肘管综合征中弓状韧带miRNAs的实验研究

发布时间：2018-08-16 15:31

【摘要】：【目的】肘管综合征(Cubital tunnel syndrome,CuTS),也称迟发性尺神经炎,是第二常见的周围神经卡压病变。肘部的创伤及慢性劳损可以导致弓状韧带出现肥厚增生,引起尺神经卡压磨损,这是CuTS最常见的病因。在此本课题组选用微阵列芯片检测弓状韧带中差异表达的microRNAs(miRNAs)并行生物信息学分析,进一步对相关通路进行验证,期望为CuTS的发病机制和病因学研究提供新的思路。【方法】1.3例弓状韧带标本及3例对照屈肌总腱旁腱性组织标本均收集自2014年8月至2014年11月期间于天津医科大学总医院行肘部尺神经松解前移手术患者。常规提取总RNA并行微阵列芯片分析,qRT-PCR用于验证部分miRNAs表达一致情况。2.miRWalk2.0软件用于预测靶基因。Cytoscape3.0软件制作miRNAs与相应靶基因关系网络图。DAVID6.7软件、Bioconductor基因注释软件用于靶基因的GO功能富集分析和KEGG通路富集分析。通过Cluster3.0软件对miRNAs及通路行聚类分析。3.构建人皮肤成纤维细胞系(Human skin fibroblast cell lines,HSF),并转染miR-146b-5p过表达(miR-146b-5p mimic)及反义干扰载体(anti-miR-146b-5p),通过qRT-PCR及Western-Blot技术检测纤维化指标COL1A1、COL3A1、Fibronectin(FN)、SMAD4及TGFB1,从而明确miR-146b-5p影响纤维化是否与TGF-β/SMAD4通路有关。【结果】在弓状韧带组织中差异表达的miRNAs共有70个(P0.05),其中上调3个(hsa-miR-422a、hsa-miR-7855-5p、hsa-miR-1343-3p);下调67个(hsa-miR-196a-5p、hsa-miR-146b-5p、hsa-miR-297、hsa-miR-21-3p、hsa-miR-595、hsa-miR-663b、hsa-miR-615-5p、hsa-miR-185-3p等)。qRT-PCR验证结果表明hsa-miR-1343-3p、hsa-miR-146b-5p和hsa-miR-21-3p表达与芯片结果一致。通过miRWalk2.0软件共发现1804个预测靶基因。对靶基因分析后发现大部分靶基因主要受7个miRNAs(hsa-miR-146b-5p,hsa-miR-21-3p,hsa-miR-185-3p,hsa-miR-615-5p,hsa-miR-659-3p,hsa-miR-663a和hsa-miR-760)的调控。通过Cluster3.0软件对miRNAs行聚类分析,结果可见hsa-miR-146b-5p与hsa-miR-196a-5p、hsa-miR-659-3p功能相聚类,hsa-miR-21-3p与hsa-miR-297功能相聚类。对全部靶基因分析发现,WNT2、TGFB1、FGF1等均为差异miRNAs的高频靶基因并参与体内蛋白质代谢过程,细胞生长调节,细胞周期过程,细胞分裂,细胞代谢过程,信号传导等生物过程。KEGG通路分析示靶基因主要集中在粘着连接、粘附斑、轴突导向、赖氨酸降解、其他多糖降解、细胞粘附分子(Cell adhesion molecules,CAMs)、丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号通路、逆行内源性大麻素信号、粘多糖的生物合成-硫酸乙酰肝素/肝素和神经营养因子的信号转导通路及其他8条通路。通过Cluster3.0软件对全部通路行聚类分析,发现粘附斑与ErbB信号通路功能相聚类,粘着连接与CAMs功能相聚。MAPK、粘附斑信号通路和预测靶基因关系最紧密。qRT-PCR及Western-Blot验证发现miR-146b-5p mimic组中COL1A1、COL3A1、FN、SMAD4及TGFB1各纤维化指标均下调,anti-miR-146b-5p组中各纤维化指标均上调。【结论】通过分析CuTS患者中退变增生性弓状韧带与对照腱性组织的miRNAs表达,发现了多个差异表达的miRNAs,并获得了潜在的靶基因及信号通路,为初步研究CuTS中弓状韧带增生肥厚的发病机制提供了线索。
[Abstract]:[Objective] Cubital tunnel syndrome (CuTS), also known as delayed ulnar neuritis, is the second most common peripheral nerve entrapment lesion. CuTS is the most common cause of ulnar nerve entrapment. CuTS is caused by the hypertrophy of the arcuate ligament and ulnar nerve entrapment wear caused by elbow trauma and chronic strain. Detection of differentially expressed microRNAs (microRNAs) in the arcuate ligament and bioinformatics analysis were performed to further validate the related pathways and provide new ideas for the pathogenesis and etiology of CuTS. [Methods] 1.3 specimens of the arcuate ligament and 3 specimens of the paratendinous tissue of the common flexor tendon were collected from August 2014 to 2011. Patients undergoing ulnar nerve release and anterior displacement in Tianjin Medical University General Hospital in January. Total RNA was routinely extracted and analyzed by microarray chip. QRT-PCR was used to verify the consistent expression of some microRNAs. 