血清miRNA在过敏性紫癜和紫癜性肾炎中的差异性表达

发布时间：2018-03-14 14:41

本文选题：miRNA　切入点：血清　出处：《南京医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：过敏性紫癜（HSP）是儿童最常见的系统性小血管炎之一，六个月之内极易出现血尿和（或）蛋白尿形成紫癜性肾炎（HSPN）。目前对该疾病的发病机制尚未完全明确。肾活检术是诊断肾脏疾病的金标准，但其有创伤性，并具有一定的风险，不易被患者接受，更不易通过重复肾活检来动态判断治疗效果和评估预后。因此，寻找方便、有效、无创伤性的生物学标志，将极大有利于疾病的诊断和治疗。微小RNA（microRNAs，miRNAs）是一组长约18-24nt的内源性非编码RNA，其表达具有组织细胞特异性、发育时序性和进化保守性，在转录后水平调节靶基因的表达，参与细胞增殖、分化、凋亡、脂肪代谢、氧化应激和人类疾病形成等生理和病理过程。众多研究证实血清中存在稳定表达的miRNA，可抵制RNase的降解，且受极端环境（如高温、极酸或极碱、10次冻融循环）的影响较小，可作为疾病诊断和检测的小侵袭性的生物学标志。本研究旨在应用miRNA基因芯片技术，从血清标本中筛选在过敏性紫癜和紫癜性肾炎中差异表达显著的miRNA，并应用生物信息学预测差异表达miRNA的靶基因，初步探讨miRNA在该疾病中发挥的作用，并为将来的临床应用奠定基础。研究材料和方法： 1.从正常对照组、过敏性紫癜组和紫癜性肾炎组中各抽出10例患儿血清标本用于芯片筛选实验。 2.采用miRNeasy Mini Ki（tQiagen）提取分离总RNA，使用NanoDrop1000(Nanodrop, Wilmington, Delaware, USA)检测RNA浓度。 3.利用AB TaqMan Human MicroRNA Array（TLDL低密度芯片）进行miRNA的筛选（北京博奥科技公司提供），应用DataAssist2.0软件进行数据分析，找出有差异表达的miRNA。 4.通过逆转录和实时定量PCR技术验证芯片结果。 5.应用TargetScan、PicTar、miRanda数据库对差异表达miRNA进行交集靶基因预测，之后采用Cytoscape v2.8.2软件和DAVID在线数据库进行GO富集分析和KEGG通路分析。实验结果： 1.所提取的总RNA经过检测符合芯片实验的要求。 2.以基因表达相差2倍为阈值，通过比较过敏性紫癜组和对照组芯片研究结果，发现96个miRNAs差异表达显著，其中7个表达显著上调，89个显著下调；比较紫癜性肾炎组和对照组miRNA的芯片表达结果，发现有68个miRNAs表达呈显著差异，其中11个表达显著上调，57个表达显著下调；进一步利用芯片比较紫癜性肾炎组和过敏性紫癜组miRNA的表达，发现53个miRNAs差异表达显著，其中36个表达显著上调，，17个表达显著下调。 3.对过敏性紫癜组和紫癜性肾炎组中差异表达显著的hsa-miR-15b，hsa-let-7d进行相对定量分析，结果显示这些miRNAs的表达与芯片结果表达趋势一致。 4.应用TargetScan、PicTar、miRanda软件对HSP/NC和HSPN/NC中有显著表达差异的miRNA进行靶基因预测，发现有47个miRNAs存在交集靶基因，预测到的靶基因数为617个，而HSPN/HSP中有19个miRNAs存在交集靶基因，预测到的靶基因数为435个。之后，对这两组交集靶基因进行GO富集分析，发现这些靶基因分别富集于224和219个群中（p0.05），与代谢、生长发育、表达调控等过程有关。KEGG通路分析显示这两组靶基因分别富集于13和11个通路中（p0.05），与细胞循环分裂、肿瘤形成和信号通路等密切相关。实验结论： 1.使用芯片从过敏性紫癜中筛选出96个显著表达差异的miRNAs；从紫癜性肾炎中筛选出68个显著表达差异的miRNAs；将紫癜性肾炎和过敏性紫癜比较时，筛选出53个差异表达显著的miRNAs，证实miRNA在过敏性紫癜和紫癜性肾炎中均存在差异性表达。 2.应用RT-PCR对其中2个差异表达显著的miRNAs（hsa-miR-15b、hsa-let-7d）进行相对定量分析，其结果与芯片表达趋势一致。 3.差异表达显著miRNA预测交集靶基因参与细胞增殖分化、信号通路和转录调控等过程，miRNA有可能在过敏性紫癜、紫癜性肾炎的发病中发挥重要的作用，作为疾病新的生物学标志。
[Abstract]:Henoch Schonlein purpura (HSP) is one of the most common systemic vasculitis in children six months, prone to hematuria and (or) the formation of proteinuria of Henoch Schonlein purpura nephritis (HSPN). The pathogenesis of the disease has not yet entirely clear. Renal biopsy is the gold standard for the diagnosis of kidney disease, but its traumatic, and has a certain risk, is not easy to be accepted, it is not easy to judge the dynamic effect of treatment and prognosis through repeated renal biopsy. Therefore, looking for convenient, effective, noninvasive biomarker, will greatly benefit the diagnosis and treatment of disease.
Small RNA (microRNAs, miRNAs) is an endogenous leader about 18-24nt encoding RNA, its expression has tissue specificity, developmental timing and evolutionary conservation, regulating the expression of target genes at the transcriptional level, involved in cell proliferation, differentiation, apoptosis, lipid metabolism, oxidative stress and human disease formation physiology and pathology process.
Many studies have confirmed the existence of the stable expression of miRNA in serum, RNase can resist degradation, and extreme environment (such as high temperature, extremely acid or alkali, 10 times of freeze-thaw cycle) have little effect, can be used as a biological small invasive disease diagnosis and detection of the logo. The purpose of this study is to use miRNA gene chip technology from serum samples, screening of differentially expressed significant miRNA in allergic purpura and purpura nephritis, and application of bioinformatics to predict differential expression of target gene miRNA, preliminary study of miRNA play in the role of the disease, and lay the foundation for future clinical application.
Research materials and methods:
