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MicroRNA-155调控在人椎间盘退变中的作用研究

Modulation of microRNA-155 in Human Intervertebral Disc Degeneration

【作者】 王海强

【导师】 罗卓荆;

【作者基本信息】 第四军医大学 , 外科学, 2011, 博士

【摘要】 研究背景及目的目前认为椎间盘退变为体内外多种因素综合作用的结果,凋亡的增加为其中的因素之一,而凋亡增加确切的分子机制尚不明确。研究表明,内在性和外源性凋亡信号通路在人椎间盘髓核细胞的凋亡中均发挥作用。越来越多的证据表明凋亡机制受新近发现的非编码小RNA,即miRNAs的调控。miRNAs广泛参与蛋白表达的调控,在机体的多种生理和病理过程中发挥重要的作用,但是目前对其在椎间盘退变中的表达谱及发挥的作用知之甚少。本研究的目的为阐述miRNAs,特别是miR-155,在椎间盘退变中的作用。方法我们收集脊柱侧弯(n=12)和椎间盘退变患者(n=12)的椎间盘髓核组织为对照和退变的髓核标本。我们首先从新鲜分离的人髓核细胞中分离提取总RNA,继以miRNA芯片上进行RNA标记和杂交、分析。miRNA的相对表达量以实时定量PCR验证。miR-155调控的靶蛋白以miRNAs网上数据库和计算机分析进行预测。miR-155和潜在靶蛋白的3’-UTRs的作用通过双荧光素酶报告系统进行分析。我们对体外培养的人髓核细胞转染表达pre-miR-155的慢病毒及表达antigomiR-155的慢病毒实现miR-155的上调和下调。转染成功后,以重组人FasL诱导凋亡后,以Western蛋白印迹检测髓核细胞中FADD和caspase-3的表达水平,以APC-Annexin V/7-AAD双染流式细胞分析髓核细胞凋亡率的变化。我们以透射电镜检测对照组和椎间盘退变组髓核组织中的凋亡。我们以原位杂交,免疫组化及双标染色检测miR-155,FADD和caspase-3于髓核中的表达和分布。结果我们发现人椎间盘退变髓核中29个miRNAs为差异表达,miR-155下调了0.56±0.17倍,实时定量PCR进一步证实了miR-155的下调。生物信息方法预测FADD和caspase-3为miR-155的潜在靶蛋白。miR-155可通过直接作用于其3’UTRs而抑制FADD和caspase-3的表达,而靶蛋白3’UTR与miR-155结合位点的突变可组织这种抑制作用。人髓核细胞中上调miR-155的表达引起FADD和caspase-3表达的下调,而下调miR-155的表达则引起FADD和caspase-3的上调。流式细胞分析证实下调miR-155后Fas介导的凋亡增加,而上调miR-155后Fas介导的凋亡减少。同时,我们提供了椎间盘退变组织髓核细胞凋亡的透射电镜下的直接依据。而且,miR-155表达定位于人髓核细胞胞浆中,FADD和caspase-3表达于髓核细胞胞浆近胞膜处,两者的表达为反相关,即表达miR-155的细胞为靶蛋白阴性,表达靶蛋白的细胞为miR-155阴性。另一方面,对照组及退变组髓核均主要由活细胞构成(87.42±1.13%; 83.27±5.00%, p>0.05),细胞形态多种多样,电镜下可分类为典型的髓核细胞,有长突起的髓核细胞及具有吞噬功能的髓核细胞。而且,有长突起的髓核细胞存在于椎间盘退变的髓核中,吞噬功能的髓核细胞既有组织巨噬细胞溶酶体的特点,又有髓核细胞多量内质网的特点。结论本研究为目前首次对人椎间盘退变中凋亡增加的潜在分子机制及miRNAs表达谱进行的研究。同时,我们确定了caspase-3为miR-155调控的新的靶蛋白。我们的研究表明,在人椎间盘退变中,下调的miR-155通过对靶蛋白FADD和caspase-3的调控促进Fas介导的凋亡,提示miR-155在椎间盘退变的病因学中的作用。而且,人退变椎间盘髓核中的细胞主要为活细胞,我们提供了直接的在体依据,证实人髓核细胞具有吞噬功能,有助于理解退变髓核中细胞簇的形成机制;有长突起的髓核细胞存在于脊柱侧弯和退变的椎间盘髓核中。

