【作者】 王靖宇；

【导师】 高颖；

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

【摘要】 背景与目的:心血管疾病是当今严重威胁人类生命健康的最主要疾病,其中,动脉粥样硬化(Atherosclerosis,AS)则是诱发心血管疾病发生发展最常见、最重要的病理基础。AS是一种非传染的慢性炎症性疾病,起始步骤通常是内皮细胞(Endothelial cell,EC)受损导致功能障碍,致使血小板和单核细胞聚集到内皮下,并释放包括血小板衍生生长因子(Platelet-derived growth factor,PDGF)在内的炎性细胞因子。各种细胞因子进一步刺激血管平滑肌细胞(Vascular smooth muscle cells,VSMCs)去分化为合成型表型,促进VSMCs增殖迁移,并从中膜层移行至内膜层,从而加速动脉粥样硬化的发展。另外,VSMCs在晚期动脉粥样硬化斑块中是有益的,它可以稳定并防止纤维帽破裂。微小RNA(microRNAs,miRNAs)是一类长约20个左右核苷酸大小的内源性非编码RNA。miRNAs通过在转录后水平与靶基因mRNA3’-非翻译区(3’-Untranslated Region,3’-UTR)结合,诱导mRNA的降解或翻译抑制,实现对特定基因的沉默,从而对生物体内基因的表达起到精细的调节作用。已报道许多miRNA在心血管系统中表达,并参与调节血管平滑肌细胞的功能。miRNA-520c-3p是miRNA-520家族的成员之一,该家族在染色体上位于19q13.42。目前研究发现,miRNA-520c在各种人类癌症中发挥重要作用,包括弥漫性大B细胞淋巴瘤、乳腺癌、纤维肉瘤和肝细胞癌。对乳腺癌的研究表明,miRNA-520c可通过抑制CD44促进癌细胞在体外和体内的迁移和侵袭。miRNA-520c-3p通过下调NLRP3抑制先兆子痫中NLRP3炎性体激活和炎症反应。然而,miRNA-520c-3p在动脉粥样硬化和血管平滑肌细胞中的作用还未见报道。本研究的目的旨在探讨miRNA-520c-3p在血管平滑肌细胞和动脉粥样硬化中的作用。体外实验是在PDGF刺激下,通过干扰miRNA-520c-3p的表达水平,研究 miRNA-520c-3p 对人主动脉血管平滑肌细胞(Human aortic smooth muscle cells,HASMCs)功能的影响及其分子机制。采用生物信息学、miRNA pulldown和萤光酶素报告基因系统寻找miRNA-520c-3p的靶基因,并对靶基因进行功能分析和恢复实验以了解miRNA-520c-3p是否通过该靶基因发挥作用。体内实验是通过构建载脂蛋白E缺失型(Apolipoprotein E,ApoE-/-)C57BL小鼠的AS模型和尾静脉注射miRNA-520c-3p agomir的AS干预模型,观察分析动脉粥样硬化斑块的大小和性质,探究miRNA-520c-3p对动脉粥样硬化的影响。以期更好的理解动脉粥样硬化的发病机制,为动脉粥样硬化的预防和治疗提供理论基础。方法:第一部分通过CCK-8法检测PDGF-BB对HASMCs增殖的影响,采用Western blot法检测PDGF-BB对HASMCs收缩型标志蛋白calponin和合成型标志蛋白OPN的影响。采用RT-qPCR法检测PDGF-BB对HASMCs中miRNA-520c-3p表达的影响。通过脂质体瞬时转染 miRNA-520c-3p mimics 或 miRNA-520c-3p inhibitor 改变miRNA-520c-3p在HASMCs中的表达水平,然后进行一系列功能实验:采用MTT和 EdU 法,检测 miRNA-520c-3p mimics 或 miRNA-520c-3p inhibitor 对 PDGF-BB介导的HASMCs增殖的影响;分别采用Boyden Chamber和流式细胞术,检测miRNA-520c-3p mimics对PDGF-BB介导的HASMCs迁移和周期的影响;利用Western blot 法检测 miRNA-520c-3p mimics 对周期蛋白 cyclin B1、CDK1、PCNA和cyclin D1表达的影响。第二部分采用 RT-qPCR 和 Western blot 法检测 miRNA-520c-3p mimics 对 RelA/p65 mRNA含量、蛋白质表达、磷酸化水平及细胞核内含量的影响。采用miRNA pull-down实验,检测HASMCs中miRNA-520c-3p与RelA/p65是否发生直接结合;采用双萤光素酶报告系统,探究HASMCs中miRNA-520c-3p是否能直接结合RelA/p65 3’-UTR 区,RelA/p65 是否是 miRNA-520c-3p 的靶基因;采用 RT-qPCR法检测不同时间点放线菌素D(actinomycinD,ActD)处理的HASMCs中RelA/p65 mRNA的相对含量,分析miRNA-520c-3p mimics对RelA/p65 mRNA半衰期的影响。通过脂质体瞬时转染siRelA/p65抑制RelA/p65在HASMCs中的表达,然后进行一系列功能实验:采用CCK-8和EdU法,检测siRelA/p65对PDGF-BB介导的HASMCs增殖的影响;分别采用Boyden Chamber和流式细胞术,检测siRelA/p65对PDGF-BB介导的HASMCs迁移和周期的影响;利用Western blot法检测siRelA/p65对周期蛋白cyclin B1、CDK1、PCNA和cyclin D1含量的影响。恢复实验将 miRNA-520c-3p mimics 和 pcDNA3.1-RelA/p65 共转染入 HASMCs 中,进行上述一系列的功能实验,探究共转染miRNA-520c-3p mimics和pcDNA3.1-RelA/p65与共转染miRNA-520c-3pmimics和pcDNA3.1对HASMCs增殖、迁移和周期及相关周期蛋白的影响。第三部分将60只雄性8周龄ApoE-/-C57BL小鼠随机均分为四组:(1)正常饮食+对照组(Normal diet+agomir negative control,ND+agomir NC),(2)正常饮食+实验组(Normal diet+agomir miRNA-520c-3p,ND+agomir miRNA-520c-3p),(3)高脂饮食+对照组(Hight-fat diet+agomir negative control,HFD+agomir NC),(4)高脂饮食+实验组(Hight-fat diet+agomir miRNA-520c-3p,HFD+agomir miRNA-520c-3p),构建ApoE-/-C57BL小鼠的AS模型和联合尾静脉注射miRNA-520c-3p agomir干预的AS模型。