1. from the normal control group, the anaphylactoid purpura group and the purpura nephritis group, 10 serum samples were extracted from each group for the chip screening test.
2. the total RNA was extracted with miRNeasy Mini Ki (tQiagen) and NanoDrop1000 (Nanodrop, Wilmington, Delaware, USA) was used to detect the RNA concentration.
The use of AB TaqMan Human MicroRNA Array 3. (TLDL low density array) screening miRNA (Beijing Boao technology company), DataAssist2.0 software was used for data analysis, to find out the differences in expression of miRNA.
4. the results of the chip were verified by reverse transcription and real-time quantitative PCR technology.
5., we used TargetScan, PicTar and miRanda database to predict the target genes of differentially expressed miRNA. Then we used Cytoscape v2.8.2 software and DAVID online database to do GO enrichment analysis and KEGG pathway analysis.
Experimental results:
The 1. extracted total RNA was detected in accordance with the requirements of the chip experiment.
2. the gene expression difference of 2 times the threshold, by comparing the allergic purpura group and control group chip results showed the expression of 96 miRNAs significantly, of which 7 up-regulated, 89 down regulated significantly; compared with Henoch Schonlein purpura nephritis group and control group miRNA microarray expression results, the expression of miRNAs was found 68 significant differences, of which 11 up-regulated, 57 down regulated expression; the further use of chip comparison purpura nephritis group and allergic purpura group miRNA, found that the expression of 53 miRNAs significantly, of which 36 up-regulated, 17 down regulated.
3., the expression of hsa-miR-15b and hsa-let-7d in Henoch Schonlein purpura group and purpuric nephritis group were analyzed by relative quantitative analysis. The results showed that these miRNAs expressions were consistent with the results of chip expression.
4. application of TargetScan, PicTar, miRanda software has a significant differential expression of miRNA target gene prediction of HSP/NC and HSPN/NC, found that 47 miRNAs overlap target gene and target gene prediction to the number 617, and HSPN/HSP 19 miRNAs in the presence of intersection target gene and target gene prediction to the number of 435. After that, the two group of intersection of target gene GO enrichment analysis found that these target genes were enriched in the 224 and 219 group (P0.05), and metabolism, growth and development, regulation of the.KEGG pathway analysis showed that the two groups of target genes were enriched in the 13 and 11 pathways in expression (P0.05), mitosis and cell cycle, tumor formation and signaling pathways are closely related.
Experimental conclusions:
1. the use of chips from allergic purpura were screened 96 differentially expressed miRNAs; screened from purpura nephritis in 68 differentially expressed miRNAs; comparison of purpura nephritis and allergic purpura, screened 53 differentially expressed miRNAs was confirmed miRNA, there are differences in the expression of allergy purpura and purpura nephritis.
2. the relative quantitative analysis of 2 differentially expressed miRNAs (hsa-miR-15b, hsa-let-7d) was carried out by RT-PCR, and the results were in accordance with the trend of chip expression.
3., differential expression is significant. MiRNA predicts that the target genes are involved in cell proliferation, differentiation, signal transduction and transcriptional regulation. MiRNA may play an important role in the pathogenesis of Henoch Schonlein purpura nephritis, and serve as a new biomarker for diseases.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.5

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