【作者】 马颖；

【导师】 温进坤； 杨展；

【作者基本信息】 河北医科大学 ， 生物化学与分子生物学， 2020， 博士

【摘要】 血管平滑肌细胞（vascular smooth muscle cell,VSMC）位于血管中膜,是构成血管壁的主要细胞成分。成熟的VSMC的主要功能是收缩以及调节血管的张力。在正常的血管中,VSMC的表型以分化型为主,当血管受到损伤后,VSMC从分化表型转化为合成型,获得增殖及迁移能力,并大量合成细胞外基质,造成血管内膜增生及管腔狭窄。相反,当VSMC衰老导致VSMC功能障碍时,往往引发高血压、动脉粥样硬化等心血管相关疾病。高血压发病率高,是心衰、心肌梗死等心血管疾病的重要危险因素。预计到2025年,高血压将会影响29.2%的世界人口,患病人数达到15.6亿。高血压的发病机制虽被广泛研究,但仍未完全阐明。高血压发生发展的病理生理学改变涉及血管内皮调节异常、VSMC功能障碍、氧化应激、慢性炎症反应、交感神经系统激活、肾素-血管紧张素-醛固酮系统（renin-angiotensin-aldosterone system,RAAS）调节活性改变等。另外,由于VSMC收缩及血管重构引起的血管直径缩小、血管阻力增加也是高血压发生的重要原因之一。深入研究和认识VSMC收缩功能的调节机制对高血压防治具有重要意义。细胞衰老是指细胞丧失生长分裂的能力,为不可逆的细胞生长阻滞。由于衰老细胞内细胞器的结构改变,衰老细胞表现为细胞体积变大,以及出现广泛空泡化现象。另外,衰老细胞还伴随有基因表达的改变,如调节细胞周期的p53、p21和p16蛋白表达上调;位于溶酶体中的衰老相关β-半乳糖苷酶（senescence-associatedβ-galactosidase,SA-β-gal）活性升高。同时,衰老细胞还表现为衰老相关分泌表型（senescence-associated secretory phenotype,SASP）,分泌促炎细胞因子等蛋白质,对衰老细胞及细胞外环境产生影响。近年来,细胞衰老与衰老相关疾病的关系引起了广泛的关注。虽然在VSMC衰老与高血压、动脉粥样硬化、动脉瘤等心血管疾病的关系的研究方面取得很大进展,但VSMC衰老的分子机制尚有待阐明。环状RNA（circular RNA,circRNA）是一类新发现的非编码RNA,对真核基因的表达具有重要的调控作用。与线性RNA（linear RNA,含5’和3’末端）不同,环状RNA为首尾共价连接的环状结构,没有游离的5’和3’末端,不能被核酸外切酶水解,可在细胞内稳定存在。环状RNA可通过多种方式调节基因的表达:与miRNA结合解除其对靶基因的抑制作用;与RNA结合蛋白结合或作为蛋白质结合的支架;细胞核转录因子的调节者等。近年来有研究报道,环状RNA与细胞衰老及高血压发生密切相关。例如,circ-Foxo3通过与衰老相关蛋白ID1和E2F1以及应激相关蛋白HIF1a和FAK结合,促进阿霉素诱发的心脏衰老。我们实验室前期在人主动脉平滑肌细胞（human aortic smooth muscle cell,HASMC）中发现来源于ACTA2基因（编码α-SMA）的环状RNA circACTA2,证明circACTA2通过吸附miR-548f-5p而上调α-SMA的表达,NRG-1/circACTA2/miR-548f-5p形成调节轴协同调节α-SMA表达及VSMC的收缩功能。然而,circACTA2调节HASMC细胞收缩的具体机制尚不清楚,circACTA2是否参与VSMC衰老相关的高血压的发生发展过程也有待于深入研究。本研究探索影响VSMC表达circACTA2的因素,研究circACTA2调节VSMC收缩及其在衰老相关的高血压发生发展中的作用及机制。第一部分FN1介导circACTA2对VSMC收缩的调节作用目的:探讨AngⅡ对circACTA2表达的影响以及circACTA2在AngⅡ诱导VSMC收缩中的作用。方法:1 AngⅡ和PDGF-BB刺激VSMC后,qRT-PCR检测circACTA2的表达。2 qRT-PCR和荧光原位杂交（fluorescence in situ hybridization,FISH）检测高血压病人血管组织中circACTA2的表达。3鬼笔环肽染色检查AngⅡ刺激和/或circACTA2过表达对细胞骨架及肌丝的影响。4 RAP（RNA antisense purification）和U-生物素标记的RNA pull-down技术结合液相色谱-质谱（liquid chromatography-mass spectrometry,LC-MS）定量分析鉴定与circACTA2结合的蛋白质。5 RNA pull-down和RNA结合蛋白免疫沉淀（RNA binding protein immunoprecipitation,RIP）检测circACTA2与FN1的相互作用。6在VSMC中过表达circACTA2、AngⅡ处理和/或敲低FN1,Western blot检测FN1的表达,鬼笔环肽染色观察肌丝及细胞骨架的变化。7在VSMC中过表达circACTA2的同时敲低FN1,观察乙酰胆碱诱导的细胞收缩的变化。8免疫荧光染色检查高血压病人血管组织中circACTA2和FN1的共定位及相互作用。结果:1 AngⅡ诱导circACTA2的形成AngⅡ在高血压的发生发展中发挥重要作用。我们首先给予VSMC AngⅡ或PDGF-BB刺激,qRT-PCR检测circACTA2表达。结果显示,在AngⅡ作用下,circACTA2的表达显著增加;而PDGF-BB下调circACTA2表达,结果具有统计学意义。另外,我们也检测高血压患者血管中circACTA2的表达情况。结果显示,与正常血管组织相比,高血压患者血管中circACTA2的表达明显升高。利用circACTA2荧光探针进行荧光原位杂交（FISH）实验也显示,circACTA2在高血压病人血管组织中的表达明显高于正常对照组。我们进一步观察circACTA2过表达对细胞骨架组构的影响。用慢病毒p WPI-circACTA2表达载体感染VSMC 24 h后,再用AngⅡ处理细胞。鬼笔环肽染色结果显示,与对照组相比,AngⅡ刺激或circACTA2过表达均能使F-肌动蛋白成束增加,应力纤维明显增粗;二者的共同作用效果更加显著。2 circACTA2与FN1和ILF3相互作用为了进一步明确circACTA2在VSMC中的作用机制,我们用RAP（RNA antisense purification）或U-生物素标记的RNA pull-down技术结合液相色谱-质谱（LC-MS）定量分析鉴定与circACTA2结合的蛋白质。将反义寡核苷酸序列或U-生物素标记的circACTA2沉降的蛋白质与它们相应的对照组进行定量比较分析（log₂>2,moderated T-test,p<0.05）,我们确认与circACTA2高度特异结合的蛋白质包括FN1、ILF3和VIM等,但不包括β-actin,说明其相互作用的特异性。RNA pull-down及RIP实验进一步验证了circACTA2与这3种蛋白质的结合。以上结果说明,circACTA2可以与FN1及ILF3相互作用。3 circACTA2通过上调FN1表达而促进VSMC收缩为了确定circACTA2是否影响FN1、ILF3和VIM表达,我们在VSMC中过表达circACTA2后进行Western blot分析,结果显示,FN1的表达明显增加,而ILF3和VIM的表达没有明显改变。加入不同浓度(0,10^-7,2×10^-7,4×10^-7mol/L)的AngⅡ刺激后,FN1的表达水平随着AngⅡ浓度升高而增加。用靶向FN1的si RNA敲低FN1的表达能够消除AngⅡ对FN1表达的诱导作用。我们进一步检查circACTA2和FN1对细胞收缩的影响。鬼笔环肽染色结果显示,过表达circACTA2促进细胞肌丝的形成,并且成束增加;而在VSMC中过表达circACTA2的同时敲低FN1则使细胞肌丝减少。细胞收缩实验结果显示,过表达circACTA2促进乙酰胆碱诱导的细胞收缩,而同时敲低FN1后消弱了其对细胞收缩的促进作用。这些结果表明,FN1介导circACTA2对细胞收缩的调节作用。为了探索circACTA2促进细胞收缩是否与circACTA2和FN1的相互作用有关,我们利用circACTA2荧光探针和FN1抗体进行FISH实验。实验结果显示,与对照组相比,AngⅡ处理使circACTA2和FN1表达增加的同时,二者之间的结合也明显增加,二者共定位于细胞的胞浆中。取高血压病人的动脉组织进行荧光染色也得到了同样的结果。结果提示,AngⅡ促进circACTA2与FN1的相互作用。小结:综上所述,AngⅡ显著诱导circACTA2的形成,后者通过与FN1相互作用促进VSMC收缩。第二部分circACTA2调节VSMC收缩的作用机制目的:揭示circACTA2上调FN1表达以及调节FN1与SPTBN1相互作用的分子机制。方法:1 RNA pull-down联合高通量测序鉴定circACTA2吸附的miRNA。