS-polyacrylamide gels copolymerized with1%gelatin. After electrophoresis, the gels were washed twice in2.5%Triton X-100and incubated in1%Triton X-100,5mM CaC12and5jiM ZnC12 (pH7.5) at37℃for36h. The gels were stained with0.1%Coomassie blue R250, and destained in10%isopropanol and10%acetic acid in H2O. MMP-9/MMP-2was detected as transparent bands on the blue background of a Coomassie blue-stained gel.
4. Results
One mouse died of liver fibrosis, and others finished this study.
4.1Blood samples and Biological Measurements
There was no significant difference in serum levels of TC, TG, LDL-C and HDL-C among the3groups of ApoE-/-mice both in established plaque group and in evolving plaque group. But the hydroxyproline serum levels was significant higher in lenti-P4Hα1group than that of mock group and lenti-EGFP group both in established plaque group and in evolving plaque group (P<0.05).
4.2Identification of Plaque
Micro-ultrasonography was used to evaluate the stage of the plaques. Small atherosclerotic plaques could be seen in the right common carotid artery in4weeks after collar surgery. And there were obvious plaques in10weeks and12weeks after collar surgery, otherwise, no plaque was detected in the left common carotid artery of animals.
4.3Transfection Efficiency Assays
GFP expression(about70%) was found in atherogenic plaque of carotid arteries in Lenti-P4Hα1and Lenti-EGFP groups. The mRNA of P4Hα1increased about five fold than Lenti-EGFP group and PBS group. All demonstrated efficient transfection.
4.4Effect of Lenti-P4Hα1transgenic therapy on P4Hal protein expression
①Evolving Plaque group:Western blot and Immunohistochemistry showed that the expression of P4Hα1protein in lenti-P4Hα1group was significant higher than saline and Lenti-EGFP groups (P<0.05).
②Established plaque group:consistent with the result of established plaque group (P<0.05).
4.5Effect of overexpression of P4Hal on procollagen Ⅰ, collagen Ⅰ and
procollagen Ⅲ, collagen Ⅲ in plaques
①Evolving Plaque group:Western blot and Immunohistochemistry showed that there was no significant difference in the expression of procollagen Ⅰ and procollagen Ⅲ in3groups, but the collagen Ⅰ and collagen Ⅲ protien expression in Lenti-P4Hal was significant higher than saline and Lenti-EGFP groups (P<0.05).
②Established plaque group:consistent with the result of evolving plaque group (P<0.05).
4.6Effect of overexpression of P4Hal on carotid plaque composition and stability
③Evolving Plaque group:Histological and immunohistochemical staining of carotid plaque in3groups were significantly different. Collagen content was increased significantly in the Lenti-P4H a1group; And VSMC content in the atherosclerotic lesion area, detected by a-actin staining, was also significantly increased (P<0.05). But the content of macrophages was significantly decreased in Lenti-P4Hal group compared with saline group and lenti-EGFP control groups (P<0.05). And there was no significant difference in lipid deposition among3groups.
②) Established plaque group:consistent with the result of evolving plaque group (P<0.05).
4.7Effect of overexpression of P4Hal on the morphology changes of the carotid plaque
①Evolving plaque group:The fibrous cap area, fibrous cap thickness and collagen content in fibrous cap were higher significantly in lenti-P4Hal group than control groups (P<0.05). And the vulnerability index was lower significantly than control groups. The plaque area was significant lager and the total plaque burden was significant higher in lenti-P4Ha1group, compared with control groups (P<0.05).
②Established plaque group:It was inconsistent with the result of evolving plaque group. The plaque area and total plaque burden in lenti-P4Ha1group had no significant difference from control groups (P<0.05), although in lenti-P4Hal group, the fibrous cap area, fibrous cap thickness and collagen content in fibrous cap were higher significantly than control groups (P<0.05). And the vulnerability index was also lower significantly than control groups. This was consistent with the result of evolving plaque group group,
4.8Effect of overexpression of P4Hal on inflammatory markers and on MMPs and TIMPs in established plaque group
The protein expression levels of IL-6, TNF-a, MCP-l,MMP-2, MMP-9in the carotid plaque of in lenti-P4Hal group were lower significantly compared with control groups (P<0.05), based on quantitative analysis of immunohistochemical staining, immunohistochemical staining. And the relative activity of MMP9, MMP2also decreased significantly in lenti-P4Hal group.
4.9Effect of overexpression of P4Hal on PTEN expression
〦volving Plaque group:Western blot and Immunohistochemistry showed that PTEN expression in lenti-P4Ha1group decreased significantly, compared with control groups (P<0.05).
②Established plaque group:It was different from established plaque group: there was no significant difference on PTEN expression among three groups (P>0.05).
5. Conclusion
P4Hal overexpression of can increase collagen Ⅰ and Ⅲ significantly in both evolving plaques and in established ones. It can increases the stability of established plaques but accelerates intimal hyperplasia and the total lesion area (%) in evolving plaques of Apolipoprotein E-knockout mice.
1. Background
miRNAs are short ribonucleic acid (RNA) molecules, approximately18-24nucleotide RNAs that modulate gene expression by base pairing with the3'-untranslated regions (3'-UTRs) of miRNAs to repress protein expression by inhibiting translation or promoting degradation of miRNAs. Untill now, several hundreds of miRNAs have been identified. They involved in cell proliferation, apoptosis, differentiation, development and metabolism. Studies on miRNAs show that about60%of mammalian genes are targeted by special miRNAs. One miRNA can target one or more genes.
P4H a1is the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Studies show P4Hal are losely related to the vulnerable atherosclerosis plaque. Our previous studies demonstrate that inflammatory cytokines, interleukin6, can downregulate P4H a1via a mitogen-activated protein kinase and c-Jun pathway to destabilize atherosclerotic plaques. And TNFa can also downregulate P4Hal via affecting the histone (H)4lysine12acetylation. Otherwise, Adiponectin can upregulate prolyl-4-hydroxylase al expression human aortic smooth muscle cells by regulating ERK1/2and Spl. But how miRNAs regulate P4Hal in atherosclerotic plaque is unknown till now.
miR-124is a brain-enriched microRNA that has been shown to be down-regulated in glioma and medulloblastoma and plays a crucial role in brain tumor progression. Keiwa Kin observed that miR-124was up-regulated in the tissue of atherosclerotic abdominal aortic aneurysm. It suggests that miR-124was related to the collagen metabolism of aortic remolding. But whether or not miR-124involves the progression of atherosclerotic plaque is unclear.
P4Hal is miR-124's target gene by the famous softwears, such as:MIRBASE, STARBASE, TARBASE, TARGETSCAN. But whether or not miR-124can bind to the3'-untranslated region (3'-UTR) of P4Hal gene mRNA to regulate the translation of P4Hal is unknown in fact. Guided by above theories, We put forward such scientific hypothesis as:
(1) The expression of miR-124is different in stressed plaques from unstressed plaques. And miR-124is up-regulated probably in stressed plaques.
(2) miR-124can bind to the wild3'-untranslated region (3'-UTR) of P4Hal gene mRNA but can not bind to the mutation one.
