【作者】 单发波；

【导师】 黄庆愿；

【作者基本信息】 第三军医大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 背景和目的：慢性缺氧所致的持续性肺动脉压升高称之为缺氧性肺动脉高压（hypoxicpulmonary hypertension，HPH），该病理生理过程是高原心脏病和慢性阻塞性肺疾患等疾病发生的中心环节。在慢性缺氧性肺动脉高压形成早期，肺血管收缩是主要因素，随着缺氧时间的延长，肺血管结构改建也参与其中。持续缺氧引起的肺血管结构改建过程中，肺中小动脉中膜增厚，管腔狭窄及肌化，从而使肺血管阻力持久升高，进而造成持续性肺动脉压力升高。长期持续的肺动脉高压所致的右心室后负荷增加可以引起右心室肥大，严重者甚至发展为右心衰竭。HPH是许多心肺疾病发生发展过程中重要的病理生理基础。因此，研究HPH的发病机理，对防治与其相关的心肺疾病具有重要的临床意义。肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells，PASMCs)是构成肺动脉壁的主要细胞，位于肺血管壁中层，其肥大增生是缺氧性肺血管结构改建的重要病理特征。与骨骼肌和心肌细胞等不同，血管平滑肌细胞(vascular smooth muscle cells，VSMCs)是一种非终末分化细胞，其表型具有较强的可塑性。正常成年动物的VSMCs主要是收缩表型。在多种刺激因素作用下，VSMCs可从具有收缩功能的分化表型转化为有较强增殖和迁移能力的去分化表型。在表型转化过程中，除了VSMCs增殖能力和分泌功能增强外，血管平滑肌细胞的分化标志基因，如平滑肌肌球蛋白重链(smoothmuscle myosin heavy chain，SM-MHC)，血管平滑肌α-肌动蛋白(SM-α-actin)，调宁蛋白(calponin)及平滑肌22α (smooth muscle22alpha，SM22α)蛋白等表达下调。有关缺氧性肺血管结构改建机制的研究，以往多聚焦在缺氧如何影响PASMCs的增殖、凋亡、迁移和分泌等功能方面，对PASMCs表型转化的机制研究较少。有研究提示，VSMCs的表型转化是增殖、迁移等事件发生的起始步骤，是血管平滑肌增生相关疾病发生的病理生理基础。最近研究发现，缺氧刺激也能诱导肺动脉平滑肌发生表型转化，但表型转化发生的具体机制不清楚。微小RNA(microRNA，miRNA)是近年来发现的一类长度约21个核苷酸的内源性非编码小RNA分子，主要通过与mRNA的3′非翻译区结合影响mRNA的稳定性或抑制蛋白翻译过程从而在转录后水平对相应的靶基因发挥负调控作用。据估计，miRNA能参与调节30%以上的已知人类基因，它们几乎可以调节机体的所有病理生理进程。最近研究发现，缺氧环境下miRNA表达改变能调节肺动脉平滑肌相应增殖、凋亡、迁移等功能，并参与缺氧肺动脉高压的形成。然而miRNA是否参与调节缺氧诱导的肺动脉平滑肌表型转化以及缺氧如何引起相关miRNA表达变化却少有报道。本课题通过建立缺氧诱导的肺动脉平滑肌表型转化模型，在前期实验中，我们采用RT-qPCR方法检测候选miRNA的差异表达情况，挑选出缺氧后上调最高的miR-9。进一步采用干扰和过表达方法探讨miR-9是否在缺氧诱导的肺动脉平滑肌表型转化中起重要作用，并对缺氧诱导miR-9表达上调的机制进行了研究。研究方法与内容：1.采用胶原酶I消化法进行大鼠原代PASMCs的培养，采用差异贴壁法纯化PASMCs，并使用特异性SM-α-actin抗体进行免疫荧光染色鉴定。2.缺氧（3%O2）处理PASMCs后，Western Blot法检测SM-MHC，SM-α-actin，Calponin-1和SM22α等血管平滑肌细胞标志基因的表达；RT-qPCR法检测各候选miRNA的差异表达情况。3.常氧培养PASMCs时转染miR-9mimics，采用Western Blot法检测血管平滑肌细胞标志基因的表达，同时用EdU染色检测PASMCs的增殖活性，探索常氧条件下过表达miR-9对血管平滑肌细胞标志基因的表达及增殖的影响。在缺氧诱导PASMCs表型改变模型中转染miR-9inhibitor，Western Blot检测血管平滑肌细胞标志基因的表达，采用EdU染色检测PASMCs的增殖活性，探讨miR-9在缺氧诱导PASMCs表型改变模型中的作用。4.缺氧条件下转染HIF-1α特异性siRNA，采用RT-qPCR法检测PASMCs中miR-9的表达，研究HIF-1α在缺氧诱导miR-9表达上调中的作用。5. RT-qPCR方法检测缺氧后miR-9各初级转录本的表达变化，观察缺氧对各初级转录本表达量的影响。缺氧处理原代培养PASMCs条件下转染HIF-1α特异性siRNA，检测miR-9各初级转录本的表达，探索HIF-1α在缺氧诱导miR-9各初级转录本的表达变化中的作用。6.采用生物信息学分析miR-9-1，miR-9-2和miR-9-3转录起始位点上游5KB范围内的调控区序列，使用chip bioinformatics mapper软件寻找各调控区域内HIF-1α的结合基序；采用ChIP方法验证缺氧后HIF-1α在各预测区域的结合情况。7.通过双荧光素酶报告基因实验探讨HIF-1与其结合序列对基因转录的作用。8.模拟海拔5000米低压缺氧21天，建立大鼠HPH模型，采用RT-qPCR方法检测miR-9的表达，观察miR-9在大鼠肺中小动脉段中的表达情况。结果：1.胶原酶消化法培养大鼠原代PASMCs，免疫荧光鉴定结果显示，经数次差异贴壁法纯化，细胞SM-α-actin阳性率高，PASMCs纯度可达到95%以上。2.与常氧对照比较，原代PASMCs经3%O2缺氧处理24h和48h后，VSMCs收缩表型标志基因蛋白SM-MHC、SM-α-actin、Calponin-1、SM22α的表达均显著下调；缺氧48h后，PASMCs的EdU染色阳性率显著增高。PASMCs缺氧24h后，miR-9在候选的miRNA中表达上调倍数最高，约升高5倍，且缺氧48h后仍能维持4倍以上。3.常氧条件下，与转染mimic control相比较，转染miR-9mimic能下调PASMCs中VSMCs收缩标志基因蛋白表达，提高EdU染色阳性率；缺氧条件下，与转染inhibitorcontrol相比较，转染miR-9inhibitor使PASMCs中VSMCs收缩标志基因蛋白表达显著回升，PASMCs的EdU染色阳性率显著回调。4.缺氧条件下转染HIF-1α特异性siRNA，PASMCs中miR-9表达显著降低。5.缺氧48h后，pri-miR-9-1与pri-miR-9-3表达量显著升高，pri-miR-9-2的表达量无显著改变；转染HIF-1α特异性siRNA能显著抑制pri-miR-9-1与pri-miR-9-3的表达水平。6.生物信息学分析发现，miR-9-1转录起始位点上游5KB范围内的调控区域内有三个HIF-1α结合位点，分别位于-1292/-1279（R1），-1008/-995（R2）和-874/-861（R3）区域；miR-9-3转录起始位点上游5KB范围内的调控区域内有一个HIF-1α结合位点，位于-43/-28（R）区域。ChIP检测发现，缺氧24h和48h后，HIF-1α与miR-9-1的调控区域内R1、R2和R3的结合均显著增加，其中HIF-1α在R1的富集最高。缺氧24h和48h后，HIF-1α与miR-9-3调控区域内R的结合显著增加。7.采用双荧光素酶报告基因检测HIF-1α及其相应结合位点对基因转录活性的影响，结果发现，对miR-9-1而言，缺氧与转染HIF-1α能增强野生型Luc-miR-9-1(WT)报告基因活性，分别提高2.1倍和1.8倍；但对突变型Luc-miR-9-1(M3)无作用。缺氧条件下如果干扰HIF-1α能显著减弱野生型Luc-miR-9-1(WT)报告基因活性。对miR-9-3而言，缺氧与转染HIF-1α均能增强野生型Luc-miR-9-3(WT)报告基因活性，分别提高2.7倍和1.9倍；但对突变型Luc-miR-9-3(M)无作用。缺氧条件下如果干扰HIF-1α则能显著减弱野生型Luc-miR-9-3(WT)报告基因活性。8.与常氧对照组相比，HPH模型大鼠肺中小动脉段的miR-9表达显著升高。结论：1. miR-9参与了缺氧诱导的PASMCs表型转化进程。2.缺氧条件下miR-9表达升高可能主要源于miR-9-1与miR-9-3的表达上调。3.缺氧诱导的miR-9表达上调受HIF-1调控。综上所述，HIF-1-miR-9调控途径是缺氧诱导肺动脉平滑肌细胞发生表型转化的重要调节机制，miR-9可能成为防治HPH的潜在治疗靶点。更多还原