2用AngⅡ和PDGF-BB处理VSMC后,qRT-PCR检测这些miRNA的表达变化。3生物信息学软件预测FN1是miR-149的靶基因,在VSMC中过表达或敲低miR-149,Western blot检测FN1的表达。4双荧光素酶报告基因验证miR-149与FN1 3’UTR的结合。5在VSMC中过表达circACTA2的同时敲低miR-149,检查乙酰胆碱诱导的细胞收缩,鬼笔环肽染色观察细胞骨架和肌丝的变化。6 RNA-蛋白质相互作用预测网站预测FN1与circACTA2的结合部位。7细胞过表达GFP-FN1_{（183-275）},Western blot检测GFP-FN1_{（183-275）}及FN1的表达。8细胞过表达GFP-FN1_{（183-275）}的同时给予AngⅡ刺激,Western blot检测FN1的表达。9细胞过表达GFP-FN1_{（183-275）},RIP检测FN1_{（183-275）}与circACTA2的结合。10细胞过表达circACTA2的同时过表达GFP-FN1_{（183-275）}或加入封闭结合位点的寡核苷酸序列,RIP检测FN1与circACTA2的结合。11细胞过表达circACTA2的同时过表达GFP-FN1_{（183-275）},鬼笔环肽染色观察细胞骨架和肌丝的变化。12 CoIP-MS鉴定与FN1结合的蛋白质。13细胞过表达GFP-FN1_{（183-275）}或给予AngⅡ刺激,免疫共沉淀（Co IP）检测FN1与SPTBN1的结合。14细胞过表达circACTA2的同时敲低FN1,免疫荧光检测FN1与SPTBN1的共定位及相互作用。结果:1 circACTA2通过吸附miR-149间接调节FN1表达利用RNA pull-down联合高通量测序方法鉴定circACTA2可以吸附的miRNA。首先向AngⅡ和PDGF-BB处理的细胞裂解液中分别加入生物素标记的circACTA2探针和对照探针进行RNA pull-down,然后提取总RNA进行miRNA测序。结果显示,有24种miRNA可以被circACTA2探针所富集。为了验证miRNA的测序结果,我们利用qRT-PCR检测RNA pull-down沉淀物中相关miRNA表达。与对照探针相比,miR-27b-3p、miR-27b-5p、miR-149、miR-197等在circACTA2探针所富集的miRNA中明显上调,结果具有统计学意义。荧光素酶报告基因证实,circACTA2能与miR-149相结合。另外,用AngⅡ或PDGF-BB刺激VSMC,qRT-PCR检测这些miRNA的表达。结果显示,miR-149和miR-487b-3p在AngⅡ刺激的细胞中明显下调,而在PDGF-BB的作用下明显上调,并且与circACTA2的变化相反,表明circACTA2可与这两种miRNA相互作用。我们进一步发现,在FN1的3’UTR具有miR-149的结合位点。为了验证miR-149对FN1表达的影响,我们设计miR-149的模拟物及抑制剂并用它们转染VSMC。Western blot结果显示,miR-149的模拟物抑制FN1蛋白在VSMC中的表达,反之,miR-149的抑制剂增加FN1蛋白的表达,说明FN1被miR-149负调控。双荧光素酶报告基因分析结果也提示,miR-149模拟物能抑制FN1 3’UTR指导的报告基因表达。这些结果说明,circACTA2通过吸附miR-149解除其对FN1表达的抑制。另外,在用慢病毒p WPI-circACTA2感染细胞的同时加入miR-149的抑制剂,抑制剂能够进一步促进乙酰胆碱诱导的细胞收缩。鬼笔环肽染色显示,circACTA2过表达的同时抑制miR-149表达,细胞肌丝形成进一步增多,肌丝成束更加明显。结果提示,抑制miR-149表达对细胞收缩具有促进作用。2干扰circACTA2与FN1相互作用可抑制circACTA2对VSMC收缩的诱导我们通过RNA-蛋白质相互作用预测网站预测circACTA2与FN1的结合位点,发现FN1的237-252aa肽段可能是circACTA2的结合部位。我们设计可以编码FN1的183-275aa肽段的DNA序列与GFP-C1空载体连接,用于表达FN1的183-275aa肽段,称为GFP-FN1_{（183-275）}。Western blot结果显示,转染该载体后细胞可表达GFP-FN1_{（183-275）}。接着,我们分别用GFP-C1和GFP-FN1_{（183-275）}转染VSMC细胞,利用抗GFP抗体进行沉淀,RIP-PCR检测两种沉淀物中circACTA2的富集情况。结果显示,GFP-FN1_{（183-275）}富集的circACTA2明显高于GFP-C1组,说明GFP-FN1_{（183-275）}能够与circACTA2相结合。重要的是,在VSMC中过表达GFP-FN1_{（183-275）}能够显著抑制FN1蛋白的表达,说明干扰FN1与circACTA2的结合可促进FN1的降解。另外,转染GFP-FN1_{（183-275）}能够消除AngⅡ对FN1表达的诱导作用。我们进一步用RIP实验研究GFP-FN1_{（183-275）}对circACTA2与FN1结合的影响。首先,在用慢病毒过表达circACTA2的同时再转染GFP-FN1_{（183-275）},细胞裂解后加入抗FN1抗体。结果显示,在过表达circACTA2的细胞中,circACTA2被FN1抗体的富集明显升高,但是,在同时转染GFP-FN1_{（183-275）}的细胞中,被FN1抗体富集的circACTA2减少,说明GFP-FN1_{（183-275）}能够干扰circACTA2与全长FN1的结合。此外,我们设计一段可以与FN1结合的寡核苷酸,封闭FN1分子上与circACTA2结合的位点后,再用抗FN1抗体检查其对circACTA2的富集作用。结果显示,这段具有封闭作用的寡核苷酸明显抑制了FN1抗体对circACTA2的富集。接着,我们观察GFP-FN1_{（183-275）}对细胞骨架的影响。鬼笔环肽染色结果显示,转染GFP-FN1_{（183-275）}使细胞肌丝明显减少,并且GFP-FN1_{（183-275）}还可以抑制过表达circACTA2引起的肌丝增加。提示GFP-FN1_{（183-275）}通过干扰circACTA2和FN1相互作用而抑制circACTA2对VSMC收缩的诱导。3 FN1通过与SPTBN1相互作用调节VSMC收缩我们进一步通过Co IP-MS实验来寻找可以与FN1相互作用的蛋白质。将VSMC分为对照组和AngⅡ处理组,收集细胞裂解后加入抗FN1抗体,然后对免疫沉淀物进行质谱分析。结果显示,在AngⅡ处理的细胞中有14种蛋白质与FN1发生特异性结合,包括SPTBN1、CALU等。其中SPTBN1引起了我们的兴趣。SPTBN1属于细胞骨架蛋白,通过与肌动蛋白结合形成细胞骨架。因此我们设想,FN1通过与SPTBN1相互作用调节细胞收缩。我们进一步用Co IP分析验证质谱分析结果,发现FN1确实与SPTBN1相互结合,并且AngⅡ促进二者的结合。免疫双荧光染色结果也显示,FN1与SPTBN1在胞浆共定位,circACTA2过表达增加FN1与SPTBN1的相互作用,敲低FN1则抑制它们的结合。另外,Co IP实验结果显示,当细胞转染GFP-FN1_{（183-275）}后,FN1与SPTBN1的结合作用减弱。这些结果表明,circACTA2通过调节FN1与SPTBN1相互作用促进VSMC收缩。小结:综上所述,一方面,circACTA2通过吸附miR-149,解除其对FN1表达的抑制而上调FN1表达;另一方面,circACTA2通过与FN1结合而调节FN1与SPTBN1的相互作用,进而促进VSMC收缩。第三部分circACTA2通过与ILF3相互作用促进AngⅡ诱导的VSMC衰老目的:揭示circACTA2介导AngⅡ诱导VSMC衰老的分子机制。方法:1用AngⅡ刺激VSMC和/或在VSMC中过表达或敲低circACTA2,SA-β-gal染色检查细胞衰老、Western blot检测p21、p16和CDK4的表达。2在VSMC中过表达或敲低ILF3,SA-β-gal染色检查细胞衰老、Western blot检测ILF3、p21、p16和CDK4的表达,Brd U掺入实验检测细胞增殖。3在VSMC中过表达或敲低ILF3,qRT-PCR检测CDK4 m RNA的表达。4在VSMC中过表达或敲低ILF3,然后加入放线菌素D,作用不同时间后,qRT-PCR检测CDK4 m RNA表达。5 GST pull-down检测CDK4、cyclin D1、cyclin E1 m RNA与ILF3的结合。6 RNA pull-down检测CDK4、cyclin D1、cyclin E1 m RNA与ILF3的结合。7细胞经AngⅡ处理后,GST pull-down和RNA pull-down检测CDK4m RNA或circACTA2与ILF3结合的变化。