(3) P4Hal is miR-124's target gene. In human aortic smooth muscle cells, miR-124mimics can inhibit P4Hal expression and down-regulate "collagen I, collagen Ⅲ. Otherwise, miR-124inhibitors can up regulate P4Hal and increase the production of collagen Ⅰ, collagen Ⅲ.
Aims:
(1) To identify miR-124alteration in atherosclerotic plaques and the different expession of miR-124between stressed plaques and unstressed ones.
(2)To clarify the relationship between miR-124and P4Hal.
(3)To clarify the mechanism on how miR-124regulate P4Hal and to identify miR-124's special binding site on3'-UTR of P4Hal.
(4) To clarify the relationship between miR-124and collagen Ⅰ and collagen3.
2. Materials and methods
(1) Animals
Male apolipoprotein E-deficient mice (n=60,8weeks old, weighing25-30g) were obtained from Peking University (Beijing, China).
60mice were divided randomly into2groups:unstressed group (n=30) and stressed group (n=30). Carotid collar placement and mental stress protocol is consistent with paper1.
(2) Carotid artery isolation
After12weeks, the carotid artery of E-deficient mice, restricted by collar placement, were isolated carefully to store at-80℃refrigerator for RT-PCR and Western blot detected.
(3) Computational targeted gene predictions
miRanda, TargetScan, RNAhybrid and PicTar etal..
(4) Plasmids, mutagenesis, transfection and luciferase reporter assays
The3'-UTR of the mouseP4Hal gene was amplified. All constructs were verified by sequencing. The wild and mutated3'-UTR of theP4Hal gene were obtained from Jinan Biolabs. The luciferase reporter vector pGL3-basic vector [luc2] was also from Jinan Biolabs.
(5) miRNA mimics, miRNA inhibitors
miRNA mimics or miRNA inhibitors were transfected using Lipofectamine2000(Invitrogen). Luciferase activity was detecked by using Dual-lo Luciferase Reporter Assay Kit (Promega) on a FLUOstar Omega luminometer (BMG Labtech, Cary, NC), after48hours of transfections.
(6) HASMCs culture
HASMCs were obtained from ScienCell. They were cultured with5%CO2at37℃in smooth muscle cell medium (ScienCell, USA). HASMCs (passages4to8) were used.
(7) RNA isolation and qRT-PCR
Total RNA was isolated from carotid artery or HASMCs using TRIzol, and cDNA was synthesized according to manufacturer's instructions (Invitrogen, San Diego, CA).
(8) Western blot analysis
Western blots were performe d using anti-P4HAl, anti-COLlA1, anti-Collagen3, and anti-β-actin antibodies (Santa Cruz Biotechnology) following our published method[10].
(9) Immunofluorescence
HASMCs were grown on chamber slides and rmiR-124transfection was carried out. Forty-eight hours following the transfection, HASMCs were fixed in4%paraformaldehyde for15min at room temperature and was rised with PBS three times.5%rabbit serum was used to block the cells, following by incubation with anti-P4H ol antibody overnight at4℃.
3. Results
3.1miR-124is upregulated in stressed plaques and in TNFa treated HVSMCs
(1) The mRNA was isolated from unstressed carotid plaques and stressed carotid plaques in vivol. The expression of miR-124was determined by real-time; PCR. The relative levels of miR-124expression in stressed plaques were significantly increased (P<0.05).
(2) The mRNA was isolated from TNFa treated HVSMCs and from control HVSMCs in vitrol. The expression of miR-124was also determined by real-time PCR. The relative levels of miR-124expression in TNFa treated HVSMCs were significantly higher (P<0.05), compared with control HVSMCs.
3.2miR-124is predicted to target P4Hal gene
miRanda, TargetScan, RNAhybrid and PicTar et al., the famous computational approaches for the identification of mammalian microRNA targets, identified that Homo sapiens prolyl4-hydroxylase, alpha polypeptide I (P4Hal), transcript variant1, mRNA. NM_000917is miR-124's targe gene and miR-124is P4Hal's target mirRNA.
3.3miR-124targetsP4Hal
The protein expression ofP4Hal was significantly reduced after miR-124mimics transfection (P<0.05). Otherwise, miR-124inhibitor can make the reduction of P4Hal induced by TNFa significantly abrogated (P<0.05).
3.4miR-124downregulates the expression of P4Hal via targeting the
3'-UTR of P4Hal mRNA
Transient co-transfection of miR-124mimics with luciferase reporter plasmids resulted in a significant repression of luciferase reporter gene expression in HVSMCs, whereas co-transfection of HVSMCs with negative control miRNA or mutations did not have any effect on the expression of luciferase.
3.5Inhibition of miR-124increases collagen maturation.
To addresse whether miR-124overexpression had an inhibitory effect on collagen protein expression. After48hours transfection of miR-124mimics in HVSMCs, collagen Ⅰ and Ⅲ were all significantly decreased (P<0.05) as expected. Whereas, miR-124inhibitor can make the reduction of collagen Ⅰ and Ⅲ induced by TNFa significantly abrogated (P<0.05).
4. Conclusions
miR-124involved in and play important roles in atherosclerotic plaques. It is upregulated in stressed plaques and TNFa induced HVSMCs. miR-124can regulate collagen maturation via targeting its target gene, the P4Ha1mRNA.
