青少年特发性脊柱侧弯相关长链非编码RNA筛选及临床意义

发布时间：2018-04-26 12:59

本文选题：青少年特发性脊柱侧弯 + 长链非编码RNA　；参考：《第二军医大学》2016年博士论文

【摘要】：背景:青少年特发性脊柱侧弯(Adolescent Idiopathic Scoliosis,AIS)属于特发性脊柱侧弯(Idiopathic Scoliosis,IS)中最常见的一类,多为女性患者,发病年龄一般在10-18岁,并会随着身体的成长呈现进行性加重,不仅会影响到患者的外观,更有甚者影响到患者的心脏和肺脏功能。尽管在临床上、流行病学及分子生物学分析AIS的发病因素有很多,但目前为止对于其发病机理仍然不清楚。关于研究Harrington的相关报道,发生了cobb角度超过了15度的患者之中,其后代有27%可能会发生脊柱侧弯。孪生子女的AIS相关基础及临床研究中,提示单卵双生的共同发生率约为73%,而双卵双生的发生可能率只有27%。Wynne通过对AIS患者随访研究发现,其具有明显的家族性发病倾向,但机制仍不明确。由此可见,基因遗传因素在发病机制中具有重要作用。随着基因技术的发展,基因组计划的完善,基因功能的发展或将成为人们研究所关注的焦点。基于转录组学(Transcriptome)的AIS分子分型逐渐成为研究的热点。其用于阐明AIS的发病机理及如何治疗方式的研究目前还是比较度却尚处于初始阶段,但临床应用具有很广阔的前景。随着基因技术的成熟和发展,二代基因测序技术(RNA-seq)以及及全基因组的表达谱芯片(Gene Expression Microarray)的转录组学分析在功能基因组学的研究中发挥了非常重要的作用,不仅仅可以用于检测编码蛋白质的mRNA,更能够检测各种的不同调节性非编码RNA(microRNA和lncRNA等)。转录组学的研究对于如何进一步筛选AIS发病可能相关的分子,揭示其发生发展机制和寻求新的AIS治疗分子靶点等具有重要的应用前景和临床价值。长链非编码RNA(Long noncoding RNA,lncRNA)是当前研究的热点,它能通过表观遗传、转录水平、转录后水平等多个方面对基因表达产生调控作用。在许多复杂的疾病如肿瘤、神经退行性疾病、心血管疾病等中发现lncRNA存在表达异常,部分lncRNA具有促进或抑制疾病发生的作用。但至今为止,关于AIS相关的lncRNA表达等方面的报道仍很少。本研究通过应用RNA-Seq测序技术得到AIS患者和正常人的lncRNA表达谱,同时得到mRNA表达谱,并进一步探讨AIS患者lncRNA表达存在的差异,为进一步研究提供方向。本次研究的主要内容概述如下:第一部分:正常青少年及AIS患者外周血样本内lncRNA和mRNA的表达谱研究。在这本部分研究中我们选取10例外周血样本(5例正常样本,5例AIS样本)。标准化处理后,将10个样本中至少有5个样本出现lncrna-mrna表达(包括模糊表达)定义为有意义的靶基因。结果发现,10例样本中,共2116个lncrnas、540个mrnas满足以上标准。质量评估:火山图法、聚类分析热图法处理上述数据,结果提示:两组样本的基因表达存在了显著差异性,ais样本内某些基因的表达水平与正常样本呈现出显著的差异。以表达水平差异倍数超过2倍且studentt检验后p0.05,我们以此为差异性的评定标准,之后对基因芯片数据进行筛选。发现,ais组中有2116个lncrnas和540个mrnas发生差异表达现象,其中lncrnas表达上调为1015个,下调为1101个。筛选的差异表达的lncrnas、mrnas中,差异超过5倍的分别为64个(其中1个上调,63个下调)和26个(0个上调,26个下调)。与正常组相对比,ais患者样本基因中有346个一致性表现为上调或下调(≥2个差异倍数)。nonhsat137367是最显著的下调lncrna基因(差异倍数=11.27617),而nonhsat103134是最显著的上调lncrna(差异倍数=5.174644)。本研究发现,与正常组相比,arc,set及tpm3基因在ais患者样本中表现出显著的差异。第二部分:选取5条lncrnas,在15例ais外周血样本和15例正常外周血样本中验证上述结果,在ais外周血样本和正常外周血样本中对其进行了qrt-pcr检测,验证结果与前述结果高度一致。由此可见,高通量的基因芯片结果具有较高的可信度。第三部分:生物信息量预测:kegg生物学通路、go分析及david功能注释簇集法的应用对差异表达的lncrna、mrna进行功能注释和预测。通过kegg法,我们发现focaladhesion、amoebiasis、adherensjunction、regulationofactincytoskeleton、leukocytetransendothelialmigration、salivarysecretion、arginineandprolinemetabolism和thyroidcancer八个生物学通路具体潜在的研究价值。对差异表达的mrna进行的david功能注释簇集分析的结果中出现了中央神经系统的发育、表皮发育、跨膜蛋白受体色氨酸激酶信号通路调节、肺部发育、细胞转移及生长因子等,提示在ais患者中,上述病理变化广泛存在。pearson分析表明,差异显著的10个lncrnas,与相关共表达的mrna构建成cnc网络关系图。go分析提示,上述lncrnas在蛋白转移、增殖或凋亡、基因表达的转录后调控、细胞分化、凋亡调控以及蛋白的代谢等多个与ais相关的病理进化过程发挥着重要的作用。基于此,这10个差异表达最为显著的lncrnas,究竟在ais发病及加重过程中发挥何种作用值得进行下一步深入的研究。第四部分:我们招募了ais患者男女各四名;正常的青少年男女各四名,研究AIS患者性别之间的遗传差异。Venn、GO分析和KEGG分析是用来筛选与AIS女性多发相关的lncRNAs。Venn分析挑选出134条与AIS女性多发相关的lncRNAs。其中NONHSAG015771基因下调4.4倍,CUST_8497_PI429545388基因上调5.5倍。在这项研究中用微阵列筛选与女性多发相关的lncRNA。共有134 lncRNAs作为候选靶基因,通过GO分析和KEGG分析得出:他们参与吞噬体,溶酶体和HTLV-1感染生物学过程,可能参与RNA聚合酶II启动子转录的负调控。这些生物过程均可能是女性AIS多发的病理机制。在本实验中,我们首次对AIS发病LncRNA的相关性进行研究,进一步发现其潜在致病因素。在未来的研究中,深入了解特定LncRNA在AIS病理机制中发的明确作用。
[Abstract]:Background: adolescent idiopathic scoliosis (Adolescent Idiopathic Scoliosis, AIS) is the most common type of idiopathic scoliosis (Idiopathic Scoliosis, IS), most of which are female patients. The age of onset is usually 10-18 years old and increases with the growth of the body. It will not only affect the appearance of the patient, but also affect the patient's appearance. The heart and lung function of the patient. Although there are many clinical, epidemiological and molecular biological analysis of the pathogenesis of AIS, the pathogenesis is still unclear. Related reports on the study of Harrington have occurred among patients with more than 15 