【Abstract】 Introduction.The etiology of intervertebral disc degeneration (IDD) is ascribed to various factors, one of which is the increase in apoptosis. The underlying molecular mechanisms of the increase in apoptosis remain largely unknown. Both the extrinsic and intrinsic apoptotic pathways play essential roles in the apoptosis of human nucleus pulpusos (NP) cells. Accumulating evidence has shown that the apoptotic machinery is regulated by the newly defined small noncoding RNAs, microRNAs (miRNAs). Despite miRNAs play an important role in a variety of physiologic and pathologic processes, little is known on their expression profiles and attendant roles in IDD. The purpose of this study was to investigate the role of miRNAs, in particular miR-155, in IDD.Methods.Human nucleus pulposus (NP) specimens were collected from patients with idiopathic scoliosis (n=12) and IDD (n=12) as control and degenerative NP samples. Following total RNA isolation from freshly isolated NP cells, RNA labeling and hybridization on miRNA microarray chips were completed on 6 samples. The relative expression of miRNAs was verified using real-time PCR. The prediction of putative targets of miR-155 was achieved using online miRNAs database search and computational analysis. The interaction of miR-155 and the 3’-UTRs of FADD and caspase-3 was performed using the dual luciferase reporter assay. In vitro over-expression and knockdown of miR-155 in human NP cells were achieved by transfection of NP cells with lentiviral Pre-miR-155 and antagomiR-155. Following treated with FasL, the expression levels of FADD and caspase-3 in NP cells with modulated miR-155 were detected using Western Blot. Flow cytometry with APC-Annexin V/7-AAD staining was used to detect the apoptosis in NP cells with modulated miR-155. Transmission electon microscopy was used to detect apoptosis in control and IDD samples. Moreover, miR-155, FADD and caspase-3 expression in human NP was validated using in situ hybridization, immunohistochemistry and a combination thereof.Results.We found that 29 miRNAs were differentially expressed and miR-155 was down-regulated in human degenerative NP. The deregulation of miR-155 (0.56±0.17-fold) was further verified using qRT-PCR. FADD and caspase-3 were predicted as putative targets of miR-155 using the bioinformatics methods. miR-155 inhibited FADD and caspase-3 expression by directly targeting their 3’UTRs, which was abolished by mutation of the predicted miR-155-binding-sites. Overexpression and knockdown of miR-155 in human NP cells resulted in significant deregulation and upregulation expression of FADD and caspase-3. As well, Fas-mediated apoptosis was increased when antagonizing miR-155 and decreased when using pre-miR-155. Furthermore, we presented direct evidence of NP cells undergoing apoptosis in IDD samples. Moreover, miR-155 expressed in the cytoplasm of human NP cells with reverse correlation with FADD and caspase-3. On the other hand, both control and degenerative human NP consisted of mainly viable cells (87.42±1.13%; 83.27±5.00%, p>0.05) with a variety of morphology, which were further categorized as typical NP cells, NP cells with long processes and phagocytic NP cells. Moreover, NP cells with long processes existed in degenerative NP. Phagocytic NP cells had the hallmarks of both stationary macrophages with lysosomes and NP cells with a large amount of the endoplasmic reticulum.Conclusion.To our knowledge, this is the first study addressing the underlying mechanism of apoptosis and the expression profiles of miRNAs in IDD. As well, caspase-3 is identified as a novel target of miR-155. Our results suggest that deregulated miR-155 promotes Fas-mediated apoptosis in human IDD by targeting FADD and caspase-3, implicating a role of miR-155 in the etiology of IDD. In addition, human degenerative NP consists of primarily viable cells. Furthermore, we present direct and in vivo evidence that human NP cells have phagocytic potential, which may have implications for the underlying mechanisms of cell clusters. Moreover, NP cells with long processes exist in both scoliosis and degenerative NP.

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