目前,心脑血管病仍然是全球引起致死致残的主要原因,而且心脑血管病的发病率仍然呈上升趋势。动脉粥样硬化斑块的不稳定性与斑块内胶原含量减少密切相关。增加斑块中胶原含量,对稳定斑块,减少斑块破裂,降低临床血管事件的发生具有重要的意义。
Ⅰ型、Ⅲ型胶原是动脉及粥样硬化斑块中主要的胶原类型,在斑块重塑过程中发挥着重要作用。成熟的Ⅰ型和Ⅲ型胶原,尤其是纤维帽中的胶原大分子可形成保护层,对抗斑块外的拉力及剪切力的应变,是防止斑块破裂的有效阻抗。除此之外,Ⅰ型和Ⅲ型胶原能抑制动脉平滑肌细胞的增生和迁移,减少基质金属蛋白酶与炎性因子释放,与斑块的稳定性密切相关。
P4Ha1胶原合成的关键酶。能通过胶原的翻译后修饰作用促进胶原的成熟与分泌。理论上,在前胶原表达丰富的前提下,过表达胶原合成的关键酶P4Ha1能够促进脯氨酸的羟化,从而增加成熟胶原的表达。
动脉粥样硬化斑块是脂质和胶原等成份在大中型动脉内中膜的异常聚集。它是一个渐进的病理过程。不同时期的动脉粥样硬化斑块有不同的组织学特征。斑块内的各种成分所占的比例也随斑块的不同阶段而有所增减。同样,动脉粥样硬化斑块发展过程中P4Ha1的表达也会有所不同,明确P4Hα1在动脉粥样硬化斑块发展过程中的表达规律,对理解动脉粥样硬化斑块病理过程及正确选择治疗干预的时间窗具有一定的意义。
研究表明,动脉粥样硬化斑块中胶原成分的减少与急性血管事件的发生密切相关。目前公认的易损斑块的组织学特征是纤维帽薄,胶原含量少,脂质含量多(>40%),炎性因子浸润,伴有血管的正性重构。文献表明,P4Hα1能有效的促进胶原的合成,动脉粥样硬化斑块中的Ⅰ型、Ⅲ型胶原能有效的稳定斑块,降低斑块的的易损指数。我们以往的许多研究措施可以间接地通过提高斑块中P4Ha1的表达而增加斑块内胶原的含量,提高斑块的稳定性。但是直接在动脉粥样硬化斑块中过表达P4Hαl观察斑块稳定性的研究,到目前为止尚无相关的报道。
此外,PTEN是近年来被发现的一个抑癌基因,具有抑癌作用,同时,还参与细胞的增殖、迁移和凋亡,与细胞体积大小及细胞稳态有密切关系。在血管病中,被称为平滑肌细胞增生的负性调节因子,能抑制早期斑块新生内膜的增生,并且胶原与PTEN的关系密切。但是,斑块中过表达P4Hα1,对PTEN有何影响,到目前为止,未见相关报道。
基于此,提出本研究的科学假说。在动脉粥样硬化斑块发展的早、晚不同期,即套管4周的斑块和套管12周的斑块中过表达P4Hα1,能增加动脉粥样硬化斑块中P4Hα1蛋白的表达,增加动脉粥样硬化斑块中Ⅰ型和Ⅲ型胶原含量,降低斑块的易损指数,发挥稳定斑块的作用。不同阶段斑块的组织学特征及形态学特征可能与过表达P4Hα1影响PTEN的表达有关。
研究目的
(1)探讨ApoE-/-小鼠动脉粥样硬化斑块发展过程中P4Hα1的表达变化规律。
(2)在ApoE-/-小鼠早、晚不同时期的动脉粥样硬化斑块中转染P4Hα1慢病毒,观察P4Ha1对斑块的组织学、形态学及易损性的影响。
(3)在分子生物学、组织学水平探讨过表达P4Hα1作用的可能机制。
2.材料和方法
2.1构建含目的基因的病毒载体
由上海inventrogen公司用化学合成的方法合成目的基因P4Ha1并携带增强型绿色荧光蛋白报告基因的慢病毒载体(Lenti-P4Hal),及仅携带增强型绿色荧光蛋白报告基因但无目标基因P4Hα1的阴性对照空病毒载体(Lenti-EGFP).
2.2构建小鼠动脉粥样硬化斑块模型
购买北京大学维通利华动物技术公司的8周龄ApoE-/-雄性小鼠,全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。
2.2.1构建小鼠主动脉根部自发粥样硬化斑块模型
ApoE-/-雄性小鼠(8周龄,80只),全程高脂饮食(含0.25%胆固醇和15%脂肪)喂养。随机分为4组：10周龄组、12周龄组、14周龄组、16周龄组；每组20只。高脂喂养至10周龄、12周龄、14周龄、16周龄,行安乐死处死动物,取材检测。见流程图1.
2.2.2构建小鼠颈总动粥样硬化斑块模型
为诱发颈总动脉斑块形成,8周龄ApoE-/-雄性小鼠行右侧颈总动脉套管术。基因转染干预后并分别于套管术后4周(套管4周的斑块)及12周(套管12周的斑块)取材,进行检测。
颈动脉套管和基因转染流程
(1)颈动脉套管：8周龄ApoE-/-雄性小鼠行右侧颈总动脉套管术,用0.08%戊巴比妥钠(40mg/Kg),术前行腹腔注射麻醉。将小鼠固定于手术台,用脱毛剂给予颈部脱毛处理后,碘酒消毒。沿颈正中线小心地切开皮肤。分离腺体与肌肉,暴露气管,将腺体上翻,清晰看到右侧颈总动脉,小心分离,避免损伤伴行的迷走神经。将预先剖开消毒处理的硅胶套管轻柔小心地套于颈总动脉上,丝线结扎固定套管。恢复组织结构层次后,缝合皮肤切口。
(2)基因转染流程：
①套管4周的斑块干预组：60只8周龄ApoE-/-雄性小鼠,右侧颈动脉套管术后2周,随机分为三个亚组：生理盐水组(Mock组),空病毒组(Lenti-EGFP组)和P4Ha1组(Lenti-P4Hal组),每组20只。分别小鼠尾静脉注射20ml生理盐水,空病毒(Lenti-EGFP,1.75x10.Tfu/ml,20u1),携带增强型绿色荧光蛋白报告基因的慢病毒载体(Lenti-P4Hal,1.75x10.Tfu/m1,20u1)的悬液。转染2周行安乐死后取材。见流程图2。
②套管12周的斑块干预组：60只右侧颈总动脉套管术后8周开始给予精神应激刺激,刺激持续到术后12周。精神应激刺激方法：将实验小鼠封闭于50m1大小、带有通气孔的塑料管中,给与强度为110dB的噪音刺激,噪音刺激间隔为5分钟,每次持续3秒,每天持续6小时,共刺激四周到实验结束。基因转染：套管术后10周,所有实验小鼠随机分为生理盐水组(Mock组),空病毒组(Lenti-EGFP组)和P4Hα1组(Lenti-P4Hal组),每组20只。分别小鼠尾静脉注射20u1生理盐水,空病毒(Lenti-EGFP,1.75x108Tfu,20ul),携带增强型绿色荧光蛋白报告基因的慢病毒载体(Lenti-P4Hal,1.75x108Tfu/ml,20ul)的悬液。转染2周后予以安乐死后取材生理盐水组。见流程图3。
2.3显微超声检测斑块：显微超声影像系统Vevo7700(RMV708探头)分别于套管术后2周、10周转染干预前和4周、12周安乐死前行套管侧颈总动脉动脉粥样硬化斑块的血管超声检测。
2.4体重及血液学指标的检测：分别于套管术后2周、10周转染干预前和4周、12周安乐死前检测小鼠体重；血脂水平；血清中羟脯氨酸水平。
2.5组织学检测：小心分离套管侧颈总动脉,常规冰冻切片后,分别行苏木素一伊红、天狼猩红、油红0染色。使用Image-Pro Plus6.0图像分析软件测量斑块面积、纤维帽面积、纤维帽厚度、纤维帽中胶原含量及脂质核面积并计算易损指数。
2.6免疫组化：
颈动脉组织冰冻切片漂洗后,常规行5%血清封闭,为检测不同指标,分别行相应的一抗二抗孵育：P4Ha1、MOMA-2,α-actin,Ⅰ型,Ⅲ型前胶原,Ⅰ型及Ⅲ型胶原,IL-6、TNFa、MCP-1、MMP9, MMP2, TIMP-1, TIMP-2。使用Image-Pro Plus6.0图像分析软件分别计算以上不同指标在相应斑块中的阳性染色面积(%)。
2.7蛋白印迹检测：
常规提取动脉斑块的组织蛋白,行凝胶电泳,转膜,孵育一抗二抗,发光。观察目的蛋白P4H α1、Ⅰ型及Ⅲ型胶原、MMP2、MMP9、PTEN的相对表达变化。Quantity-one图像分析软件分析计算三组中各种不同的目的蛋白的相对含量。