8在VSMC中过表达circACTA2的同时过表达ILF3,然后加入放线菌素D,作用不同时间后,qRT-PCR检测CDK4 m RNA表达。9在VSMC中过表达circACTA2的同时过表达ILF3,SA-β-gal染色检查细胞衰老。结果:1 AngⅡ通过上调circACTA2表达促进VSMC衰老我们前部分研究证实,AngⅡ显著上调circACTA2在VSMC中的表达,许多研究也报道,AngⅡ能够诱导VSMC的衰老。为此,我们想进一步研究circACTA2是否参与AngⅡ诱导的VSMC衰老的发生与发展过程。HASMC被10^-7mol/L AngⅡ刺激72h,用β-半乳糖苷酶（SA-β-gal）染色试剂盒检查SA-β-gal活性。结果显示,与对照组相比,AngⅡ处理的细胞被染成蓝色的衰老细胞明显增多,结果具有统计学意义。接着,我们利用Western blot检测衰老标志基因p21和p16的表达。结果显示,AngⅡ处理后,VSMC中p21和p16蛋白表达明显增加。而且,细胞周期特异性蛋白CDK4表达明显下调,这些结果说明,AngⅡ促进VSMC的衰老。我们进一步检查circACTA2过表达对细胞衰老的影响。结果显示,在circACTA2过表达的细胞中SA-β-gal活性升高,衰老标志基因p21和p16的表达明显上调。反之,用sh RNA敲低circACTA2表达后,SA-β-gal活性降低,p21和p16表达明显下降。这些结果表明,circACTA2过表达促进细胞衰老。接着,我们在给予VSMC AngⅡ处理的同时,敲低circACTA2的表达。SA-β-gal染色和衰老标志基因表达检测结果显示,敲低circACTA2部分抵消了AngⅡ对细胞衰老的促进作用。这些结果说明,circACTA2介导AngⅡ对细胞衰老的诱导作用。2 circACTA2通过与ILF3相互作用促进AngⅡ诱导的细胞衰老我们进一步研究circACTA2介导AngⅡ诱导的细胞衰老是否与circACTA2结合蛋白有关。在前面的实验中,我们发现,circACTA2可以与ILF3结合,于是我们检测ILF3对细胞衰老的影响。用sh ILF3敲低ILF3表达后,SA-β-gal染色阳性细胞增多,衰老标志基因p21和p16表达明显增加,CDK4表达减少。我们还检测了ILF3对细胞增殖的影响。Brd U掺入实验结果显示,敲低ILF3抑制细胞增殖。反之,当过表达ILF3后,SA-β-gal染色阳性细胞减少,p21和p16表达下调,CDK4表达上调。结果说明,ILF3抑制VSMC衰老。接下来,我们在给予细胞AngⅡ刺激的同时过表达ILF3。结果显示,过表达ILF3部分抵消了AngⅡ对细胞衰老的诱导作用。这些结果说明,circACTA2通过与ILF3相互作用促进AngⅡ对细胞衰老的诱导作用。3 ILF3通过稳定CDK4 m RNA而上调CDK4表达,进而抑制细胞衰老我们进一步研究ILF3如何影响细胞衰老。在前面的实验中我们发现,ILF3上调CDK4的表达。因此我们推测,ILF3可能通过影响CDK4的表达抑制细胞衰老。qRT-PCR结果显示,ILF3过表达促进VSMC中CDK4 m RNA的表达,而敲低ILF3则作用相反。ILF3是一种RNA结合蛋白,可以结合不同种类的RNA。有文献报道,ILF3可以结合cyclin E1的3’UTR,发挥稳定m RNA的作用。因此我们推测,ILF3可能与CDK4 m RNA的3’UTR结合,对其m RNA起稳定作用。我们的结果显示,敲低ILF3增强放线菌素D对CDK4 m RNA水平的下调作用,相反,ILF3过表达部分抑制了放线菌素D对CDK4 m RNA水平的下调。提示ILF3通过稳定CDK4 m RNA而上调CDK4表达。我们进一步验证ILF3是否确实能够结合CDK4 m RNA。首先,Co IP实验证明,抗ILF3抗体能够沉降足够的内源性ILF3。然后,我们通过体外的RIP实验,检测被带有GST标签的ILF3（GST-ILF3）所富集的m RNA。qRT-PCR结果显示,cyclin E1和CDK4 m RNA被显著富集,而cyclin D1m RNA含量很少。我们再通过RNA pull-down实验,在细胞内加入cyclin D1、cyclin E1和CDK4 m RNA的3’UTR探针后,检测它们与ILF3的结合。结果显示,cyclin E1和CDK4 m RNA的3’UTR能与ILF3结合,而cyclin D1m RNA无明显结合,与RIP结果相一致。4被AngⅡ上调的circACTA2通过与CDK4 m RNA竞争性结合ILF3而促进细胞衰老由于circACTA2与CDK4 m RNA都可以与ILF3结合,我们推测它们之间的结合可能存在竞争关系。为了证实这一推测,我们用AngⅡ处理细胞后进行RIP实验。利用GST-ILF3检测其与cyclin D1和CDK4 m RNA及circACTA2的结合。qRT-PCR结果显示,与对照组相比,AngⅡ刺激明显增加GST-ILF3对circACTA2的富集,而对CDK4 m RNA的富集减少,对cyclin D1 m RNA的富集无明显影响。进一步,我们在给予细胞AngⅡ刺激后,进行RNA pull-down实验,检测AngⅡ对CDK4 m RNA和circACTA2沉降ILF3蛋白的影响。结果显示,AngⅡ作用后,circACTA2与ILF3的结合明显增加,而CDK4 m RNA与ILF3的结合显著减少。这些结果提示,circACTA2和CDK4 m RNA与ILF3之间的结合存在竞争性关系,即AngⅡ促进circACTA2与ILF3结合的同时,抑制CDK4 m RNA与ILF3的结合。我们进一步研究circACTA2与ILF3结合对CDK4 m RNA稳定性的影响。细胞分别过表达ILF3或circACTA2,或者同时过表达ILF3和circACTA2后,加入放线菌素D抑制转录过程。我们发现,与对照组相比,过表达circACTA2显著抑制放线菌素D对CDK4 m RNA水平的下调,而过表达ILF3作用相反;同时过表达ILF3和circACTA2时,circACTA2部分抵消了ILF3对CDK4 m RNA的稳定作用。这些结果提示,circACTA2过表达通过减少ILF3与CDK4 m RNA的结合而降低CDK4 m RNA的稳定性。我们最后检查circACTA2和ILF3对VSMC衰老的影响。SA-β-gal染色结果显示,circACTA2过表达使SA-β-gal染色阳性的衰老细胞增多;单独过表达ILF3减少衰老细胞的数量;当circACTA2和ILF3同时过表达时,ILF3部分抵消circACTA2对细胞衰老的诱导作用。小结:综上所述,AngⅡ促进circACTA2与ILF3结合的同时,抑制CDK4m RNA与ILF3结合,进而使CDK4 m RNA稳定性降低;CDK4因表达下调而导致VSMC衰老。结论:1在VSMC,AngⅡ显著诱导circACTA2的形成,后者通过与FN1相互作用促进VSMC收缩。2一方面,circACTA2通过吸附miR-149,解除其对FN1表达的抑制而上调FN1表达;另一方面,circACTA2通过与FN1结合而调节FN1与SPTBN1的相互作用,进而促进VSMC收缩。3 AngⅡ促进circACTA2与ILF3结合的同时,抑制CDK4 m RNA与ILF3结合,进而使CDK4 m RNA稳定性降低;CDK4因表达下调而导致VSMC衰老。更多还原

【Abstract】 Vascular smooth muscle cells（VSMC）play important roles in the physiological functions of vessels.VSMCs and the excellular matrix synthesized and secreted by VSMCs are the main regulators of vascular contraction.They can maintain the peripheral vascular resistance,keep the blood pressure and control the distribution of blood flow pumped by heart.VSMCs are not only the regulators,but also the effectors.They are the sensors of vasoactive substances and mechanical forces,and then transduce them into various signals which can be recognized by the cells.Therefore,VSMCs are the focus of vascular studies due to their vital structures and functions.Hypertension,which has affected more than a quarter of the world’s