degrees of Cobb, and 27% of their descendants may have scoliosis. In the AIS related basic and clinical study of twin children, the common occurrence rate of monozygotic twins is about 73%, and the possibility rate of double egg twins is only 27%.Wynne through follow-up study of AIS patients. It has obvious familial tendency, but the mechanism is still not clear. Therefore, the genetic factors are found to be in the pathogenesis. The development of gene technology, the perfection of the genome project, the development of gene function will be the focus of attention. The AIS molecular typing based on Transcriptome has gradually become the focus of research. The research on the pathogenesis of AIS and how to treat it is still still more than yet. At the initial stage, the clinical application has a broad prospect. With the maturation and development of gene technology, the transcriptional analysis of the two generation gene sequencing technology (RNA-seq) and the whole genome Gene Expression Microarray plays a very important role in the study of functional genomics, not only for use in the study of functional genomics. A variety of different regulatory noncoding RNA (microRNA and lncRNA, etc.) can be detected in the detection of mRNA encoded proteins. The study of transcriptional studies has important application prospects and clinical value on how to further screen the possible molecules associated with AIS pathogenesis, reveal its development mechanism and seek new molecular targets for AIS treatment. Chain non coding RNA (Long noncoding RNA (lncRNA)) is a hot spot of current research. It can regulate gene expression through epigenetic, transcriptional and post transcriptional levels. In many complex diseases such as tumor, neurodegenerative disease, cardiovascular disease, the expression of lncRNA is abnormal, and part of lncRNA is promoted. But so far, there are few reports on the expression of AIS related lncRNA. This study has obtained the lncRNA expression profiles of AIS patients and normal people by using RNA-Seq sequencing technology and obtained the mRNA expression profiles, and further explored the differences in the expression of lncRNA in AIS patients, providing a further study for further research. The main contents of this study are summarized as follows: the first part: Study on the expression of lncRNA and mRNA in the peripheral blood samples of normal adolescents and AIS patients. In this part of the study, we selected 10 cases of peripheral blood (5 normal samples, 5 AIS samples). After standardization, at least 5 samples of the 10 samples appeared lncrna-mrna The expression (including fuzzy expression) was defined as a meaningful target gene. The results showed that in 10 samples, 2116 lncrnas and 540 mRNAs met the above criteria. Quality assessment: Volcano mapping and cluster analysis thermograph method to deal with the above data. The results showed that there were significant differences in gene tables in the two groups, and the expression of some genes in AIS samples. There was a significant difference between the level and the normal sample. In order to express the horizontal difference multiplier more than 2 times and P0.05 after studentt test, we used this as the standard of differential evaluation and then screened the gene chip data. It was found that there were 2116 lncrnas and 540 mRNAs expressions in the AIS group, and the lncrnas expression was up to 1015, The down regulation was 1101. The differential