强直性肌营养不良1型骨骼肌组织中microRNA的表达及靶基因预测

发布时间：2018-06-01 03:32

本文选题：强直性肌营养不良1型 + miR-146a　；参考：《中国人民解放军医学院》2015年硕士论文

【摘要】：目的：基于前期建立的强直性肌营养不良1型(myotonic dystrophy type 1, DM1)患者骨骼肌组织microRNA表达谱,筛选出差异表达的microRNA,检测其中4个microRNA (miR-146a、miR-182、miR-200c 和 miR-885)在DM1患者及正常对照骨骼肌组织中的表达水平,同时检测与肌肉组织发育和再生相关的niR-1、miR-133、 miR-206及种子区含有CAG重复序列的miR-103、miR-107勺表达,比较其在DM1患者和对照骨骼肌组织中的表达差异,分析差异表达microRNA与DM1患者基本临床参数之间的相关性。同时利用生物信息学方法,预测差异表达microRNA的靶基因,分析靶基因的功能,为DM1发病机制中microRNA作用的研究提供新的思路和依据。方法：1)选取2012年9月至2014年6月在解放军总医院神经内科经基因检测确诊的DM1患者12例,收集并整理DM1患者的临床资料。通过肌肉活检获取肌肉标本,酶组织化学染色进行病理诊断;对照组肌肉标本取自12例无神经肌肉疾病且年龄和性别大致匹配的骨科手术患者。2)采用实时荧光定量 PCR (real-time polymerase chain reaction)技术检测病例组及对照组骨骼肌组织中microRNA(miR-146a、miR-182、miR-200c、miR-885、miR-1、miR-133、miR-206、miR-103和miR-107)的相对表达水平。3)统计学分析：利用SPSS 20.0软件,数据以均数±标准差表示,通过两独立样本的T检验方法比较病例组和对照组microRNA的表达差异,用方差分析比较microRNA的表达与临床参数之间的相关性,取双侧检验p0.05作为判断有统计学意义的标准。4)利用生物信息学软件TargetScan、 miRanda及PicTar预测nicroRNA的靶基因,并与前期筛选出的差异基因取交集,通过GO-analysis对交集基因进行显著性功能分析,根据P值进行相关性排序。结果：1)DM1患者骨骼肌组织中miR-146a的相对表达量明显低于对照组,其差异有统计学意义(p=0.0030.05),其他microRNA的表达在两组间均无统计学差异。DM1患者骨骼肌组织中niR-146a的表达水平与性别、病程、MIRS评分之间无明显相关性。2)利用TargetScan、miRanda及PicTar三种软件预测miR-146a靶基因并与前期实验筛选出的1950个差异基因比对,对17个交集基因做GO-analysis,根据p值(p0.05)经过显著性筛选,结果显示交集基因主要参与神经元分化、细胞凋亡的负性调节等生物学过程。结论：miR-146a在DM1患者骨骼肌组织中低表达,但其差异水平与患者性别、病程和MIRS评分均无相关性。通过生物信息学分析,miR-146a可能通过参与神经元分化、细胞死亡等重要的生物学过程而在DM1的致病过程中发挥作用,为DM1发病机制中microRNA作用的研究提供新的思路和依据。
[Abstract]:Objective: to investigate the expression of microRNA in skeletal muscle of patients with myotonic dystrophy type 1 (DM1). The differentially expressed microRNAs were screened out, and the expression levels of four microRNA miR-146aanmiR-182 miR-200c and miR-885) were detected in skeletal muscle tissues of DM1 patients and normal controls. The expression of niR-1 miR-133 related to muscle tissue development and regeneration, miR-206 and miR-103 miR-103miR-107 in the seed region were detected. To compare the difference of expression of microRNA between DM1 patients and control group, and to analyze the correlation between the differential expression of microRNA and the basic clinical parameters of DM1 patients. At the same time, bioinformatics was used to predict the differential expression of target genes of microRNA, and to analyze the function of target genes, so as to provide a new idea and basis for the study of the role of microRNA in the pathogenesis of DM1. Methods from September 2012 to June 2014, 12 patients with DM1 diagnosed by gene test in Department of Neurology, General Hospital of PLA were selected and collected and sorted out the clinical data of DM1 patients. Muscle specimens were obtained by muscle biopsy and pathological diagnosis was performed by enzyme histochemical staining. Muscle specimens of control group were collected from 12 patients without neuromuscular disease and matched roughly with age and sex.) Real-time PCR real-time polymerase chain reaction) technique was used to detect miR-146amiR-182 miR-200cmiR-885mcmiR-885 and miR-133miR-206 miR-103 and miR-107 in skeletal muscle tissue of patients and control group. Statistical analysis: using SPSS 20.0 software, The data were expressed as mean 卤standard deviation. The difference of microRNA expression was compared between the two independent samples by T test, and the correlation between the expression of microRNA and clinical parameters was compared by ANOVA. The target genes of nicroRNA were predicted by the bioinformatics software Target Scan. miRanda and PicTar. The target genes were intersected with the differentially selected genes, and the significant sexual function of the intersecting genes was analyzed by GO-analysis. The correlation was sorted according to P value. Results the relative expression of miR-146a was significantly lower in skeletal muscle tissue of patients than that of control group (P < 0.05). There was no significant difference in the expression of other microRNA between the two groups. The expression level and sex of niR-146a in skeletal muscle of patients with DM1 were significantly lower than those of the control group, and there was no significant difference in the expression of other microRNA between the two groups. There was no significant correlation between Mirs scores. 2) Target ScanmiRanda and PicTar were used to predict miR-146a target genes and compared with 1950 differentially selected genes in previous experiments. GO-analysis was performed on 17 intersecting genes according to p value (p < 0.05). The results showed that the intersecting genes were mainly involved in the biological processes such as neuronal differentiation, negative regulation of apoptosis and so on. Conclusion the low expression of miR-146a was found in skeletal muscle of patients with DM1, but there was no correlation between the difference level and gender, course of disease and MIRS score. Through bioinformatics analysis, miR-146a may play a role in the pathogenesis of DM1 by participating in important biological processes such as neuronal differentiation and cell death, which provides a new idea and basis for the study of the role of microRNA in the pathogenesis of DM1.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R746.2

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