尾悬吊诱导骨骼肌萎缩过程中miRNA表达谱的变化研究

发布时间：2018-05-20 13:23

  本文选题：miRNAs + 基因芯片　； 参考：《北京体育大学》2017年硕士论文

【摘要】：研究目的:miRNA是真核生物中广泛存在的一种长度约为21-23nt的RNA分子,可通过与mRNA的结合抑制其转录,在众多生物学过程中发挥重要作用。研究显示miRNA在肌萎缩过程中发挥重要调控作用,已有研究发现miRNAs在多种类型的肌萎缩中存在差异表达,但在失重性肌萎缩及恢复过程中miRNAs表达谱的变化未见报道。本实验拟通过尾悬吊诱导小鼠失重性肌萎缩研究miRNAs表达谱的变化,寻找失重性肌萎缩过程中具有重要调控作用的miRNAs。研究方法:以C57BL/6小鼠为实验对象,尾悬吊14天诱导失重性肌萎缩,动态分析尾悬吊及恢复过程中小鼠后肢背侧骨骼肌肌纤维横截面积及肌萎缩标志蛋白和基因表达的变化;在此基础上对小鼠比目鱼肌miRNAs进行基因芯片检测,寻找差异表达的基因,并通过qPCR对部分未见报道的miRNAs进行验证。研究结果:(1)14天尾悬吊过程中,小鼠后肢背侧肌重显著下降,恢复过程中逐渐升高;其中比目鱼肌重量丢失程度明显高于腓肠肌和跖肌,肌纤维横截面积显著下降。(2)小鼠比目鱼肌中肌萎缩标志基因FoxO3a、MuRF1和Atrogin-1的蛋白和mRNA表达随尾悬吊时间逐渐升高。14天尾悬吊后,FoxO3a的蛋白含量显著升高(P0.01),Atrogin-1的蛋白含量也显著升高(P0.01),MuRF1的蛋白含量在尾悬吊7天升高最明显(P0.05)。(3)FoxO3a、Atrogin-1和MuRF1的基因表达在尾悬吊过程中有升高趋势。FoxO3a mRNA在尾悬吊3天最高(P0.01),Atrogin-1 mRNA在尾悬吊7天水平最高(P0.01),MuRF1 mRNA在尾悬吊7天表达最高(P0.01)。(4)芯片结果显示,尾悬吊过程中14个miRNAs上调,2个miRNAs在尾悬吊3天和7天上调,4个miRNAs先下调后上调,22个miRNAs下调。(5)qRT-PCR对肌肉萎缩过程中差异表达的miRNAs进行验证,筛选出8个符合芯片结果的miRNAs。研究结论:本研究通过尾悬吊成功构建了小鼠后肢骨骼肌萎缩模型,具有废用性肌萎缩的特征,包括肌肉重量下降、肌纤维横截面积的减小和肌萎缩相关基因的升高。通过基因芯片对失重性肌萎缩形成及恢复过程中差异表达的miRNAs进行了筛选,并进一步利用qRT-PCR对差异表达的miRNAs进行了验证和分析,筛选到了在失重性肌萎缩中具有潜在调控作用的miRNAs。
[Abstract]:Objective\ 21-23nt is a kind of RNA molecule widely existing in eukary@@ Studies have shown that miRNA plays an important regulatory role in the process of muscle atrophy. It has been found that there is a differential expression of miRNAs in various types of muscle atrophy, but the changes of miRNAs expression profile during the course of weightlessness and recovery have not been reported. The purpose of this study was to investigate the changes of miRNAs expression profile in mice with weightlessness muscle atrophy induced by tail suspension, and to search for miRNAs that play an important role in the process of weightlessness muscle atrophy. Methods: C57BL/6 mice were used to induce weightlessness muscle atrophy after 14 days of tail suspension. The changes of muscle fiber cross-sectional area, muscle atrophy marker protein and gene expression during tail suspension and recovery were analyzed. On the basis of this, the miRNAs of mouse soleus muscle was detected by gene chip, and the differentially expressed genes were found, and some unreported miRNAs were verified by qPCR. Results the weight loss of soleus muscle was significantly higher than that of gastrocnemius muscle and metatarsal muscle during 14 days of tail suspension. The expression of protein and mRNA of muscle atrophy marker genes FoxO3a and MuRF1 and Atrogin-1 in soleus muscle of mice increased with the tail suspension time. 14 days after tail suspension, the protein content of FoxO3a increased significantly, and the protein content of P0.01 and Atrogin-1 also showed significant increase in mice soleus muscle atrophy marker gene FoxO3a and Atrogin-1. The protein content of P0.01Atrogin-1 increased most obviously during the 7th day of tail suspension. The gene expression of FoxO3a Atrogin-1 and MuRF1 increased during tail suspension. The highest level of P0.01Atrogin-1 mRNA in tail suspension was P0.01Atrogin-1 mRNA at 7 days of tail suspension. The highest level of P0.01Atrogin-1 mRNA was found at 7 days after tail suspension. The gene expression of FoxO3a and MuRF1 was increased during tail suspension. The highest level of P0.01Atrogin-1 mRNA in tail suspension was P0.01Atrogin-1 mRNA during 7 days of tail suspension. The result of the chip showed that, During tail suspension, 14 miRNAs were up-regulated, 2 miRNAs were up-regulated 3 and 7 days after tail suspension, 4 miRNAs were down-regulated and then up-regulated, 22 miRNAs down-regulated .5 miRNAs RT-PCR was used to verify the differential expression of miRNAs during muscle atrophy, and 8 miRNAs were screened out according to the results of microarray. Conclusion: the model of skeletal muscle atrophy in hind limb of mice was successfully constructed by tail suspension. It has the characteristics of disused muscle atrophy, including the decrease of muscle weight, the decrease of cross sectional area of muscle fiber and the increase of gene related to muscle atrophy. The differentially expressed miRNAs during the formation and recovery of weightlessness muscle atrophy were screened by gene microarray, and the differentially expressed miRNAs was verified and analyzed by qRT-PCR. MiRNAs with potential regulatory role in weightlessness muscle atrophy were screened.
【学位授予单位】：北京体育大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R85

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