adult population in 2000,and the proportion is expected to rise to 29%by 2025,is a worldwide public-health problem.As a leading risk factor for mortality,hypertension can result in the development of various related complications,such as stroke and vascular diseases.The mechanisms that underlie hypertension are widely investigated but still remain unclear.VSMC contraction which leads to increased vascular resistance is one of the mechanisms of hypertension.Cellular senescence is traditionally defined as a permanent and irreversible proliferative arrest state of cells.Senescent cells exhibit large and flattened morphological characteristics,increased gene expression of p53,p21and p16,which are negative regulators of the cell cycle,as well as increased activity of senescence-associatedβ-galactosidase（SA-β-gal）,a biomarker of cellular senescence.Senescence-associated secretory phenotype（SASP）is also included.Emerging data indicate that hypertension and atherosclerosis are significantly associated with senescence of VSMCs.Circular RNAs（circRNA）are a novel type of noncoding RNAs,characterized by covalently closed loop structures without free terminals.The circular structure,which is formed by the backsplicing,makes circRNAs more stable than linear m RNAs.circRNAs play key roles in many biological and pathological processes.Most of circRNAs located in the cytoplasm act as miRNA sponges,protein sponges,or protein scaffolds,a few circRNAs stay in the nucleus regulating splicing and transcription.Furthermore,circRNAs containing particular sequences can even be translated into proteins or peptides.Emerging studies demonstrate that circRNAs are implicated in cardiovascular diseases,such as hypertension.For instance,the circulating levels of c ZNF609 were down-regulated in patients with hypertension.circRNAs are also involved in cellular senescence.circ-Foxo3 promotes cardiac senescence by binding to anti-senescent proteins.circACTA2 is a circular RNA found in VSMCs by our laboratory,and the mechanism underlying circACTA2-regulated VSMC functions is still unknown.In this study,we focused on the roles of circACTA2 in VSMC contraction and senescence and investigated the underlying mechanisms of circACTA2 action.PartⅠFN1 mediates circACTA2-regulated VSMC contractionObjective:To explore the effect of AngⅡstimulation on circACTA2expression and the roles of circACTA2 in VSMC contraction.Methods:1 VSMCs were treated with AngⅡor PDGF-BB and the expression of circACTA2 was detected by qRT-PCR.2 The expression of circACTA2 in vessels of hypertension patients was detected by qRT-PCR and fluorescence in situ hybridization（FISH）.3 VSMCs were transfected with circACTA2 and/or treated with AngⅡ.The actin stress fibers were observed by confocal microscopy after phalloidin staining.4 circACTA2-interacting proteins were captured and identified by combining RNA antisense purification（RAP）or U-biotin-labeled RNA pull-down with quantitative liquid chromatography-mass spectrometry（LC-MS）.5 circACTA2 and FN1 interaction was examined by RNA pull-down and RNA binding protein immunoprecipitation（RIP）.6 VSMCs were transfected with circACTA2,or treated with AngⅡaccompanied by si FN1 transfection.The expression of FN1 was examined by Western blot,and actin stress fibers were observed by confocal microscopy after phalloidin staining.7 VSMCs were transfected with circACTA2 accompanied by si FN1transfection.Cell contraction induced by acetyl choline（Ach）was examined.8 Cells were treated with AngⅡ,and vessels were obtained from hypertension patients.Immunofluorescence staining was performed to detect the co-localization and interaction between circACTA2 and FN1.Results:1 The expression of circACTA2 is upregulated by AngⅡWe first demonstrated that circACTA2 expression was up-regulated by AngⅡtreatment and down-regulated by PDGF-BB treatment.Both