【Abstract】 Background and ObjectiveChronic hypoxia-induced pulmonary hypertension is called hypoxic pulmonaryhypertension (HPH), which plays a key role in the development of high altitude heartdisease and chronic pulmonary heart disease such as chronic obstructive pulmonary disease.HPH is a severe disease characterized by pulmonary vasoconstriction and vascularremodeling, leading to increased vascular resistance and right ventricular dysfunction.Thickening of the media occurs consistently at all levels of the pulmonary arterial tree,especially in small pulmonary arteries, and there is extension of new smooth muscle into thepartially muscular and non-muscular peripheral arteries. The changes of vascular narrowingmay lead to a progressive increase in pulmonary vascular resistance and right ventricleafterload, which in turn induce right ventricular hypertrophy, or even cause the rightventricular failure and death. HPH is believed to be the major pathophysiological process ofmany cardiovascular diseases. Therefore, it is worthy to elucidate the pathogenesis of HPHfor clinical significance of prevention and treatment in cardiovascular diseases.Pulmonary arterial smooth muscle cells (PASMCs) are the cellular components of thenormal pulmonary arterial wall that provides structural integrity and regulates the diameterby contracting and relaxing dynamically in response to vasoactive stimuli. Hypertrophy andhyperplasia of PASMCs are important pathological features during hypoxic pulmonaryvascular structural remodeling. Unlike the skeletal and cardiac muscle cells, vascularsmooth muscle cells (VSMCs) are non-terminally differentiated cells and retain remarkableplasticity. The differentiated VSMCs are characterized by specific contractile proteins, ionchannels, and cell surface receptors that regulate the contractile process and are thus termedcontractile phenotype cells. VSMCs can modulate their phenotype from contractile featureto proliferative feature under certain environmental cues. The phenotype-switched VSMCs always exhibit increased proliferation, migration and matrix synthesis, characterized bydecreased expression of contractile marker proteins, including smooth muscle (SM) α-actin(SM-α-actin), SM-myosin heavy chain (SM-MHC), h1-calponin (calponin1) and SM22α.Previous studies suggested that phenotypic switch of VSMCs is the initial step of somemolecular events such as proliferation and migration and is the pathophysiological basis incardiovascular diseases. Recently, several studies have demonstrated that PASMCs caninduce a phenotype switch from contractile to proliferative activity and induce excessiveproliferation when exposed to hypoxia. These changes represent the majorpathophysiological characteristics in HPH. However, the mechanisms involved in theseevents are poorly understood.MicroRNA (miRNA) is a class of small endogenous non-coding RNA