2.8实时定量检测：收集主动脉根部或右侧套管近心端颈总动脉各组斑块组织,常规提取总的mRNA,实时荧光定量RT-PCR反应检测各组斑块干预后P4H α1、Ⅰ型及Ⅲ型胶原mRNA的表达水平。
2.9明胶酶谱法：检测MMP2/MMP9的活性。常规方法分别制备10%分离胶、4%浓缩胶；在10%的分离胶中加入明胶储存液,至终浓度为0.1%；然后常规上样、电泳。取下电泳后的凝胶,放入100m1酶谱复性缓冲液(2.5%Triton X-100)中,室温条件下振荡30min;再在酶谱显影缓冲液中平衡30mim;将凝胶置于新鲜的酶谱显影缓冲液中,37℃过夜；凝胶染色、脱色；分析条带大小并测定灰度；计算相对量的变化。
3.结果
3.1实验动物一般情况
实验过程中生理盐水组中1只小鼠死于麻醉,套管12周的斑块P4Hα1组中1只小鼠死于转染后第13天,解剖后发现明显的肝硬化。余实验小鼠未观察到局部和全身性的不良反应,均完成实验。套管4周的斑块组中的3组,与套管12周的斑块组中3组的ApoE-/-小鼠的体重、及血脂指标：TC、TG、LDL-C和HDL-C的血清水平无显著统计学差异。而套管4周的斑块组与套管12周的斑块组中P4Hal组中血清羟脯氨酸水平显著高于生理盐水组与空病毒组。
3.2显微超声检测斑块形成
小鼠显微血管超声检测显示,套管2周套管侧颈动脉内膜增厚,4周见小斑块形成；10周、12周套管侧的颈总动脉有明显斑块形成。而对侧未套管的颈总动脉管腔光滑。
3.3动脉粥样硬化斑块的转染效率的检测
慢病毒转染预实验于慢病毒转染7天、10天、14天、21天、28天、35天后,分别取材,检测粥样硬化斑块内绿色荧光表达。慢病毒转染14天P4Hα1组和空病毒组动脉粥样硬化斑块内GFP表达率最高(70%),而生理盐水组的颈总动脉粥样硬化斑块内无GFP表达。证实体内转染有效。所以取慢病毒转染2周取材作为本实验的慢病毒转染干预时间点。
3.4过表达P4Ha1对套管4周的斑块组织病理学的影响
(1)过表达P4Hα1对套管4周的斑块中P4Hα1表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：三组比较,P4Hα1组P4Hαl的mRNA和蛋白表达显著增加(P<0.05)。
(2)过表达P4Hα1对套管4周的斑块中内胶原表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：三组比较,Ⅰ型和Ⅲ型胶原的mRNA水平无显著性差异(P>0.05)；但P4Hα1组Ⅰ型和Ⅲ型胶原的蛋白表达显著增加(P<0.05)。
(3)过表达P4Hα1对套管4周的斑块组份和易损指数的影响
病理学染色显示：三组比较,P4Hα1组胶原含量、平滑肌细胞含量显著增加(P<0.05)；脂质含量三组间无显著性差异(P>0.05)：P4Hα1组巨噬细胞含量显著降低(P<0.05)；易损指数显著显著降低(P<0.05)。
(4)过表达P4HαI对套管4周的斑块纤维帽的影响
HE及天狼星红染色显示：三组比较,P4Hα1组斑块平均纤维帽厚度显著性增厚(P<0.05)；纤维帽面积显著增大(P<0.05)；纤维帽中胶原的含量显著增多(P<0.05)。
(5)过表达P4Hα1对套管4周的斑块形态学的影响
HE及油红0染色显示：三组比较,P4Hα1组斑块的面积及主动脉表面AS病变范围显著增大(P<0.05)。
(6)过表达P4Hα1对套管4周的斑块中PTEN的表达影响
免疫组化、Western blot显示,P4Hα1组与空病毒组和生理盐水组比较PTEN的阳性染色面积,及PTEN的蛋白相对表达量显著性减少(P<0.05)。
3.5过表达P4Hα1对套管12周的斑块组织病理学的影响
(1)过表达P4Hα1对套管12周的斑块中P4Hα1表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：P4Hα1组与空病毒组和生理盐水组比较P4Hα1的mRNA和蛋白表达显著增加(P<0.05)。
(2)P4H α1过表达对套管12周的斑块内胶原表达的影响
RT-PCR、Western blot检测及免疫组化染色显示：P4Hα1组与空病毒组和生理盐水组比较Ⅰ型和Ⅲ型胶原的蛋白表达显著增加(P<0.05)；三组间Ⅰ型和Ⅲ型前胶原(I型和III型胶原)的mRNA的表达,及Ⅰ型和Ⅲ型前胶原的蛋白表达均无显著性差异(P>0.05)。
(3)过表达P4Hα1对套管12周的斑块组份和易损指数的影响
病理学染色显示：
①P4Hα1组与空病毒组和生理盐水组比较胶原含量显著增加(P<0.05)；
②P4Hα1组与空病毒组和生理盐水组比较平滑肌细胞含量显著增加(P<0.05)；
③P4Hα1组与空病毒组和生理盐水组比较脂质含量三组间无显著性差异(P>0.05)。
④P4Hα1组与空病毒组和生理盐水组比较巨噬细胞含量显著降低(P<0.05)；
⑤P4Hα1组与空病毒组和生理盐水组比较易损指数显著显著降低(P<0.05)；
(4)过表达P4HαI对套管12周的斑块内纤维帽的影响
HE及天狼星红染色显示：
①P4Hα1组与空病毒组和生理盐水组比较平均纤维帽厚度显著性增厚(P<0.05)；
②P4Hα1组与空病毒组和生理盐水组比较斑块的纤维帽面积显著增大(P<0.05)；
③P4Hα1组与空病毒组和生理盐水组比较斑块的纤维帽中胶原的含量显著性增大(P<0.05)；
(5)过表达P4Hα1对套管12周的斑块形态学的影响
①P4Hα1组与空病毒组和生理盐水组比较斑块的面积三组之间无显著地差异(P>0.05)；
②P4Hα1组与空病毒组和生理盐水组比较主动脉表面AS病变范围三组之间无显著地差异(P>0.05)。
(6)过表达P4Hα1对套管12周的斑块内炎性因子的影响
斑块内炎性因子IL-6、TNF-a和MCP-I的免疫组化染色显示：
①P4Hα1组与空病毒组和生理盐水组比较斑块内工L-6阳性染色面积显著性减小(P<0.05)；
②P4Hα1组与空病毒组和生理盐水组比较斑块内TNF-a阳性染色面积显著性减小(P<0.05)；
③P4Hα1组与空病毒组和生理盐水组比较斑块内MCP-1阳性染色面积显著性减小(P<0.05)；
(7)过表达P4Hα1对套管12周的斑块中基质金属蛋白酶表达的影响
斑块内MMP9、MMP2、TIMP-1、TIMP-2的免疫组化染色及Western blot显示：
①P4Hα1组与空病毒组和生理盐水组比较斑块内MMP9、MMP2的阳性染色面积显著性减小(P<0.05)；相反,TIMP-1.TIMP-2的阳性染色面积显著性增大(P<0.05)；
②Western blot显示,P4Hα1组与空病毒组和生理盐水组比较MMP9、MMP2的蛋白相对表达量显著性减少(P<0.05)；
③基质金属蛋白酶原位酶谱法检测显示：P4Ha1组与空病毒组和生理盐水组比较MMP9、MMP2活性显著性降低(P<0.05)；
(8)过表达P4Ha1对套管12周的斑块中PTEN的表达影响
①免疫组化显示,三组中PTEN的阳性染色面积无统计学差异(P>0.05)。
②Western blot显示出相同的趋势：三组中PTEN的蛋白表达无统计学差异(P>0.05)。
4.结论
(1)在ApoE-/-小鼠脉粥样硬化斑块发展过程中,P4Hα1的表达逐渐减少。
(2)在ApoE-/-小鼠套管4周的斑块及套管12周的斑块中,过表达P4Hα1均能升高斑块中胶原的含量,降低易损指数,但在套管4周的斑块模型中过表达P4Hα1却促使斑块增大和主动脉表面斑块总负荷的增多.