qRT-PCR and fluorescence in situ hybridization（FISH）indicated that circACTA2expression in the vessels of hypertension patients was significantly higher than that in normal controls.Then we explored whether circACTA2 influences cytoskeleton organization.VSMCs were treated with AngⅡor transfected with lentivirus encoding circACTA2 alone,or treated with them together,then phalloidin staining was performed.We found a strong F-actin cable formation in AngⅡ-treated or circACTA2-overexpressed cells,and the effect was further strengthened in cells treated with them together.2 circACTA2 interacts with FN1 and ILF3To identify specific interactors with circACTA2,we quantitatively compared the proteins captured with antisense oligonucleotides or U-biotin-labeled circACTA2 with their corresponding control-captured proteins.We analyzed biological replicate purifications in the two methods,quantifying 181 proteins that were identified by more than two unique peptides in each method.We reproducibly identified the part of strongly enriched proteins that met our enrichment criteria（mean log2>2.0,P<0.05,moderated t-test）,and confirmed that circACTA2 interaction with FN1,VIM and ILF3,but notβ-actin,was highly specific.Their interaction was further validated by RNA-binding protein immunoprecipitation（RIP）.3 circACTA2 regulates VSMC contraction by stabilizing FN1To explore whether circACTA2 affects the expression of the proteins interacting with it,VSMCs were transfected with circACTA2.The results showed that the expression of FN1 significantly increased while the expression of ILF3 and VIM did not.When cells were treated with different concentrations of AngⅡ(0,10^-7,2×10^-7,4×10^-7mol/L),the expression of FN1 was increased in an Ang II dose-dependent manner.The results indicate that FN1 is stabilized by circACTA2.Then we designed a si FN1 to knock down FN1 expression.As shown by Western blot,the elevation of FN1expression caused by AngⅡwas abrogated by FN1 knockdown,and VSMCs transfected with circACTA2 exhibited the same results.We further examined the effect of FN1 on VSMC contraction.We found that increased F-actin cable formation in circACTA2-overexpressing cells was decreased by knockdown of FN1,as shown by phalloidin staining.And the Ach-induced cell contraction was alleviated by FN1 knockdown.Then we explore the mechanism whereby VSMC contraction is regulated by circACTA2.FISH experiments showed that circACTA2 and FN1 were co-localized in the cytoplasm,and AngⅡtreatment enhanced their interaction.Summary:AngⅡinduces circACTA2 formation and the latter regulates VSMC contraction by interacting with FN1.PartⅡcircACTA2 promotes VSMC contraction via regulating FN1 and SPTBN1 interactionObjective:To explore the molecular mechanism whereby circACTA2upregulates FN1 expression and regulates the interaction of FN1 with SPTBN1.Methods:1 RNA pull-down and high throughput sequencing were used to detect miRNAs binding to circACTA2.2 miRNAs binding to circACTA2 were confirmed by RNA pull-down and qRT-PCR.3 The binding sites of miR-149 on the FN1 3’UTR were predicted by bioinformatic analysis.The expression of FN1 in cells transfected with miR-149 mimic or inhibitor was detected by Western blot analysis.4 Dual luciferase reporter gene assay was used to confirm the interaction between miR-149 and FN1 3’UTR.5 VSMCs were transfected with circACTA2 accompanied by knockdown of miR-149,cell contraction induced by Ach was examined,and the formation of actin stress fibers was observed by confocal microscopy after phalloidin staining.6 The binding sites between FN1 and circACTA2 were predicted by bioinformatic analysis.7 The expression of GFP-FN1_{（183-275）}and FN1 in VSMCs transfected with GFP-FN1_{（183-275）}was examined by Western blot analysis.8 VSMCs were transfected with GFP-FN1_{（183-275）}accompanied by AngⅡtreatment,and