通过RT-qPCR检测小鼠主动脉中miRNA-520c-3p的表达。通过HE(Hematoxylin-eosin,HE)染色来观察血管病变情况。采用免疫组织化学方法检测小鼠主动脉瓣切片中RelA/p65的蛋白表达,探究miRNA-520c-3pagomir对血管壁中RelA/p65表达的影响。采用酶标仪比色法检测小鼠血清中CHO、TG、LDL-C、HDL-C的表达情况。通过马松(Masson)染色来观察血管壁胶原纤维含量。通过组织免疫荧光(Immunohistofluorescence,IHF)染色PCNA检测血管壁中增殖细胞的含量。结果:第一部分CCK-8结果显示,PDGF-BB可以促进HASMCs增殖,且10ng/mL或20ng/mL PDGF-BB的促增殖能力较为明显。为明确PDGF-BB对HASMCs表型的影响,Western blot检测表型标志蛋白,结果显示PDGF-BB可增加增殖型标志蛋白OPN的表达,OPN的增加趋势在12h时较为明显;PDGF-BB可减少收缩型标志蛋白calponin的表达,calponin在各个时间点均呈现明显的递减趋势,5ng/mLPDGF-BB已足够减少calponin。RT-qPCR结果显示,PDGF-BB刺激可以抑制HASMCs中miRNA-520c-3p的表达,且10ng/mL PDGF-BB刺激HASMCs 12h时抑制效果最明显。MTT和EdU增殖实验结果显示,miRNA-520c-3p mimics能够抑制PDGF-BB介导的 HASMCs 增殖,而 miRNA-520c-3p inhibitor 则对 PDGF-BB 介导的 HASMCs增殖没有影响。Boyden Chamber实验结果显示,miRNA-520c-3p mimics能够抑制PDGF-BB介导的HASMCs迁移。流式细胞周期结果显示,miRNA-520c-3p mimics能抑制细胞G1期向S期的进展,同时使细胞阻滞在G2/M期。Western blot结果显示,miRNA-520c-3p mimics 能够抑制 PDGF-BB 诱导的 cyclin B1、CDK1、PCNA的蛋白表达,而不影响cyclin D1。第二部分RT-qPCR 和 Western blot 结果显示,miRNA-520c-3p mimics 显著降低了HASMCs中RelA/p65 mRNA和蛋白的表达,降低了磷酸化RelA/p65(S536)的表达,同时减少了细胞核中RelA/p65的含量。miRNA pull-down实验结果表明,miRNA-520c-3p mimics 对 RelA/p65 mRNA 的富集是对照组的 2 倍多,miRNA-520c-3p可以直接结合RelA/p65 mRNA。双萤光素酶报告实验结果显示,miRNA-520c-3p通过种子区直接结合RelA/p65 3’-UTR位点2,而不是位点1,说明RelA/p65是HASMCs中miRNA-520c-3p的靶基因。mRNA稳定性实验表明,miRNA-520c-3p降低了 HASMCs中RelA/p65 mRNA的稳定性,缩短了其半衰期。CCK-8、EdU细胞增殖和Boyden Chamber细胞迁移实验结果显示,siRelA/p65能够抑制PDGF-BB诱导的HASMCs增殖和迁移。流式细胞周期结果显示,siRelA/p65减少处于S期的细胞比例。Western blot结果显示,转染siRelA/p65可下调HASMCs中cyclin B1、CDK1、PCNA蛋白的表达。功能恢复实验结果显示,共转染miRNA-520c-3p mimics 和 pcDNA3.1-RelA/p65 可逆转共转染 miRNA-520c-3p mimics 和 pcDNA3.1对HASMCs增殖、迁移和细胞周期分布的抑制作用。第三部分RT-qPCR结果显示,注射miRNA-520c-3p agomir上调了小鼠主动脉中miRNA-520c-3p 的表达。HE 染色结果显示,miRNA-520c-3p agomir 能够减少 ApoE-/-C57BL小鼠的动脉粥样硬化斑块面积。RelA/p65的免疫组化结果显示,miRNA-520c-3p agomir可抑制小鼠VSMCs中RelA/p65的表达。miRNA-520c-3p agomir降低正常饮食组小鼠血清中TG的含量,但是没有显著影响小鼠血清中T-CHO、LDL、HDL的含量。Masson染色结果显示,miRNA-520c-3p agomir降低了小鼠血管壁胶原纤维的含量。PCNA组织免疫荧光结果显示,miRNA-520c-3p agomir减少了小鼠血管壁及斑块内PCNA阳性细胞数。结论:1.miRNA-520c-3p mimics抑制PDGF-BB介导的HASMCs增殖、迁移和周期进程,而miRNA-520c-3p inhibitor对细胞增殖功能和周期进程没有影响。2.miRNA-520c-3p 通过直接结合靶基因 RelA/p65 mRNA 3’-UTR 促进 HASMCs中RelA/p65 mRNA的降解,减少RelA/p65蛋白的表达。3.siRelA/p65抑制PDGF-BB介导的HASMCs增殖、迁移和周期进程。4.miRNA-520c-3p通过靶向抑制RelA/p65来调节HASMCs的增殖、迁移和周期进程。5.miRNA-520c-3p agomir可下调小鼠主动脉瓣RelA/p65的表达,减少小鼠动脉粥样硬化斑块面积、胶原含量和PCNA阳性细胞数。更多还原

【Abstract】 Background and Objective:Cardiovascular disease is the most important disease that seriously threatens human life and health.Among them,atherosclerosis(AS)is the most common and important pathological basis of cardiovascular disease.Atherosclerosis is a non-infectious chronic inflammatory disease.The initial step in the pathologic process of AS is generally endothelial cells(ECs)damaged and endothelial dysfunction.Damaged endothelial cells recruit platelets and monocytes into subendothelial and release the