模拟微重力对小鼠成纤维细胞miRNA表达的影响

Effects of simulated microgravity on miRNA expression in mouse fibroblasts Song Xiangwei

目的探讨微重力环境对小鼠成纤维细胞应激损伤相关miRNA表达的影响。方法体外培养L929细胞，应用简单随机方法分为模拟微重力（SMG）组（SMG组）和正常重力（NG）对照组（NG组）。细胞培养7d，提取样本总RNA行荧光标记和芯片杂交，采用Feature Extraction软件处理杂交图像提取原始数据，应用Genespring软件进行分位数标准化和后续处理，筛选差异表达显著的miRNA，qRT—PCR验证芯片结果准确性。通过miRNA靶基因预测数据库TargetScan和microRNAorg对差异miRNA进行靶基因预测，其交集作为潜在调节靶基因，对预测靶基因分别进行基因本体和京都基因与基因组百科全书（KEGG）富集分析，判定差异miRNA主控的生物学过程和（或）信号通路。结合mRNA差异表达谱行miRNA—mRNA联合分析，并构建miRNA—mRNA功能网络和miRNA—mRNA调控网络。结果miRNA芯片检测发现，L929细胞在模拟微重力环境下较正常重力环境下差异表达的miRNA有21个，4个显著上调，包括mlnu—miR-669j、-122—5p、-30a-3p和-6516—3p，其中mmu—miR-669i上调最为显著（52．84倍，P〈0．05）；17个显著下调，包括ttlmu—miR-21a-3p、-miR-28a-5p、-218—5p、-210—3p、-miR-19a-3p、-miR-31—3p—miR-19b-3p等，其中mmu—miR-28a-5p下调最为显著（15．47倍，P〈0．05）。qRT—PCR结果与miRNA芯片结果-致（P〈0．05）。靶基因预测和功能富集分析提示多种创伤修复相关生物学过程和信号通路显著富集（P〈0．05）。通过miRNA—mRNA联合分析构建的miRNA—mRNA功能网络和miRNA—mRNA调控网络涵盖了所有具有显著差异的miRNA。结论微重力环境下小鼠成纤维细胞多种应激损伤相关miRNA表达发生显著变化，可能在微重力环境下创伤修复过程中发挥重要作用。基于芯片技术的miRNA靶基因预测和功能富集分析可为失重应激损伤机制探讨和创伤修复措施提供理论依据。

Objective To investigate the effects of simulated microgravity by rotary cell culture system （RCCS） on expression profiles of miRNA in mouse fibroblasts L929 cell line. Methods L929 cells were cultured in vitro and divided into simulated microgravity （SMG） group and normal gravity （NG） group according to the simple random method. Samples of two groups were collected on 7th day of culture and the total RNAs were extracted, labeled, and hybridized in sequence. Feature Extraction Software was used to collect the array images and get raw data, which were analyzed by Genespring Software. Differentially expressed miRNAs were identified and then validated by qRT-PCR. Target genes of differentially expressed miRNAs were predicted by the databases of Targetscan and microRNAorg, and the intersections of databases were identified as potential regulatory target genes. Gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） analysis were applied to determine the roles of these target genes. Relevant biological functions and/or signaling pathways of the regulated genes especially related with wound healing process were categorized based on their enrichments. Then integration predictions of the miRNA and mRNA expression profiles had been proposed to refine the functional miRNA-mRNA relationships. The miRNA-mRNA functional network and miRNA-mRNA control network were constructed. Results Four miRNA genes were up-regulated significantly including mmu- miR-669j, -122-5p, -30a-3p, -6516-3p, among which mmu-miR-669j was up-regulated at 52.84 folds with the greatest significance （ P 〈 0.05 ）. Seventeen miRNA genes were down-regulated significantly including mmu-miR-21a-3p, -miR-28a-5p, -218-5p, -210-3p, -miR-19a-3p, -miR-31-3p, and-miR- 19b-3p, among which mmu-miR-28a-5p was down-regulated at 15.47 folds with the greatest significance （ P 〈 0. 05 ）. The qRT-PCR showed a high concordance with the microarray results （ P 〈 0.05 ）. Target gene prediction and functional enrichment analysis suggested that a variety of biological processes and signaling pathways involved in wound repair were significantly enriched （ P 〈 0.05 ）. Function network and regulation network of miRNA-mRNA covered all the differentially expressed miRNAs, which suggested that miR-21 a-3p and predicted target gene Smad3 might play an important role in wound healing under mierogravity. Conclusions Simulated microgravity by RCCS can significantly affect the expression of stress-related miRNAs in mouse fibroblasts L929. The miRNA target gene prediction and functional enrichment analysis based on gene chip technology may provide theoretical basis for illustrating the mechanism and management of weightlessness stress injury.