the expression of FN1 was examined by Western blot analysis.9 VSMCs were transfected with GFP-FN1_{（183-275）},and circACTA2binding to FN1_{（183-275）}was detected by RIP.10 VSMCs were transfected with circACTA2 accompanied by GFP-FN1_{（183-275）}or blocking oligonucleotide transfection,circACTA2 binding to FN1 was detected by RIP.11 VSMCs were transfected with circACTA2 accompanied by GFP-FN1_{（183-275）}transfection,actin stress fibers were observed by confocal microscopy after phalloidin staining.12 Proteins binding to FN1 were detected by Co IP-MS.13 VSMCs were transfected with GFP-FN1_{（183-275）}or treated with AngⅡ,the interaction of FN1 with SPTBN1 was detected by coimmunoprecipitation（Co IP）.14 VSMCs were transfected with circACTA2 accompanied by FN1knockdown,the co-localization and interaction of FN1 with SPTBN1 were examined by immunofluorescence staining.Results:1 circACTA2 upregulates the expression of FN1 by sponging miR-149RNA pull-down and high throughput sequencing were used to identify miRNAs sponed by circACTA2.There were 24 miRNAs enriched by circACTA2 probe.Then qRT-PCR was used to examine the expression of miRNAs in the precipitates of oligo pull-down.The results showed that miR-27b-3p,miR-27b-5p,miR-149,and miR-197 were significantly elevated in the precipitates of circACTA2 probe.The interaction of circACTA2 and miR-149 was further verified by dual luciferase reporter assay.Furthermore,the expression of miR-149 was upregulated in AngⅡ-treated VSMCs and downregulated in PDGF-BB-treated cells.We found that there is a binding site of miR-149 on FN1 3’UTR.Then we designed miR-149 mimic and inhibitor and confirmed that miR-149 mimic inhibited the expression of FN1 while the inhibitor promoted its expression.The interaction of FN1 and miR-149 was further verified by dual luciferase reporter assay.These results suggested that circACTA2 upregulates FN1expression via sponing miR-149.Moreover,increased F-actin cable formation in circACTA2-overexpressing VSMCs was further increased by miR-149inhibitor,as shown by phalloidin staining.And the Ach-induced cell contraction in circACTA2-overexpressing VSMCs was enhanced by miR-149inhibitor.2 VSMC contraction induced by circACTA2 is inhibited by interfering with circACTA2 and FN1 interactionWe predicted that the oligo-peptide residing in 237-252aa of FN1 might be a binding site of circACTA2.To test this,a DNA sequence,which could be translated into this oligo-peptide,was recombined with GFP-C1,and named GFP-FN1_{（183-275）}.After transfected with GFP-FN1_{（183-275）},cells could express the oligo-peptide containing 183-275aa of FN1,as confirmed by Western blot.Then VSMCs were transfected with GFP-C1 or GFP-FN1_{（183-275）},and RIP-PCR was performed to examine circACTA2 enrichment in the precipitates pulled down by anti-GFP antibody.circACTA2 enrichment in GFP-FN1_{（183-275）}–overexpressing cells was much higher than that in GFP-C1 control cells,indicating that GFP-FN1_{（183-275）}binds to circACTA2.Importantly,transfection of GFP-FN1_{（183-275）}inhibited the expression of FN1.And transfection of GFP-FN1_{（183-275）}alleviated the upregulation of FN1 expression induced by AngⅡ.RIP experiment showed that transfection of GFP-FN1_{（183-275）}reduced circACTA2 enrichment which was enhanced by circACTA2 overexpression.These results indicated that GFP-FN1_{（183-275）}disturbed the interaction of circACTA2 with FN1.Oligonucleotide was used to block the binding site in another RIP experiment,and it did inhibit circACTA2 enrichment which was enhanced by circACTA2 overexpression.Then we examined the cytoskeleton organization after GFP-FN1_{（183-275）}transfection.After phalloidin staining,we found that increased F-actin cable formation in circACTA2-overexpressing cells was abolished by GFP-FN1_{（183-275）}transfection.3 The contraction of VSMCs is regulated