inflammatory cytokines,including platelet-derived growth factor(PDGF).Various cytokines stimuli further promotes vascular smooth muscle cells(VSMCs)from a differentiated,contractile state to a differentiated,synthetic phenotype.Aberrant VSMCs proliferate and migrate from medial arterial wall into the intimal layer and then accelerate atherosclerosis.In addition,VSMCs are beneficial in advanced AS plaques,promoting the stability and preventing rupture of the fibrous cap.MicroRNAs(miRNAs)are a class of endogenous non-coding RNA molecules with a length of about 20 nucleotides.miRNAs have been found to silence the expression of specific genes by binding to the 3’-untranslated region(3’-UTR)of target mRNAs to induce mRNA degradation and/or inhibit mRNA translation at the post-transcriptional level,and therefore regulation of gene expression can be finely controlled in the organisms.Many miRNAs have been reported to be expressed in the cardiovascular system and to carry out regulatory roles in the function of vascular smooth muscle cells.miRNA-520c-3p belongs to a family of miR-520/373 located on human chromosome 19ql3.42.Previous studies have shown that miRNA-520c plays an important role in various types of human cancers including diffuse large B-cell lymphoma,breast cancer,fibrosarcoma and hepatocellular carcinoma.Study of breast cancer showed that miRNA520c could promote cancer cell migration and invasion in vitro and in vivo by suppression of CD44.miRNA-520c-3p suppresses NLRP3 inflammasome activation and inflammatory cascade by downregulating NLRP3 in preeclampsia.However,the role of miRNA-520c-3p in atherosclerosis and vascular smooth muscle cells has not been reported.The purpose of the present study was to investigate the role of miRNA-520c-3p in vascular smooth muscle cells and atherosclerosis.In vitro,we studied the effects of miRNA-520c-3p on the function of PDGF-BB-stimulated human aortic smooth muscle cells(HASMCs)and its molecular mechanism by interfering the expression of miRNA520c-3p.Bioinformatics,miRNA pull-down and dual luciferase reporting system were used to find the target gene of miRNA-520c-3p in HASMCs.Then,functional experiments and recovery experiments were performed to find out whether miRNA-520c3p regulating the function of HASMCs by the target gene.In vivo,atherosclerotic mouse model was built with ApoE-/-mice and intervention group AS mouse model established with ApoE-/-mice by intravenous injection of miRNA-520c-3p agomir.The size and characterization of atherosclerotic plaques were observed and analyzed in ApoE-/-mice to explorer the effect of miRNA-520c-3p on atherosclerosis.Our results may provide a theoretical basis for a better understanding of the pathological process of atherosclerosis as well as potential prevention and therapeutic strategies in atherosclerosis.Methods:Part ⅠThe effect of PDGF-BB on the HASMCs