旋转细胞培养系统模拟微重力环境对小鼠成纤维细胞lncRNA表达的影响

Effect of rotary cell culture system-simulated microgravity environment on the expression of lncRNA in mouse fibroblasts

目的研究旋转细胞培养系统(RCCS)模拟微重力环境对小鼠成纤维细胞株L929 lncRNA表达的影响。方法体外培养L929细胞,随机分为模拟微重力组(SMG组)和正常重力组(NG组),每组3个样本。SMG组回转器轴心与地面平行旋转,NG组回转器轴心与地面垂直旋转,两组转速一致。RCCS培养7d,收集样本,提取样本总RNA,进行荧光标记和芯片杂交。利用Agilent Mouse lncRNA芯片分别检测SMG组和NG组L929细胞的lncRNA和mRNA表达,筛选差异表达显著的lncRNA,RT-q PCR验证芯片结果;利用GO和Pathway分析差异表达lncRNA的功能分布,结合mRNA差异表达谱,进行lncRNA-mRNA联合分析。结果 lncRNA芯片检测分析发现,RCCS模拟微重力环境下小鼠成纤维细胞L929共有238条差异表达的lncRNA,其中134条表达上调,104条表达下调;差异表达的mRNA共有237条,其中53条表达上调,184条表达下调。获取差异表达lncRNA的聚类分析图,对差异表达显著的4条lncRNA芯片结果进行RT-q PCR验证,结果相吻合。GO分析结果显示差异表达的lncRNA与巨噬细胞分化、伤口愈合的负性调节等生物学过程相关,Pathway分析结果显示差异表达的lncRNA与系统性红斑狼疮、TGF-β等信号通路相关。同时成功构建了lncRNA-mRNA-TF可视化网络图。结论 RCCS模拟微重力环境显著影响L929细胞的lncRNA及mRNA表达谱,基于芯片技术的lncRNA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修复措施建立提供理论依据。

Objective To investigate the effects of simulated microgravity by rotary cell culture system（RCCS） on expression profiles of long non-coding RNA（lncRNA） in mouse fibroblasts L929 cell line. Methods L929 cells were cultured in vitro and randomly divided into simulated microgravity（SMG） group and normal gravity（NG） group. Each group had three samples, the rotator axis of SMG group was paralleled to the ground rotation, while the rotator axis of NG group was vertical to the ground rotation, and the speed of rotation was consistent for the two groups. The samples of two groups were collected on 7 th day of culture and the total RNAs were extracted, labeled and hybridized in sequence. The lncRNA and mRNA were detected by Agilent Mouse lncRNA Chips respectively. Differentially expressed lncRNA were identified and then validated by RT-q PCR. GO and Pathway analysis were applied to determine the functional distribution of these target genes. The integration predictions of the lncRNA and mRNA co-expression had been proposed to refine the functional lncRNA-mRNA relationships. Results There were 238 differentially expressed lncRNAs including 134 lncRNAs up-regulated and 104 lncRNAs down-regulated, and 237 differentially expressed mRNAs including 53 mRNAs up-regulated and 184 mRNAs down-regulated significantly in mouse fibroblasts L929 cell line under simulated microgravity by RCCS. The RT-q PCR showed a high concordance with chip microarray results in 4 differentially expressed lncRNA. GO analysis showed that the differentially expressed lncRNAs were related to the biological processes such as negative regulation of megakaryocyte differentiation and negative regulation of wound healing. Pathway analysis showed that these target genes were related to the signal pathways of systemic lupus erythematosus and TGF-β. The lncRNA-mRNA co-expression networks were also established. Conclusion The simulated microgravity by RCCS could significantly affect the expression profiles of lncRNA and mRNA in mouse fibroblasts L929. The lncRNA target genes prediction and functional enrichment analysis based on gene chip technology may provide the theoretical basis for illustrating the mechanism and management of weightlessness stress injury.