by FN1 and SPTBN1interactionTo identify the proteins interacted with FN1,we treated VSMCs with or without AngⅡ.There were 14 proteins interacted with FN1 in AngⅡtreated-VSMCs,including SPTBN1 and CALU among them.As SPTBN1belongs to a cytoskeletal protein,it is of great interest to us.Co IP experiment confirmed that FN1 and SPTBN1 interacted with each other,and AngⅡtreatment enhanced their interaction.Immunofluorescent staining exhibited the colocalization of FN1 with SPTBN1 in the cytoplasm,and their interaction was enhanced by circACTA2 overexpression while inhibited by si FN1.Moreover,Co IP experiment also showed that the interaction of FN1 with SPTBN1 was reduced by GFP-FN1_{（183-275）}transfection.Summary:circACTA2 promotes the expression of FN1 via sponging miR-149,and FN1 interacts with SPTBN1 to regulate VSMC contraction.PartⅢ circACTA2 promotes AngⅡ-induced VSMC senescence via interacting with ILF3Objective:To explore the molecular mechanism whereby circACTA2medietes AngⅡ-induced VSMC senescence.Methods:1 VSMCs were treated with AngⅡ,and/or were transfected with circACTA2 or sh-circACTA2,VSMC senescence was examined by SA-β-gal staining.The senescent cells were observed by light microscope.The expressions of p21,p16 and CDK4 were detected by Western blot analysis.2 VSMCs were transfected with ILF3-expressing vector or sh-ILF3accompanied by AngⅡtreatment,VSMC senescence was examined by SA-β-gal staining.The senescent cells were observed by light microscope.The expressions of p21,p16 and CDK4 were detected by Western blot analysis.3 VSMCs were transfected with ILF3-expressing vector or sh-ILF3,and the expression of CDK4 m RNA was detected by qRT-PCR.4 VSMCs were transfected with ILF3-expressing vector or sh-ILF3accompanied by actinomycin D treatment,and the expression of CDK4m RNA was detected by qRT-PCR.5 The interaction of CDK4,cyclin D1,and cyclin E1 m RNA with ILF3was detected by GST pull-down.6 The interaction of CDK4,cyclin D1,and cyclin E1 m RNA with ILF3was detected by RNA pull-down.7 The interaction between CDK4 m RNA or circACTA2 and ILF3 was detected by GST pull-down and RNA pull-down.8 VSMCs were transfected with circACTA2 accompanied by ILF3overexpression,the expression of CDK4 m RNA was detected by qRT-PCR.9 VSMCs were transfected with circACTA2 accompanied by ILF3overexpression,and VSMC senescence was examined by SA-β-gal staining.The senescent cells were observed by light microscope.Results:1 circACTA2 mediates AngⅡ-induced VSMC senescenceVSMCs were treated with AngⅡ,then SA-β-gal staining was used to detect cellular senescence.The results showed that there were more senescent cells in AngⅡ-treated group than in control group.And the expression of p21and p16 was upregulated.The expression of CDK4 was downregulated.These results indicated that AngⅡinduces VSMC senescence.Then we explored the function of circACTA2 in cellular senescence.We found that the expression of p21 and p16 was also upregulated in circACTA2-overexpressing VSMCs.These results suggested that circACTA2 also induces VSMC senescence.Then circACTA2 expression in VSMCs was knocked down,and at the same time VSMCs were treated with AngⅡ.SA-β-gal staining showed that circACTA2 knockdown abrogated the cellular senescence induced by AngⅡtreatment.The upregulation of p21 expression was reduced,while the expression of CDK4 exhibited the opposite alteration.These results suggested that circACTA2 is involved in VSMC senescence induced by AngⅡtreatment.2.AngⅡ-induced VSMC senescence is mediated by circACTA2 and ILF3 interactionWe speculated that circACTA2 could exert its function in VSMC senescence through interaction with proteins.Because ILF3 was one of the proteins that interact with circACTA2,we examined its effect on cellular senescence.When VSMCs were transfected with sh ILF3 to knock down the expression of