proliferation was analyzed by CCK-8 assay.The effect of PDGF-BB on the expression of contractile phenotype marker proteins calponin and synthetic phenotypic marker protein osteopontin(OPN)in HASMCs was detected by Western blot.RT-qPCR was used to detect the expression of miRNA-520c3p in PDGF-BB-treated HASMCs.RT-qPCR analysis verified that miRNA-520c-3p was successfully overexpressed or knockdowned in HASMCs transiently transfected with miRNA-520c-3p mimics or miRNA-520c-3p inhibitor using lipofectamine 2000,and then perform a series of functional experiments in vitro.The effect of miRNA-520c-3p mimics or miRNA-520c-3p inhibitor on the proliferation of PDGF-BB-treated HASMCs were evaluated by MTT and EdU assay.The effect of miRNA-520c-3p mimics on the migration and cell cycle distribution of PDGF-BB-treated HASMCs were detected by Boyden chamber and Flow cytometry assay,respectively.Western blot was used to detect the effects of miRNA-520c-3p mimics on the expression of cell-cycle-related proteins,including cyclin B1,CDK1,PCNA and cyclin D1 in HASMCs.Part ⅡRT-qPCR and Western blot were used to detect the effects of miRNA-520c-3p mimics on mRNA and protein expression level of RelA/p65,phosphorylation of RelA/p65 at S536 and the amount of RelA/p65 protein in nucleus.Biotinylated miRNA pull-down assay was used to detect whether miRNA-520c-3p directly bind to RelA/p65 mRNA in HASMCs.Dual luciferase reporting system was used to investigate whether miRNA-520c-3p could direct targeting RelA/p65 mRNA 3’-UTR and verify whether RelA/p65 was the target gene of miRNA-520c-3p in HASMCs.The expression of RelA/p65 mRNA at different time points were detected by RT-qPCR in HASMCs treated with actinomycin D(Act D)to analyze the effect of miRNA-520c-3p mimics on the halflife of RelA/p65 mRNA.RT-qPCR analysis verified that RelA/p65 was successfully knockdowned in HASMCs transiently transfected with siRelA/p65 using lipofectamine 2000,and then perform a series of functional experiments in vitro.CCK-8 and EdU assay were used to evaluate the effect of siRelA/p65 on the proliferation of PDGF-BB-treated HASMCs.The effect of siRelA/p65 on the migration and cell cycle distribution of PDGF-BB-treated HASMCs were detected by Boyden chamber and Flow cytometry assay,respectively.Western blot was used to detect the effects of siRelA/p65 on the expression of cell-cyclerelated proteins,including cyclin B1,CDK1,PCNA and cyclin D1 in HASMCs.The same set of functional recovery experiments as described above were performed to detect cell proliferation,cell migration,cell cycle distribution and the expression of cell-cyclerelated proteins,when miRNA-520c-3p mimics were co-transfected into HASMCs with the pcDNA3.1 or pcDNA3.1-RelA/p65,respectively.Part ⅢSixty male 8-week-old apolipoprotein E-deficient(ApoE-/-)mice with a C57BL/6 background were randomly divided into four groups:(1)Normal