(3)过表达P4Hα1促进套管4周的斑块的增大及主动脉表面斑块总负荷的增多,与过表达P4Hα1抑制了PTEN的表达有关。
1.背景
MicroRNA (miRNA)是近年来发现的一类内源性的非编码单链小RNA分子,长度约为18-24个核苷酸,能够特异地结合于靶基因mRNA的3’端非翻译区(3’-UTR),干扰靶基因的翻译或促进靶基因mRNA的降解。从而抑制靶蛋白在体内的表达。MicroRNA与其靶基因之间构成网络调控关系,每个MicroRNA可以调控多个靶基因,而多个miRNAs也可以调节同一个靶基因。据推测,miRNA调节着人体内约60%以上的基因,在人体细胞的发育、增殖、分化和凋亡等生理及病理过程中发挥着重要作用。
研究表明,胶原的减少与动脉粥样硬化斑块的不稳定性有关。P4Ha1是胶原合成的限速酶,能使前胶原(procollagen)肽链上的脯氨酸羟化成为羟基脯氨酸,促进胶原的成熟与分泌。本实验室以往的研究表明,炎性因子如IL-6通过MAPK和c-Jun分子通路,下调P4Hal的表达,降低动脉粥样硬化斑块的稳定性；TNFa通过影响转录因子Nono的甲基化等表观遗传学的调控而降低P4Hal的表达。相反,脂连素则可以通过MAPK-AP1分子传导通路上调P4Hal的表达,增加动脉粥样硬化斑块的稳定性。但是动脉粥样硬化斑块中MicroRNA分子与调节胶原合成的关键酶P4Ha1的关系如何目前尚无相关的研究报道。
MicroRNA124(miR-124)最早在神经系统肿瘤-神经胶质瘤中发现其参与肿瘤的病理调控过程。以后,又发现其在动脉粥样硬化性腹主动脉瘤中高表达。提示miR-124可能与动脉重构过程中胶原的代谢有关。Micro.org.等多个MicroRNA应用软件均推测miR-124能特异的与P4Hα1的3’端非翻译区(3’-UTR)结合,并且结合能低,P4Ha1是miR-124理论上的靶基因。那么miR-124是否在动脉粥样硬化斑块中发挥作用?是否与斑块的胶原代谢有关?能否调控P4Ha1,能否进一步影响胶原的合成?到目前为止尚无相关的报道。
本研究提出如下假说：
(1)miR-124在应激条件下的动脉粥样硬化斑块与非应激条件下的斑块中的表达不同,在应激条件下的粥样硬化斑块中表达上调。
(2)P4Ha1是miR-124特定的靶基因。在人主动脉平滑肌细胞中上调miR-124能特异的抑制P4Hal蛋白的表达,并进一步降低Ⅰ、Ⅲ型胶原的合成。相反,抑制主动脉平滑肌细胞中miR-124的表达,促进Ⅰ、Ⅲ型胶原的合成。
研究目的
(1)观察ApoE-/-小鼠的动脉粥样硬化斑块应激组与非应激组中MicroRNA124(miR-124)与P4Ha1表达变化关系。
(2)验证miR-124能特异地结合于P4Hα13’端非翻译区,明确P4Ha1是miR-124作用靶基因的分子机制。
(3)探讨miR一124是否能调节P4Hal,进而影响主动脉平滑肌细胞中胶原的成熟和表达。
2.材料和方法
(1)构建动脉粥样硬化斑块模型：动物来源和颈动脉套管术同论文一
60只套管后8周的ApoE-/-小鼠,随机分成两组：
①应激斑块组：颈动脉套管术后给予脂多糖与应激刺激以诱发斑块中的炎症反应,应激刺激的方法同论文一。
②非应激组：不给予脂多糖与应激刺激,仅持续高脂喂养。见流程图1。
(2)设计与合成miR-124特异性引物
miR-124特异性引物的设计与合成由丹麦Exiqon公司提供(本公司不提供引物序列)。
(3)设计与合成miR-24特异性引物模拟物及抑制物
使用化学合成的方法合成miR-124mimics及miR-124inhibitors。由上海吉玛生物技术有限公司提供。
(4)构建含有P4Hal-3'-UTR的荧光素酶报告基因质粒
分别构建含有野生型P4Hα1基因的3’端非编码区序列3'-UTR(P4Hal-3'-UTR-WT)、突变型P4Ha1基因的3’端非编码区序列3'-UTR(P4Hal-3'-UTR-MUT)的荧光素酶报告基因质粒(pGL3)：PGL3-P4H al-3'-UTR-WT和pGL3-P4H a1-3'-UTR-MUT。通过脂质体2000和miR-124mimic共转染人主动脉平滑肌细胞,检测荧光素酶荧光表达强度。验证软件预测结果。明确miR-124与P4Hα1基因的3’端非编码区特异的碱基互补结合序列。探讨miR-124调控的下游靶基因P4Hα1的分子机制。
(5)miRNA提取和实时荧光定量qRT-PCR
提出颈动脉斑块及人主动脉平滑肌细胞中总的MicroRNAs,通过qRT-PCR检测miR-124在两组斑块中及主动脉平滑肌细胞不同干预组中的相对表达量。
(6)人主动脉平滑肌细胞培养
原代人主动脉平滑肌细胞,购自美国Sciencell,应用VSMC培养液常规培养至80-90%融合后,传代再培养。第4-8代平滑肌细胞用于本实验。
(7)人主动脉平滑肌细胞培养的转染与刺激
①miRNAs转染人主动脉平滑肌细胞
应用脂质体2000进行miRNA (miR-con、miR-124mimics或miR-124inhibitor)转染。具体步骤按GenePORTER2的说明书进行转染操作,转染后37℃,5%CO2培养箱中培养6-8h,更换为常规的细胞培养液,继续培养24-48小时,进行下一步检测。
②TNFa刺激人主动脉平滑肌细胞
六孔板中平滑肌细胞的密度达到80%-90%时,按100ng8mlTNFa的剂量浓度,将TNFa加入到细胞培养基刺激平滑肌细胞,37℃,5%CO2温箱中培养24小时后进行下一步实验。
(8)生物信息软件分析
应用miRanda, TargetScan, RNAhybrid and PicTar等著名的MicroRNA分析预测软件,确定P4Hα1是miR-124用的靶基因。并且miR-124是调控P4Ha1的MicroRNA分子之一。进一步确定miR-124与P4Ha13’非转录区(3’-UTR)特异结合的互补结合位点。
(9)免疫印迹(Western Blot)检测
同论文一
(10)细胞免疫荧光
人主动脉平滑肌细胞干预后,4%甲醛固定,PBS漂洗,常规行5%兔血清封闭,分别行抗P4Hal的一抗及带荧光标记的二抗孵育(避光),DAPI染核后,避光拍照,使用Image-Pro Plus6.0图像分析特异荧光的表达含量。
3.结果
3.1P4Ha1在斑块中的表达
P4Hα1在应激组斑块中的蛋白表达显著低于非应激组(P<0.05)。
3.2miR-124在斑块中的表达
miR-124在应激组斑块中的表达显著高于非应激组(P<0.05)。
3.3P4Hα1在人主动脉平滑肌细胞中的表达
P4Hα1在TNFa刺激组中的蛋白表达显著低于无TNFa刺激组(P<0.05)。
3.4mi R-124在人主动脉平滑肌细胞中的表达
qRT-PCR显示,miR-124在TNFa刺激的人主动脉平滑肌细胞中的表达是无TNFa刺激组的2.57倍(U6标准化后)。两组间的差异有显著性(P<0.05)。
3.5生物信息软件分析结果
P4Hal (NM_000917)是miR-124的理论靶基因,并且结合能低。miR-124与P4Ha1的结合位点位于P4Ha1基因的3’端非翻译区(3,-UTR)的第834-841个碱基。此碱基序列位点在生物界的各物种之间有高度保守性。
3.6miR-124对P4Ha1表达的影响
miR-124mimics转染人主动脉平滑肌细胞48小时后,P4Ha1的蛋白表达量较转染前显著降低(P<0.05)。miR-124inhibitors转染人主动脉平滑肌细胞后,TNFa再刺激主动脉平滑肌细胞24小时后的P4Ha1的蛋白表达量较无miR-124inhibitors预转染组上调(P<0.05)。
3.7miR-124通过与P4Ha1的3’端非翻译区(3’-UTR)的结合位点的特异结合作用
miR-124mimics与含有野生型P4Hαl基因的3’端非编码区序列P4Hα13’UTR(P4Hα1-WT)荧光素酶报告质粒瞬时共转染人主动脉平滑肌细胞48小时后,荧光素荧光表达显著地被抑制(P<0.05)。
3.8miR-124对胶原的合成与成熟的影响
miR-124mimics转染人主动脉平滑肌细胞48小时后,胶原Ⅰ和胶原Ⅲ的蛋白表达量较转染前显著减少(P<0.05)。miR-124inhibitors预转染能使TNFa诱导人主动脉平滑肌细胞降低胶原Ⅰ和胶原Ⅲ的作用部分地消失,差异具有显著性(P<0.05)。
4.结论
(1)在应激条件下,动脉粥样硬化斑块中miR-124表达显著上调,P4Hα1的表达显著下调。
(2)miR-124与P4Hα1的3’端非翻译区(3’-UTR)特异的结合位点结合,抑制P4Hα1蛋白的表达,进而影响Ⅰ型与Ⅲ型胶原的合成过程。
1. Backgroud
The rapture of atherosclerotic plaque is responsible for the clinical acute events of vascular, such as stroke and myocardial infarction, which is the most common cause of mortality and morbidity in developed countries. To stabilize atherosclerotic plaques is an essential strategy to reduce the clinical acute events of vascular.
