Proteome Profiling of Mitotic Clonal Expansion during 3T3-L1 Adipocyte Differentiation Using iTRAQ-2DLC-MS/MS
Proteome Profiling of Mitotic Clonal Expansion during 3T3-L1 Adipocyte Differentiation Using iTRAQ-2DLC-MS/MS
复制标题
使用 iTRAQ-2DLC-MS/MS 对 3T3-L1 脂肪细胞分化过程中的有丝分裂克隆扩张进行蛋白质组分析。
DOI：
10.1021/pr401292p
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发表时间：
2014-03-01
影响因子：
4.4
通讯作者：
Tang, Qi-Qun
中科院分区：
文献类型：
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作者：
Jiang, Yan;Guo, Liang;Tang, Qi-Qun
有丝分裂克隆扩增（Mitotic clonal expansion， MCE）是发生在3T3-L1脂肪细胞分化早期的重要事件之一。为了研究这一过程的机制，我们进行了时间蛋白质组学分析，以描述MCE的动态变化。使用8-plex- itraq - 2lc -MS/MS分析，3152个蛋白在3T3-L1脂肪形成的最初28小时内被定量。我们对595种可能参与MCE的蛋白进行了功能分析，这些蛋白在诱导脂肪形成20 h时的量最大或最小，确定了PI3K/AKT/mTOR是最相关的途径。在595个蛋白中，我们选择PKM2 （Pyruvate kinase M2）作为进一步的研究，PKM2是我们之前的工作中发现的C/EBPβ的潜在靶基因。网络分析显示C/EBPβ、PIN1和PKM2呈正相关，这可能与PI3K-AKT通路有关。siRNA敲低PKM2抑制3T3-L1细胞的MCE和脂肪细胞分化。此外，当C/EBPβ表达下调时，PKM2的mRNA水平和蛋白水平均下调。过表达PKM2能部分恢复MCE，但不能恢复终末脂肪细胞分化，而终末脂肪细胞分化被siC/EBPβ抑制。因此，PKM2可能受C/EBPβ调控，参与脂肪细胞分化过程中的MCE。定量的动态蛋白质组变化为揭示脂肪形成的分子机制提供了有希望的基础。
Mitotic clonal expansion (MCE) is one of the important events taking place at the early stage during 3T3-L1 adipocyte differentiation. To investigate the mechanism underlying this process, we carried out a temporal proteomic analysis to profile the dynamic changes in MCE. Using 8-plex-iTRAQ-2DLC-MS/MS analysis, 3152 proteins were quantified during the initial 28 h of 3T3-L1 adipogenesis. Functional analysis was performed on 595 proteins with maximum or minimum quantities at 20 h of adipogenic induction that were potentially involved in MCE, which identified PI3K/AKT/mTOR as the most relevant pathway. Among the 595 proteins, PKM2 (Pyruvate kinase M2), a patterned protein identified as a potential target gene of C/EBPβ in our previous work, was selected for further investigation. Network analysis suggested positive correlations among C/EBPβ, PIN1, and PKM2, which may be related with the PI3K-AKT pathway. Knockdown of PKM2 with siRNA inhibited both MCE and adipocyte differentiation of 3T3-L1 cells. Moreover, PKM2 was down-regulated at both the mRNA level and the protein level upon the knockdown of C/EBPβ. And overexpressed PKM2 can partially restore MCE, although it did not restore terminal adipocyte differentiation, which was inhibited by siC/EBPβ. Thus, PKM2, potentially regulated by C/EBPβ, is involved in MCE during adipocyte differentiation. The dynamic proteome changes quantified here provide a promising basis for revealing molecular mechanism regulating adipogenesis.