diet+agomir negative control(ND+agomir NC),(2)Normal diet+agomir miRNA-520c-3p(ND+agomir miRNA-520c-3p),(3)Hight-fat diet+agomir negative control(HFD+agomir NC),(4)Hight-fat diet+agomir miRNA-520c-3p(HFD+agomir miRNA-520c-3p).Atherosclerotic mouse model was built with ApoE-/-mice and intervention group AS mouse model was established with ApoE-/-mice by intravenous injection of miRNA520c-3p agomir.RT-qPCR was applied to test the expression of miRNA-520c-3p in mouse aorta tissue.The pathological change of AS in aorta valve were observed by hematoxylin-eosin staining(HE).The protein expression of RelA/p65 in mouse aortic valve sections was measured by immunohistochemical(IHC)to explore the effect of miRNA-520c-3p agomir on the expression of RelA/p65 in vascular wall.The level of serum CHO,TG,LDL-C,and HDL-C in ApoE-/-mice were detected by a microplate reader colorimetric method.Masson staining was used to measure collagen content in the aortic valve of mice.Immunohistofluorescence(IHF)for the detection of PCNA was used to detect the number of proliferating cells in mouse aortic valve tissue.Results:Part ⅠThe results of CCK-8 assay indicated that PDGF-BB promoted the proliferation of HASMCs and had a dramatical effect on proliferation at concentration of 10ng/mL or 20ng/mL.The protein level of VSMCs phenotypic marker were measured by Western blot to clarify the effect of PDGF-BB on HASMCs phenotype.The results showed that PDGF-BB increase the expression of OPN(VSMCs synthetic phenotype marker)and the increasing trend of OPN reached the peak at 12h.PDGF-BB decrease the expression of calponin(VSMCs contractile phenotype marker).The decreasing trend of calponin is significant at all time points and 5ng/mL PDGF-BB was sufficient to reduce the expression of calponin.The results of RT-qPCR showed that PDGF-BB suppressed the expression of miRNA-520c-3p in HASMCs,and reached the lowest point at 12h stimulated by 1Ong/mL PDGF-BB.The results of MTT and EdU assay showed that miRNA-520c-3p mimics mitigated PDGF-BB-induced proliferation of HASMCs,while miRNA-520c-3p inhibitor has no effects on PDGF-BB-induced proliferation of HASMCs.These results of Boyden chamber revealed that miRNA-520c-3p mimics diminished PDGF-BB-induced migration of HASMCs.The results of flow cytometry demonstrated that miRNA-520c-3p mimics decreased the percentage of cells at S phase and increased the percentage of cells arrested at G2/M phase.The results of Western blot showed that miRNA-520c-3p mimics reduced PDGF-BB-induced the expression of cyclin B1,CDK1 and PCNA protein in HASMCs,without affecting the expression of cyclin D1.Part ⅡThe results of RT-qPCR and Western blot showed that miRNA-520c-3p mimics significantly decreased mRNA and protein level of RelA/p65,reduced the expression of phosphorylated RelA/p65 at S536 in parallel with the content of RelA/p65 protein in nucleus.The