Collagen is one kind important composition in atherosclerotic plaque. Type I and type III collagen are the predominant collagen in atherosclerotic plaques and arterial wall. The vulnerable plaque is characterized by less collagen and more lipids. Otherwise, the stable one with more collagen and less lipids.
Prolyl-4-hydroxylase (P4H) is a key intracellular enzyme involved in the synthesis of collagens. It catalyzes the proline residues repeating X-Pro-Gly triplets located in the procollagens to format hydroxyproline. It is essential for collagen posttranslational modification, and this modification facilitate folding the polypeptide procollagen chains into stable triple-helical molecules.
P4H is composed of a and P subunits; The a subunit is a rate-limiting enzyme.The active enzyme is a tetramer composed of two pairs of non-identical subunits (a2p2). P4Hal is the most important form of all kinds of P4Hs. It is responsible for the posttranslational modifications of procollagens. Studies demonstrated that less P4Hal in plaques is closely related to destability of plaques. So to increase the expression of P4Hal in plaques is a good strategy to avoid the plaque rupture.
In theory, overexpression of P4Hal can make over production of collagen under the condition of sufficient procollagen. But whether or not overexpression of P4Ha1influences the collagens and other components in atherosclerotic plaques, and whether or not overexpression of P4Hal influences the vulnerable index are unknown.
Collagen metabolism is composed of collagen synthesis and collagen degradation modulated by extracellular matrix metallopeptidase (MMPs), along with their endogenous tissue inhibitors (TIMPs) constituting an important system for regulating extracellular matrix (ECM), especially, collagen turnover. But whether or not overexpression of P4Hal influences the MMPs and TIMPs is also unknown.
Guided by above theories, We put forward such scientific hypothesis as:
(1) The expession of P4Hal is developmental decrease in the pathological course of atherosclerotic plaques in ApoE-/-mice.
(2) Overexpression of P4Ha1can increase the product of Type I and type III collagen both in evolving plaques and in established ones.
(3) Overexpression of P4Hal can increase the stability of evolving plaques and established ones. But there may be some different influence in evolving plaques from established ones.
Objectives
(1) To investigate the developmental expession of P4Hal in the pathological course of atherosclerotic plaques of ApoE-/-mice.
(2) To investigate effects of overexpression of P4H a1on the collagen metabolism both in evolving plaques and in established ones of ApoE-/-mice.
(3) To elucidate the effect of overexpression of P4Hal on the stability of evolving plaques and established ones of ApoE-/-mice.
(4) Clarify the different influence of overexpression of P4Hal on evolving plaques from established ones and explore the mechanism under the appearance.
2. Material and Methods
(1) Plasmids and Lentivirus-P4Ha1Construction The Lentivirus-P4Haland empty Lentivirus (control) were constructed by Inventrogen, Shang hai.
(2) Animal Model
Ethics Statement
The animal experimental protocol complied with the Animal Management Rules of the Chinese Ministry of Health (document No55,2001) and was approved by Animal Care Committee of Shandong University.
Spontaneous plaque of aortic root
Male apolipoprotein E-deficient mice (n=80,8weeks old, weighing25-30g) were obtained from Peking University (Beijing, China), and were divided into randomly4groups:10weeks old group;12weeks old group;14weeks old group and16weeks old group (n=20). All of them had high-fat diet. At the end of10,12,14,16week, they were killed kindly. Histopathology-Immunohistochemistry and Western, Blot, were carried on for detecting the expression of P4Hal in Spontaneous plaque of aortic root.
Carotid Collar Placement and Transgenic therapy
Male apolipoprotein E-deficient mice (n=120,8weeks old, weighing25-30g) m were obtained from Peking University (Beijing, China). Atherosclerotic lesions were induced by placing a perivascular collar on the right common carotid artery of mice. The Lenti-EGFP groups were used as controls.120mice were divided randomly into two groups:established plaques and early ones.