moleculesranging from21-23nucleotides in length that controls gene expression inpost-transcriptional level by targeting mRNAs for translational repression or cleavage. It isestimated that miRNA can directly regulate at least30%of the genes in the human genomeand are therefore believed to be involved in regulating almost all physiological andpathological cellular processes. Recent studies have found that several miRNA are involvedin the changes of proliferation, apoptosis, migration and other functions in pulmonary arterysmooth muscles during hypoxia and participate in HPH, however, the underlyingmechanisms remain obscure.In an initial quantitative RT-qPCR-based screen for differentially expressed miRNA,we identified miR-9as the most significantly up-regulated miRNA in primary PASMCs inresponse to hypoxia. The objective of our current study is to determine whether miR-9playsan important role in phenotypic switch of PASMCs and, if so, to determine the epigeneticregulatory mechanism for the up-regulation of miR-9in phenotypically modulated PASMCsunder hypoxia.Methods and contents:1. Collagenase digestion methods were applied for culturing primary PASMCs of rats,PASMCs were purified by differential adherence and were identified by immunofluorescence staining for SM-α-actin antibody.2. The marker gene expressions of vascular smooth muscle cells were detected byWestern Blot; and miRNA expressions were measured by quantitative RT-PCR (RT-qPCR). 3. To explore the roles of miR-9in normoxia, PASMCs were transfected miR-9mimics.To explore the roles of miR-9under hypoxia, PASMCs were transfected with miR-9inhibitor. The protein expressions of marker genes were measured by Western Blot and theproliferation ability was detected by EdU assay.4. To investigate the role of HIF-1α in miR-9expression under hypoxic conditions,PASMCs were transfected with HIF-1α-specific siRNA before miR-9expression wasdetected by RT-qPCR.5. RT-qPCR was performed to determine the expressions of three primary transcripts ofmiR-9under hypoxic conditions and HIF-1α-specific siRNA were applied to investigate therole of HIF-1α in the differential expressions of three primary transcripts of miR-9underhypoxia.6. The putative HIF-1binding motifs (5′-RCGTG-3′) located within the5-KB regionsupstream of TSSs of miR-9-1, miR-9-2and miR-9-3loci were predicted with the MAPPERdatabase. ChIP assays were performed to determine whether HIF-1α was recruited to theputative binding sites under hypoxia.7. To investigate the functional consequences of each binding sites, wild type anddeletion mutant reporters were constructed and dual-luciferase reporter assays wereperformed.8. To investigate miR-9expression in small pulmonary arteries of HPH, the HPHmodel of rats was established and then the miR-9expression in pulmonary arterial sectionwas detected by RT-qPCR.Results:1. After several times of purification for PASMCs by differential adherence, theSM-α-actin-positive cells can reach95%.2. Hypoxia treatment down-regulated several VSMCs-specific genes at protein levels,including SM-MHC, SM-α-actin, calponin1and SM22α, compared with the normoxiacontrol group. Cells exposed to hypoxia for48h showed an increased EdU positivePASMCs. Among the candidate miRNAs in HPH, miR-9showed the highest increase(5.3-fold) after24h