results of miRNA pull-down experiments showed that biotin-miRNA-520c3p mimics enriched RelA/p65 mRNA more than twice compared to control group,suggesting miRNA-520c-3p could directly combine with RelA/p65 mRNA.Luciferase reporter assay showed that the seed sequences of miRNA-520c-3p directly binds to RelA/p65 mRNA 3’-UTR site 2 instead of site 1,confirming that RelA/p65 mRNA is a direct target of miRNA-520c-3p in HASMCs.The results of mRNA stability experiments showed that miRNA-520c-3p mimics decreased the stability of RelA/p65 mRNA and thus shortened the RelA/p65 mRNA half-life in HASMCs.CCK-8,EdU and Boyden chamber experiments showed that siRelA/p65 inhibited PDGF-BB-induced proliferation and migration of HASMCs.The results of flow cytometry demonstrated that siRelA/p65 reduced the proportion of cells at S phase.The results of Western blot showed that the expression of cyclin B1,CDK1,and PCNA proteins were down-regulated in HASMCs transfected with siRelA/p65.The results of functional recovery experiments showed that the group co-transfected with miRNA-520c-3p mimics and pcDNA3.1-RelA/p65 reverse the inhibiting effect of the group co-transfected with miRNA-520c-3p mimics and pcDNA3.1 on proliferation,migration and cell cycle distribution of HASMCs.Part ⅢRT-qPCR results showed that the expression of miRNA-520c-3p was up-regulated in aorta tissue of mice injected miRNA-520c-3p agomir.The HE staining results showed that miRNA-520c-3p agomir limited the atherosclerotic plaque size of ApoE-/-C57BL mice.The results of IHC demonstrated that the expression of RelA/p65 was suppressed in aortic valve of miRNA-520c-3p agomir group mice.miRNA-520c-3p agomir reduced the serum level of TG in normal diet mice,but did not significantly affect the serum level of CHO,TG,LDL-C,and HDL-C in ApoE-/-mice.Masson staining results showed that miRNA-520c-3p agomir reduced the collagen fibers content in the vessel wall of mice.IHF staining of PCNA emonstrated that miRNA-520c-3p agomir reduced the number of PCNA-positive cells in the vessel wall and AS plaques of mice.Conclusions:1.miRNA-520c-3p mimics inhibited PDGF-BB-induced HASMCs cell proliferation,migration and cell cycle progression,while miRNA-520c-3p inhibitor had no effect on cell proliferation and cell cycle progression.2.miRNA-520c-3p mimics could promote RelA/p65 mRNA degradation and decrease the expression of RelA/p65 protein in HASMCs by directly binding to RelA/p65 mRNA3’-UTR.3.siRelA/p65 could suppress PDGF-BB-induced HASMCs cell proliferation,migration and cell cycle progression.4.miRNA-520c-3p regulates HASMCs proliferation,migration and cell cycle progression at least in part by directly targeting RelA/p65.5.The expression of RelA/p65,atherosclerotic plaque size,collagen content and a number of PCNA-positive cell in aortic valve of miRNA-520c-3p agomir treated mice were significantly decreased.更多还原