成骨不全差异性miRNA与之相互作用的非编码RNA和靶基因分析

发布时间：2018-09-09 10:28

【摘要】：环状RNA(circularRNA,circRNA)是广泛存在于各种生物细胞中一类区别于传统线性RNA的新型形式,富集在胞浆中具有牢固的环状结构,大量存在于真核转录组中。大部分环状RNA是由外显子构成,具有良好的物种保守性以及组织特异性,不易被核酸酶裂解。这使得环状RNA在作为新型临床诊断标记物的应用上具有明显的优势。在功能方面,环状RNA作为一类新型的ceRNA调节物,充当RNA海绵功能,竞争性抑制miRNA的表达,阻断miRNA对其靶标表达的抑制作用。研究人员发现有11种miRNAs(miR-21、miR-26a、miR-29a、miR-29b、miR-30e、miR-34c、miR-133a、miR-145、miR-210、miR-489与miR-1297)在成骨不全症中出现差异表达(P0.05)。本课题旨在通过生物信息学技术,进一步分析出与这些差异性miRNA相互作用的circRNAs、lncRNAs、靶基因,并初步进行实验验证。目的:预测与已知成骨不全差异表达miRNAs相互作用的circRNAs、lncRNAs以及靶基因,分析它们相互之间以及与信号通路之间的联系。在生物信息学预测基础上,初步分析预测的circRNA、靶基因与miRNA等在成骨不全中的表达。研究方法:采用starbase与miRWalk数据库对成骨不全症中出现差异表达十一种miRNA进行circRNAs、lncRNAs、靶基因预测,并通过DAVID、Cytoscape软件对miRNAs、circRNAs、lncRNAs、靶基因进行生物信息学分析。通过Trizol提取8例成骨不全患者矫形手术中产生的废弃,以8例先天性髋关节错位患者作为对照。通过RT-QPCR方法进行has_circ_000713、has_circ_001439与has_circ_001499等共三个circRNA,NTN4、PPP2CA、SRGAP1、YES1、FAT1、KIAA1586、LMNB2、RFC1等八个靶基因表达分析。结果:通过starbase数据库对miR-21、miR-26a、miR-29a、mi R-29b、miR-30e、m-iR-34c、miR-133a、miR-145、miR-210、miR-489与miR-129711种miRNA相关circR-NAs、lncRNAs预测,得到与其相互作用的circRNAs总数量为222个,其中非重叠ci rcRNAs为141个,与其相互作用的lncRNAs总数量为359个,其中非重叠lncRNAs为215个。DAVID、Cytoscape软件对miRNAs、circRNAs、lncRNAs、靶基因进行生物信息学分析得到miR-29a/miR-29b/miR-133a与KIAA1586_hsa_circ_001439、靶基因PPP2CA存在相互作用,且包括在TGF-beta、Tight junction与WNT信号通路中。MiR-133a/miR-145与lincRNA RP11-156E6.1、FAT1_hsa_circ_000713、LMNB2_hsa_circ_001499以及YES1存在相互作用,且可能与Tight junction信号通路相关。MiR-26a/miR-145与LncRNA分子AL589743.1、HNRNPU-AS1、MALAT1、RP11-478C19.2等四种LncRNA以及,RFC1_hsa_circ_001649 circRNA,NTN4与SRGAP1基因间存在相互作用,且可能与Axon guidance信号通路相关。在以上生物信息学预测基础上,RT-QPCR结果表明预测的hsa_circ_000713、hsa_circ_001439、hsa_circ_001499等三种circRNA在检测的骨组织中无显著性差异表达。LMNB2、NTN4、RFC1与SRGAP1基因存在显著性差异表达。结论:预测分析并构建了11种已知报道的成骨不全差异性表达miRNA及与之相互作用的circRNAs/lncRNAs及靶基因之间的相互作用关系,及其在特定信号通路中的定位。为进一步深入研究它们之间的相互作用奠定了基础。
[Abstract]:Cyclic RNA (circularRNA,circRNA) is a new type of cell which is different from the traditional linear RNA in a variety of biological cells. It is enriched in the cytoplasm and has a firm ring structure and a large number of eukaryotic transcriptome. Most of the cyclic RNA is composed of exons with good species conservation and tissue specificity and is not easily cleavage by nuclease. This makes annular RNA have obvious advantages in the application of new clinical diagnostic markers. As a new type of ceRNA regulator, cyclic RNA acts as a RNA sponge, which competently inhibits the expression of miRNA and blocks the inhibitory effect of miRNA on its target expression. The researchers found that 11 miRNAs (miR-21,miR-26a,miR-29a,miR-29b,miR-30e,miR-34c,miR-133a,miR-145,miR-210,miR-489 and miR-1297) were differentially expressed in osteogenic insufficiency (P0.05). The purpose of this study is to further analyze the circRNAs,lncRNAs, target genes interacting with these differential miRNA by bioinformatics, and to carry out preliminary experimental verification. Aim: to predict the interaction of circRNAs,lncRNAs and target genes with known osteogenic imperfections, and to analyze the relationship between them and signal pathways. On the basis of bioinformatics prediction, the expression of circRNA, target gene and miRNA in osteogenic insufficiency were preliminarily analyzed. Methods: starbase and miRWalk databases were used to predict the circRNAs,lncRNAs, target genes in the differential expression of 11 kinds of miRNA in osteogenic insufficiency, and the bioinformatics analysis of miRNAs,circRNAs,lncRNAs, target genes was carried out by DAVID,Cytoscape software. Eight cases of osteogenic insufficiency were extracted by Trizol and 8 cases of congenital dislocation of hip were used as control. Eight target genes of has_circ_000713,has_circ_001439 and has_circ_001499 were analyzed by RT-QPCR method. Results: miR-21,miR-26a,miR-29a,mi R-29bmmiR-30eanm-iR-34cmmiR-133aanmiR-145miR-210miR-489 was predicted by starbase database. The total number of circRNAs interacting with circR-NAs,lncRNAs was 222, in which there were 141 non-overlapping ci rcRNAs and 359 lncRNAs interacting with it. 鍏朵腑闈為噸鍙爈ncRNAs涓，

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