2. MicroWalk 2.0 software was used to predict target genes. Cytoscape 3.0 software was used to make a network diagram of the relationship between microRNAs and the corresponding target genes. DAVID 6.7 software, B. Ioconductor gene annotation software was used for GO function enrichment analysis and KEGG pathway enrichment analysis of target genes.Cluster 3.0 software was used for clustering analysis of microRNAs and pathways.3. Human skin fibroblast cell lines (HSF) were constructed and transfected with overexpression of microRNAs-146b-5p and antisense interference vector (anti-m-m). IR-146b-5p, COL1A1, COL3A1, Fibronectin (FN), SMAD4 and TGFB1 were detected by qRT-PCR and Western-Blot techniques to determine whether the effect of microwave-146b-5p on fibrosis was related to the TGF-beta/SMAD4 pathway. [Results] There were 70 differentially expressed microRNAs in arcuate ligament tissues (P 0.05). Three of them were up-regulated (hsa-microwave-422a, hsa-microwave-78). 55-5p, hsa-microRNA-1343-3p; 67 down-regulated (hsa-microRNA-196a-196a-5p, hsa-microRNA-196a-196a-5p, hsa-microRNA-146b-5p, hsa-microRNA-297, hsa-microRNA-297, hsa-microRNA-microRNA-21-21-3p, hsa-microRNA-595, hsa-microRNA-595, hsa-microRNA-microRNA-595, hsa-microRNA-microRNA-66-663b, hsa-microRNA-615-5p, hsa-microRNA-microRNA-185-3p and so on). qRT-PCR validation results showed that hsa-microRNA-microRNA-microRNA-1343-The expression of -3p was consistent with the result of microarray. A total of 1804 predictive targets were found by using the microarray Walk 2.0 software. Most of the target genes were mainly regulated by seven microRNAs (hsa-microRNA-146b-5p, hsa-microRNA-146b-5p, hsa-microRNA-21-3p, hsa-microRNA-185-3p, hsa-microRNA-185-3p, hsa-microRNA-615-5p, hsa-microRNA-659-3p, hsa-microRNA-microRNA-663a and hsa-microRNA-663a-760). Cluster3.0 software was used to cluster analysis of microRNA. The results showed that hsa-microRNA-microRNA-146b-146b-5p and hsa-microRNA-5p and hsa-microRNA-1965-5p, hsa-196a-196a-196a-659-3p function The analysis of all target genes showed that WNT2, TGFB1 and FGF1 were high-frequency target genes of differentially expressed microRNAs and were involved in biological processes such as protein metabolism, cell growth regulation, cell cycle process, cell division, cell metabolism and signal transduction. Because of the main focus on adhesion junction, adhesion plaque, axon-directed, lysine degradation, other polysaccharide degradation, cell adhesion molecules (CAMs), mitogen-activated protein kinase (MAPK) signaling pathway, retrograde endocannabinoid signal, mucopolysaccharide biosynthesis - heparan sulfate / heparin Cluster analysis of all the pathways by Cluster 3.0 software showed that adhesion plaques were clustered with ErbB signaling pathway function, adhesion junctions were clustered with CAMs function. MAPK, adhesion plaque signaling pathway and predictive target genes were most closely related. QRT-PCR and Western-Blot validation showed that microRNA146b was found. COL1A1, COL3A1, FN, SMAD4 and TGFB1 were down-regulated in the -5p mimic group, and all fibrosis indexes were up-regulated in the anti-microRNA-146b-5p group. [Conclusion] By analyzing the expression of microRNAs in the degenerative proliferative arcuate ligament and the control tendinous tissue in CuTS patients, multiple differentially expressed microRNAs were found and potential target genes and messages were obtained. The pathway provides clues for the preliminary study of the pathogenesis of arcuate ligament hyperplasia and hypertrophy in CuTS.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R688

【参考文献】