①Evolving Plaques:60mice (male,8weeks old) were induced carotid atherosclerotic lesions by placement of a restraint perivascular silica collar as above. After surgery2weeks,60were divided into randomly3groups:saline group; Lenti-EGFP group and Lenti-P4H a1group. Three group mice were given intravenously saline, Lentivirus-P4Ha1and empty Lentivirus transgenic therapy.2weeks later, all mice were given euthanasia for research.
②Established plaques:60mice (male,8weeks old) were induced carotid atherosclerotic lesions by placement of a restraint perivascular silica collar, and fed with high-fat diet combined stimulation of mental to create a vulnerable plaque model. After surgery10weeks,60were divided into randomly3groups:saline group; Lenti-EGFP group and Lenti-P4Hal group. Three group mice were also given intravenously saline, Lentivirus-P4Hal and empty Lentivirus transgenic therapy.2weeks later, all mice were given euthanasia for research.
(3) Micro-ultrasonography
Vevo770system was used to measure the baseline ultrasonography parameters of plaques
(4) Blood samples and Biological Measurements
Blood samples were taken to detect:total cholesterol? high-density lipoprotein cholesterol,triglycerides,low-density lipoprotein cholesterol, and hydroxyproline.Weight was also detected before surgery and euthanasia.
(5) Histopathology and Immunohistochemistry
Cryosections were stained with hematoxylin and eosin (H&E), oil O and picosirius red as previous.For immunohistochemistry, Sections were incubated with different primary antibodies and second antibodies for immunohistochemistry.
(6) Western Blot
Proteins were extracted from carotid artery specimens, separated on14%SDS-PAGE and transferred to nitrocellulose membranes. After incubation with primary antibodies and second antibodies and exposure, protein expression was determined.
(7) RT-PCR
Carotid artery specimens were pooled for RNA isolation with TriZol to detect the mRNA of P4Ha1, collagen I (procollagen I) and collagen III (procollagen III).
(8) Gelatin zymography assay
MMP-9and MMP-2activity was detected using the gelatin zymography assay. The tissue proteins were extracted using lysis buffer. The protein concentration was determined. The sample was electrophoresed on SDS-polyacrylamide gels copolymerized with1%gelatin. After electrophoresis, the gels were washed twice in2.5%Triton X-100and incubated in1%Triton X-100,5mM CaC12and5jiM ZnC12 (pH7.5) at37℃for36h. The gels were stained with0.1%Coomassie blue R250, and destained in10%isopropanol and10%acetic acid in H2O. MMP-9/MMP-2was detected as transparent bands on the blue background of a Coomassie blue-stained gel.
4. Results
One mouse died of liver fibrosis, and others finished this study.
4.1Blood samples and Biological Measurements
There was no significant difference in serum levels of TC, TG, LDL-C and HDL-C among the3groups of ApoE-/-mice both in established plaque group and in evolving plaque group. But the hydroxyproline serum levels was significant higher in lenti-P4Hα1group than that of mock group and lenti-EGFP group both in established plaque group and in evolving plaque group (P<0.05).
4.2Identification of Plaque
Micro-ultrasonography was used to evaluate the stage of the plaques. Small atherosclerotic plaques could be seen in the right common carotid artery in4weeks after collar surgery. And there were obvious plaques in10weeks and12weeks after collar surgery, otherwise, no plaque was detected in the left common carotid artery of animals.
4.3Transfection Efficiency Assays
GFP expression(about70%) was found in atherogenic plaque of carotid arteries in Lenti-P4Hα1and Lenti-EGFP groups. The mRNA of P4Hα1increased about five fold than Lenti-EGFP group and PBS group. All demonstrated efficient transfection.
4.4Effect of Lenti-P4Hα1transgenic therapy on P4Hal protein expression
①Evolving Plaque group:Western blot and Immunohistochemistry showed that the expression of P4Hα1protein in lenti-P4Hα1group was significant higher than saline and Lenti-EGFP groups (P<0.05).
②Established plaque group:consistent with the result of established plaque group (P<0.05).
4.5Effect of overexpression of P4Hal on procollagen Ⅰ, collagen Ⅰ and
procollagen Ⅲ, collagen Ⅲ in plaques
①Evolving Plaque group:Western blot and Immunohistochemistry showed that there was no significant difference in the expression of procollagen Ⅰ and procollagen Ⅲ in3groups, but the collagen Ⅰ and collagen Ⅲ protien expression in Lenti-P4Hal was significant higher than saline and Lenti-EGFP groups (P<0.05).
②Established plaque group:consistent with the result of evolving plaque group (P<0.05).
4.6Effect of overexpression of P4Hal on carotid plaque composition and stability
③Evolving Plaque group:Histological and immunohistochemical staining of carotid plaque in3groups were significantly different. Collagen content was increased significantly in the Lenti-P4H a1group; And VSMC content in the atherosclerotic lesion area, detected by a-actin staining, was also significantly increased (P<0.05). But the content of macrophages was significantly decreased in Lenti-P4Hal group compared with saline group and lenti-EGFP control groups (P<0.05). And there was no significant difference in lipid deposition among3groups.
②) Established plaque group:consistent with the result of evolving plaque group (P<0.05).
4.7Effect of overexpression of P4Hal on the morphology changes of the carotid plaque
①Evolving plaque group:The fibrous cap area, fibrous cap thickness and collagen content in fibrous cap were higher significantly in lenti-P4Hal group than control groups (P<0.05). And the vulnerability index was lower significantly than control groups. The plaque area was significant lager and the total plaque burden was significant higher in lenti-P4Ha1group, compared with control groups (P<0.05).
②Established plaque group:It was inconsistent with the result of evolving plaque group. The plaque area and total plaque burden in lenti-P4Ha1group had no significant difference from control groups (P<0.05), although in lenti-P4Hal group, the fibrous cap area, fibrous cap thickness and collagen content in fibrous cap were higher significantly than control groups (P<0.05). And the vulnerability index was also lower significantly than control groups. This was consistent with the result of evolving plaque group group,
4.8Effect of overexpression of P4Hal on inflammatory markers and on MMPs and TIMPs in established plaque group
The protein expression levels of IL-6, TNF-a, MCP-l,MMP-2, MMP-9in the carotid plaque of in lenti-P4Hal group were lower significantly compared with control groups (P<0.05), based on quantitative analysis of immunohistochemical staining, immunohistochemical staining. And the relative activity of MMP9, MMP2also decreased significantly in lenti-P4Hal group.
4.9Effect of overexpression of P4Hal on PTEN expression
〦volving Plaque group:Western blot and Immunohistochemistry showed that PTEN expression in lenti-P4Ha1group decreased significantly, compared with control groups (P<0.05).
②Established plaque group:It was different from established plaque group: there was no significant difference on PTEN expression among three groups (P>0.05).
5. Conclusion
P4Hal overexpression of can increase collagen Ⅰ and Ⅲ significantly in both evolving plaques and in established ones. It can increases the stability of established plaques but accelerates intimal hyperplasia and the total lesion area (%) in evolving plaques of Apolipoprotein E-knockout mice.
1. Background
miRNAs are short ribonucleic acid (RNA) molecules, approximately18-24nucleotide RNAs that modulate gene expression by base pairing with the3'-untranslated regions (3'-UTRs) of miRNAs to repress protein expression by inhibiting translation or promoting degradation of miRNAs. Untill now, several hundreds of miRNAs have been identified. They involved in cell proliferation, apoptosis, differentiation, development and metabolism. Studies on miRNAs show that about60%of mammalian genes are targeted by special miRNAs. One miRNA can target one or more genes.