ILF3,the proportion of cells positive for SA-βgal activity was increased.Western blot analysis showed an increased expression of p21 and p16 and reduced level of CDK4.Brd U incorporation assay indicated that cell proliferation was inhibited by ILF3 knockdown.On the contrary,the proportion of cells positive for SA-βgal activity was decreased by ILF3overexpression.The expression of p21 and p16 in ILF3-overexpressing VSMCs was downregulated,and the expression of CDK4 was increased.These results indicated that ILF3 inhibits cellular senescence.Then ILF3-overexpressing VSMCs were treated with AngⅡ.We found that ILF3overexpression significantly inhibited cellular senescence promoted by AngⅡ.These results suggested that circACTA2 mediates AngⅡ-induced VSMC senescence via interacting with ILF3.3 ILF3 inhibits VSMC senescence via stabilizing CDK4 m RNAThen we explored the molecular mechanism whereby ILF3 inhibits cellular senescence.Because ILF3 increased the expression level of CDK4,we speculated that CDK4 could be involved in cellular senescence induced by AngⅡ.The level of CDK4 m RNA was upregulated by ILF3 overexpression while downregulted by ILF3 knockdown.As a RNA-binding protein,ILF3can bind various types of RNA molecules.It was reported that ILF3 was able to bind to the 3’UTR of cyclin E1,thus stabilizing cyclin E1 m RNA.We speculated that ILF3 was also able to bind to the 3’UTR of CDK4 m RNA.After actinomycin D was used to block transcription,ILF3 knockdown obviously decreased CDK4 m RNA level,while ILF3 overexpression increased CDK4 m RNA level.We further examined whether ILF3 bound to 3’UTR of CDK4 m RNA.Firstly,Co IP was performed to confirm that enough endogenous ILF3 was pulled down by anti-ILF3 antibody.Next,in vitro RIP experiments were performed to examine m RNA binding to ILF3.qRT-PCR showed that m RNA of cyclin E1 and CDK4 was enriched in the precipitates,but cyclin D1 m RNA was hardly detected in the precipitates.The results were further confirmed by RNA pull-down.These results indicated that 3’UTR of cyclin E1 and CDK4m RNA,but not cyclin D1 was able to bind to ILF3.4 circACTA2 promotes VSMC senescence by competing with CDK4m RNA for ILF3 bindingBecause both circACTA2 and CDK4 m RNA binds to ILF3,we speculated that circACTA2 could compete with CDK4 m RNA for ILF3binding.Thus,RIP experiments were performed after AngⅡtreatment.The results showed that increased enrichment of circACTA2 but reduced enrichment of CDK4 m RNA could be found in the precipitations of AngⅡ-treated VSMCs.The enrichment of cyclin D1 m RNA did not change.Then RNA pull-down was performed.The results demonstrated that the interaction of circACTA2 with ILF3 was enhanced while the interaction of CDK4 m RNA with ILF3 was reduced in AngⅡ-treated VSMCs.These indicated that circACTA2 competes with CDK4 m RNA for ILF3 binding.We then examined the stability of CDK4 m RNA affected by circACTA2 and ILF3and found that circACTA2 overexpression reduced CDK4 m RNA stability while ILF3 overexpression facilitated CDK4 m RNA stability.These findings indicated that circACTA2 decreases CDK4 m RNA stability via reducing interaction of ILF3 with CDK4 m RNA.Finally,we examined VSMC senescence affected by circACTA2 and ILF3.The results showed that ILF3overexpression significantly suppressed cellular senescence promoted by circACTA2.Summary:In summury,circACTA2 competes with CDK4 m RNA for ILF3 binding,then decreases the stability of CDK4 m RNA and thus promotes VSMC senescence.Conclusion:1 AngⅡinduces circACTA2 formation and the latter regulates VSMC contraction by interacting with FN1.2 circACTA2 promotes the expression of FN1 via sponging miR-149,and FN1 interacts with SPTBN1 to regulate VSMC contraction.3 circACTA2 competes with CDK4 m RNA for ILF3 binding,then decreases the stability of CDK4 m RNA and thus promotes VSMC senescence.更多还原