expression of lncrnas and mRNAs were 64 (1 up, 63 down-regulation) and 26 (0 up-regulated, 26 down-regulation). Compared with the normal group, there were 346 congruence in the AIS patient's sample gene, up or down (more than 2 differential multiple).Nonhsat137367 was the most significant down-regulation of L The ncRNA gene (differential multiple =11.27617) was the most significant up-regulated lncrna (differential multiplier =5.174644). This study found that the arc, set, and TPM3 genes were significantly different in the AIS patient samples compared with the normal group. Second part: selected 5 lncrnas, in 15 AIS peripheral blood samples and 15 normal peripheral blood samples. The above results were detected by qRT-PCR in AIS peripheral blood samples and normal peripheral blood samples. The results were highly consistent with the previous results. Thus, high throughput gene chip results have high reliability. Third parts: bioinformatics prediction: KEGG biologic pathway, go analysis and the application of David function annotation cluster method Functional annotation and prediction for differentially expressed lncrna, mRNA. Through KEGG, we found specific potential research prices for eight biological pathways: focaladhesion, amoebiasis, adherensjunction, regulationofactincytoskeleton, leukocytetransendothelialmigration, salivarysecretion, arginineandprolinemetabolism and thyroidcancer. The results of the David functional annotation cluster analysis of differentially expressed mRNA showed the development of the central nervous system, the development of the epidermis, the regulation of tryptophan kinase signaling pathway, lung development, cell metastasis and growth factor, suggesting that in the AIS patients, the above pathological changes were widely found by.Pearson analysis. 10 different significant lncrnas, and related co expressed mRNA constructed into CNC network diagram.Go analysis, it is suggested that the lncrnas plays an important role in the process of AIS related pathogenesis, such as protein transfer, proliferation or apoptosis, post transcriptional regulation of gene expression, cell differentiation, apoptosis regulation and protein metabolism. Lncrnas, the most significant difference expression, is worthy of further research in the course of the pathogenesis and aggravation of AIS. The fourth part: We recruited four men and women in AIS patients; four normal young men and women, studied the genetic difference between sex of AIS patients.Venn, GO analysis and KEGG analysis were used to screen. The lncRNAs.Venn analysis of multiple hair related to AIS women selected 134 lncRNAs. associated with AIS women, in which the NONHSAG015771 gene was down 4.4 times, and the CUST_8497_PI429545388 gene was up 5.5 times. In this study, microarrays were used to screen a total of 134 lncRNAs as a candidate target gene for lncRNA.. KEGG analysis shows that they participate in the phagocytic, lysosome and HTLV-1 infection biological processes, and may be involved in the negative regulation of RNA polymerase II promoter transcription. These biological processes may be the pathological mechanism of the multiple AIS in female AIS. In this experiment, we first studied the phase of LncRNA in the pathogenesis of AIS and further found its underlying cause. In future research, we will further understand the specific role of specific LncRNA in the pathogenesis of AIS.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R726.8

【相似文献】