P4H a1is the rate-limiting isoform of P4H responsible for procollagen hydroxylation, maturation, and organization. Studies show P4Hal are losely related to the vulnerable atherosclerosis plaque. Our previous studies demonstrate that inflammatory cytokines, interleukin6, can downregulate P4H a1via a mitogen-activated protein kinase and c-Jun pathway to destabilize atherosclerotic plaques. And TNFa can also downregulate P4Hal via affecting the histone (H)4lysine12acetylation. Otherwise, Adiponectin can upregulate prolyl-4-hydroxylase al expression human aortic smooth muscle cells by regulating ERK1/2and Spl. But how miRNAs regulate P4Hal in atherosclerotic plaque is unknown till now.
miR-124is a brain-enriched microRNA that has been shown to be down-regulated in glioma and medulloblastoma and plays a crucial role in brain tumor progression. Keiwa Kin observed that miR-124was up-regulated in the tissue of atherosclerotic abdominal aortic aneurysm. It suggests that miR-124was related to the collagen metabolism of aortic remolding. But whether or not miR-124involves the progression of atherosclerotic plaque is unclear.
P4Hal is miR-124's target gene by the famous softwears, such as:MIRBASE, STARBASE, TARBASE, TARGETSCAN. But whether or not miR-124can bind to the3'-untranslated region (3'-UTR) of P4Hal gene mRNA to regulate the translation of P4Hal is unknown in fact. Guided by above theories, We put forward such scientific hypothesis as:
(1) The expression of miR-124is different in stressed plaques from unstressed plaques. And miR-124is up-regulated probably in stressed plaques.
(2) miR-124can bind to the wild3'-untranslated region (3'-UTR) of P4Hal gene mRNA but can not bind to the mutation one.
(3) P4Hal is miR-124's target gene. In human aortic smooth muscle cells, miR-124mimics can inhibit P4Hal expression and down-regulate "collagen I, collagen Ⅲ. Otherwise, miR-124inhibitors can up regulate P4Hal and increase the production of collagen Ⅰ, collagen Ⅲ.
Aims:
(1) To identify miR-124alteration in atherosclerotic plaques and the different expession of miR-124between stressed plaques and unstressed ones.
(2)To clarify the relationship between miR-124and P4Hal.
(3)To clarify the mechanism on how miR-124regulate P4Hal and to identify miR-124's special binding site on3'-UTR of P4Hal.
(4) To clarify the relationship between miR-124and collagen Ⅰ and collagen3.
2. Materials and methods
(1) Animals
Male apolipoprotein E-deficient mice (n=60,8weeks old, weighing25-30g) were obtained from Peking University (Beijing, China).
60mice were divided randomly into2groups:unstressed group (n=30) and stressed group (n=30). Carotid collar placement and mental stress protocol is consistent with paper1.
(2) Carotid artery isolation
After12weeks, the carotid artery of E-deficient mice, restricted by collar placement, were isolated carefully to store at-80℃refrigerator for RT-PCR and Western blot detected.
(3) Computational targeted gene predictions
miRanda, TargetScan, RNAhybrid and PicTar etal..
(4) Plasmids, mutagenesis, transfection and luciferase reporter assays
The3'-UTR of the mouseP4Hal gene was amplified. All constructs were verified by sequencing. The wild and mutated3'-UTR of theP4Hal gene were obtained from Jinan Biolabs. The luciferase reporter vector pGL3-basic vector [luc2] was also from Jinan Biolabs.
(5) miRNA mimics, miRNA inhibitors
miRNA mimics or miRNA inhibitors were transfected using Lipofectamine2000(Invitrogen). Luciferase activity was detecked by using Dual-lo Luciferase Reporter Assay Kit (Promega) on a FLUOstar Omega luminometer (BMG Labtech, Cary, NC), after48hours of transfections.
(6) HASMCs culture
HASMCs were obtained from ScienCell. They were cultured with5%CO2at37℃in smooth muscle cell medium (ScienCell, USA). HASMCs (passages4to8) were used.
(7) RNA isolation and qRT-PCR
Total RNA was isolated from carotid artery or HASMCs using TRIzol, and cDNA was synthesized according to manufacturer's instructions (Invitrogen, San Diego, CA).
(8) Western blot analysis
Western blots were performe d using anti-P4HAl, anti-COLlA1, anti-Collagen3, and anti-β-actin antibodies (Santa Cruz Biotechnology) following our published method[10].
(9) Immunofluorescence
HASMCs were grown on chamber slides and rmiR-124transfection was carried out. Forty-eight hours following the transfection, HASMCs were fixed in4%paraformaldehyde for15min at room temperature and was rised with PBS three times.5%rabbit serum was used to block the cells, following by incubation with anti-P4H ol antibody overnight at4℃.
3. Results
3.1miR-124is upregulated in stressed plaques and in TNFa treated HVSMCs
(1) The mRNA was isolated from unstressed carotid plaques and stressed carotid plaques in vivol. The expression of miR-124was determined by real-time; PCR. The relative levels of miR-124expression in stressed plaques were significantly increased (P<0.05).
(2) The mRNA was isolated from TNFa treated HVSMCs and from control HVSMCs in vitrol. The expression of miR-124was also determined by real-time PCR. The relative levels of miR-124expression in TNFa treated HVSMCs were significantly higher (P<0.05), compared with control HVSMCs.
3.2miR-124is predicted to target P4Hal gene
miRanda, TargetScan, RNAhybrid and PicTar et al., the famous computational approaches for the identification of mammalian microRNA targets, identified that Homo sapiens prolyl4-hydroxylase, alpha polypeptide I (P4Hal), transcript variant1, mRNA. NM_000917is miR-124's targe gene and miR-124is P4Hal's target mirRNA.
3.3miR-124targetsP4Hal
The protein expression ofP4Hal was significantly reduced after miR-124mimics transfection (P<0.05). Otherwise, miR-124inhibitor can make the reduction of P4Hal induced by TNFa significantly abrogated (P<0.05).
3.4miR-124downregulates the expression of P4Hal via targeting the
3'-UTR of P4Hal mRNA
Transient co-transfection of miR-124mimics with luciferase reporter plasmids resulted in a significant repression of luciferase reporter gene expression in HVSMCs, whereas co-transfection of HVSMCs with negative control miRNA or mutations did not have any effect on the expression of luciferase.
3.5Inhibition of miR-124increases collagen maturation.
To addresse whether miR-124overexpression had an inhibitory effect on collagen protein expression. After48hours transfection of miR-124mimics in HVSMCs, collagen Ⅰ and Ⅲ were all significantly decreased (P<0.05) as expected. Whereas, miR-124inhibitor can make the reduction of collagen Ⅰ and Ⅲ induced by TNFa significantly abrogated (P<0.05).
4. Conclusions
miR-124involved in and play important roles in atherosclerotic plaques. It is upregulated in stressed plaques and TNFa induced HVSMCs. miR-124can regulate collagen maturation via targeting its target gene, the P4Ha1mRNA.
引文
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