Fu Yuting, Zheng Xu, Jia Xiaoyang, Binderiya Uyanga, Wang Yanfeng, Bao Wenlei, Bao Lili, Zhao Keyu, Fu Yu, Hao Huifang, Wang Zhigang
College of Life Sciences, Inner Mongolia University, Hohhot, 010021, China.
Clinical Laboratory, The Hulunbuir People's Hospital, Hailaer, 021008, China.
BMC Genomics. 2016 Nov 7;17(1):879. doi: 10.1186/s12864-016-3151-y.
Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that is a central regulator of cell growth and metabolism. CCI-779 is a specific inhibitor of the mTORC1 signaling pathway.
We performed comparative transcriptome profiling on Inner Mongolia Cashmere goat fetal fibroblasts (GFbs) that were treated with CCI-779 and untreated cells. A total of 365 differentially expressed genes (DEGs) appeared between untreated and CCI-779-treated GFbs, with an FDR ≤0.001 and fold-change ≥2. These 365 DEGs were associated with mTOR signaling; 144 were upregulated in CCI-779-treated cells, and 221 were downregulated. Additionally, 300 genes were annotated with 43 Gene Ontology (GO) terms, and 293 genes were annotated with 194 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three RNA polymerase II and polymerase III subunits, 3 transcription factors, and 5 kinases in mTOR signaling were differentially expressed in CCI-779-treated GFbs. Further 6 DEGs were related to amino acid metabolism, 11 mediated lipid metabolism, 11 participated in carbohydrate metabolism, and 5 were involved in single-nucleotide metabolism. Based on our quantitative transcriptomic analysis, 40 significant DEGs with important function related to metabolism, RNA polymerase, transcription factors and mTOR signaling were selected for qPCR analysis, and the quantitative results between the two analysis methods were concordant. The qPCR data confirmed the differential expression in the RNA-Seq experiments. To validate the translational significance of the findings in certain differentially expressed genes, S6K1 and VEGF were detected by western blot, and these two proteins showed a differential expression between non-treated and treated with CCI-779 groups, which were consistent with mRNA abundance. The data showed a preliminary significance of the findings in the protein levels.
CCI-779 induces transcriptomic changes, and mTOR signaling might have significant function in the activation of RNA polymerase and certain transcription factors and in the metabolism of amino acids, lipids, carbohydrates, and single nucleotides in GFbs. These data filled the vacancy in the systematical profiling of mTOR signaling on Cashmere goat fetal fibroblasts.
雷帕霉素哺乳动物靶蛋白（mTOR）是一种进化上保守的丝氨酸/苏氨酸激酶，是细胞生长和代谢的核心调节因子。CCI-779是mTORC1信号通路的特异性抑制剂。
我们对用CCI-779处理的内蒙古绒山羊胎儿成纤维细胞（GFbs）和未处理的细胞进行了比较转录组分析。在未处理和经CCI-779处理的GFbs之间共出现365个差异表达基因（DEG），错误发现率（FDR）≤0.001且变化倍数≥2。这365个DEG与mTOR信号相关；在经CCI-779处理的细胞中有144个上调，221个下调。此外，300个基因被注释到43个基因本体论（GO）术语，293个基因被注释到194条京都基因与基因组百科全书（KEGG）通路。mTOR信号中的3个RNA聚合酶II和聚合酶III亚基、3个转录因子和5个激酶在经CCI-779处理的GFbs中差异表达。另外，6个DEG与氨基酸代谢相关，11个介导脂质代谢，11个参与碳水化合物代谢，5个参与单核苷酸代谢。基于我们的定量转录组分析，选择40个与代谢、RNA聚合酶、转录因子和mTOR信号相关的具有重要功能的显著DEG进行qPCR分析，两种分析方法的定量结果一致。qPCR数据证实了RNA测序实验中的差异表达。为了验证某些差异表达基因中发现的翻译意义，通过蛋白质免疫印迹法检测S6K1和VEGF，这两种蛋白质在未处理组和经CCI-779处理组之间表现出差异表达，与mRNA丰度一致。数据显示了这些发现在蛋白质水平上的初步意义。
CCI-779诱导转录组变化，mTOR信号可能在激活RNA聚合酶和某些转录因子以及在绒山羊胎儿成纤维细胞的氨基酸、脂质、碳水化合物和单核苷酸代谢中具有重要功能。这些数据填补了绒山羊胎儿成纤维细胞mTOR信号系统分析的空白。
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