of hypoxia and was maintained at above4-fold increase after48h ofhypoxia.3. Compared with the negative control group，transfection with the miR-9inhibitor significantly increased the expression of contractile phenotypic marker genes at proteinlevels and reduced the percentage of EdU positive PASMCs after48h of hypoxia exposure.By contrast, miR-9mimic, but not mimic control, significantly reduced VSMC markergenes at protein levels and increased the percentage of EdU positive PASMCs in normoxia.4. Transfection of HIF-1α specific siRNA significantly reduced miR-9expressionunder hypoxia.5. After48h hypoxia exposure, both pri-miR-9-1and pri-miR-9-3significantlyelevated, while pri-miR-9-2had no significant difference. In addition, silencing HIF-1αnearly abolished the hypoxia-induced increase in pri-miR-9-1and pri-miR-9-3comparedwith scrambled siRNA group.6. Using in silico analysis, we found the presence of putative HIF-1binding motifs(5′-RCGTG-3′) located within the5-KB regions upstream of TSSs of both miR-9-1andmiR-9-3loci. The miR-9-1locus contains three putative HIF-1binding sites, located at-1292/-1279(R1),-1008/-995(R2) and-874/-861(R3), respectively, and miR-9-3locus hasa putative HIF-1binding site located at-43/-28(R). The HIF-1α enrichment increased in allthe three HIF-1motifs upstream of miR-9-1after24h and48h of hypoxia; among them,miR-9-1R1showed the most significant increase. HIF-1α enrichment upstream of miR-9-3also increased after24h and48h of hypoxia exposure.7. The functional consequences of HIF-1binding sites were assessed by dual luciferasereporter assays. For miR-9-1, hypoxia exposure (1%O2) or transfection of HIF-1α vectorenhanced the luciferase activity of wild-type reporter Luc-miR-9-1(WT) to2.1-fold or1.8-fold, respectively, but there were no significant difference on the luciferase activity ofdeletion mutant reporter Luc-miR-9-1(M3). Conversely, depletion of HIF-1α using siRNAfollowed by hypoxia exposure attenuated the luciferase activity of wild-type reporter (WT)but had no effect on the deletion mutant reporter (M3). For miR-9-3, the luciferase activityof wild-type reporter Luc-miR-9-3was increased2.7-fold by hypoxia exposure and wasincreased1.9-fold by transfection of HIF-1α vector. However, neither hypoxia nor HIF-1αoverexpression had any effect on the luciferase activity of the deletion mutant reporterLuc-miR-9-3M (M). Conversely, siRNA-mediated depletion of HIF-1α followed byhypoxia exposure attenuated the luciferase activity of wild-type reporter (WT) but not ofdeletion mutant reporter (M). 8. Rats were exposed to5000m simulated high altitude in hypobaric chamber toestablish the HPH model. Compared with normoxic control group, the miR-9expression ofchronic hypoxia group in pulmonary arterial section increased significantly.Conclusion:1. miR-9is involved in hypoxia-induced phenotypic modulation in PASMCs of rats.2. The up-regulation of miR-9under hypoxia mainly originates from the transcriptionalactivation of miR-9-1and miR-9-3.3. The hypoxia-induced upregulation of miR-9expression is regulated by HIF-1.In summary, HIF-1-miR-9regulatory pathway is an important regulatory mechanismof hypoxia-induced phenotypic modulation in PASMCs of rats; miR-9could be a potentialtherapeutic target for prevention and treatment of HPH.更多还原