【摘要】：環(huán)狀RNA(circularRNA,circRNA)是廣泛存在于各種生物細(xì)胞中一類(lèi)區(qū)別于傳統(tǒng)線性RNA的新型形式,富集在胞漿中具有牢固的環(huán)狀結(jié)構(gòu),大量存在于真核轉(zhuǎn)錄組中。大部分環(huán)狀RNA是由外顯子構(gòu)成,具有良好的物種保守性以及組織特異性,不易被核酸酶裂解。這使得環(huán)狀RNA在作為新型臨床診斷標(biāo)記物的應(yīng)用上具有明顯的優(yōu)勢(shì)。在功能方面,環(huán)狀RNA作為一類(lèi)新型的ceRNA調(diào)節(jié)物,充當(dāng)RNA海綿功能,競(jìng)爭(zhēng)性抑制miRNA的表達(dá),阻斷miRNA對(duì)其靶標(biāo)表達(dá)的抑制作用。研究人員發(fā)現(xiàn)有11種miRNAs(miR-21、miR-26a、miR-29a、miR-29b、miR-30e、miR-34c、miR-133a、miR-145、miR-210、miR-489與miR-1297)在成骨不全癥中出現(xiàn)差異表達(dá)(P0.05)。本課題旨在通過(guò)生物信息學(xué)技術(shù),進(jìn)一步分析出與這些差異性miRNA相互作用的circRNAs、lncRNAs、靶基因,并初步進(jìn)行實(shí)驗(yàn)驗(yàn)證。目的:預(yù)測(cè)與已知成骨不全差異表達(dá)miRNAs相互作用的circRNAs、lncRNAs以及靶基因,分析它們相互之間以及與信號(hào)通路之間的聯(lián)系。在生物信息學(xué)預(yù)測(cè)基礎(chǔ)上,初步分析預(yù)測(cè)的circRNA、靶基因與miRNA等在成骨不全中的表達(dá)。研究方法:采用starbase與miRWalk數(shù)據(jù)庫(kù)對(duì)成骨不全癥中出現(xiàn)差異表達(dá)十一種miRNA進(jìn)行circRNAs、lncRNAs、靶基因預(yù)測(cè),并通過(guò)DAVID、Cytoscape軟件對(duì)miRNAs、circRNAs、lncRNAs、靶基因進(jìn)行生物信息學(xué)分析。通過(guò)Trizol提取8例成骨不全患者矯形手術(shù)中產(chǎn)生的廢棄,以8例先天性髖關(guān)節(jié)錯(cuò)位患者作為對(duì)照。通過(guò)RT-QPCR方法進(jìn)行has_circ_000713、has_circ_001439與has_circ_001499等共三個(gè)circRNA,NTN4、PPP2CA、SRGAP1、YES1、FAT1、KIAA1586、LMNB2、RFC1等八個(gè)靶基因表達(dá)分析。結(jié)果:通過(guò)starbase數(shù)據(jù)庫(kù)對(duì)miR-21、miR-26a、miR-29a、mi R-29b、miR-30e、m-iR-34c、miR-133a、miR-145、miR-210、miR-489與miR-129711種miRNA相關(guān)circR-NAs、lncRNAs預(yù)測(cè),得到與其相互作用的circRNAs總數(shù)量為222個(gè),其中非重疊ci rcRNAs為141個(gè),與其相互作用的lncRNAs總數(shù)量為359個(gè),其中非重疊lncRNAs為215個(gè)。DAVID、Cytoscape軟件對(duì)miRNAs、circRNAs、lncRNAs、靶基因進(jìn)行生物信息學(xué)分析得到miR-29a/miR-29b/miR-133a與KIAA1586_hsa_circ_001439、靶基因PPP2CA存在相互作用,且包括在TGF-beta、Tight junction與WNT信號(hào)通路中。MiR-133a/miR-145與lincRNA RP11-156E6.1、FAT1_hsa_circ_000713、LMNB2_hsa_circ_001499以及YES1存在相互作用,且可能與Tight junction信號(hào)通路相關(guān)。MiR-26a/miR-145與LncRNA分子AL589743.1、HNRNPU-AS1、MALAT1、RP11-478C19.2等四種LncRNA以及,RFC1_hsa_circ_001649 circRNA,NTN4與SRGAP1基因間存在相互作用,且可能與Axon guidance信號(hào)通路相關(guān)。在以上生物信息學(xué)預(yù)測(cè)基礎(chǔ)上,RT-QPCR結(jié)果表明預(yù)測(cè)的hsa_circ_000713、hsa_circ_001439、hsa_circ_001499等三種circRNA在檢測(cè)的骨組織中無(wú)顯著性差異表達(dá)。LMNB2、NTN4、RFC1與SRGAP1基因存在顯著性差異表達(dá)。結(jié)論:預(yù)測(cè)分析并構(gòu)建了11種已知報(bào)道的成骨不全差異性表達(dá)miRNA及與之相互作用的circRNAs/lncRNAs及靶基因之間的相互作用關(guān)系,及其在特定信號(hào)通路中的定位。為進(jìn)一步深入研究它們之間的相互作用奠定了基礎(chǔ)。
[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，

本文編號(hào)：2232122