没食子酸表没食子儿茶素酯靶向 FTO 并通过 mRNA mA-YTHDF2 依赖性方式抑制脂肪生成。
Epigallocatechin gallate targets FTO and inhibits adipogenesis in an mRNA mA-YTHDF2-dependent manner.
机构信息
College of Animal Sciences, Key Laboratory of Animal Nutrition & Feed Sciences, Ministry of Agriculture, Zhejiang Provincial Laboratory of Feed and Animal Nutrition, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou, Zhejiang, 310058, China.
出版信息
Int J Obes (Lond). 2018 Jul;42(7):1378-1388. doi: 10.1038/s41366-018-0082-5. Epub 2018 May 24.
BACKGROUND/OBJECTIVE: N-methyladenosine (mA) modification of mRNA plays a role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, plays a critical role in anti-obesity and anti-adipogenesis.
METHODS
High-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS) was performed to determine the mA levels in 3T3-L1 preadipocytes. The effects of EGCG on the mA levels in specific genes were determined by methylated RNA immunoprecipitation coupled with quantitative real-time PCR (meRIP-qPCR). Several adipogenesis makers and cell cycle genes were analyzed by quantitative real-time PCR (qPCR) and western blotting. Lipid accumulation was evaluated by oil red O staining. All measurements were performed at least for three times.
RESULTS
Here we showed that EGCG inhibited adipogenesis by blocking the mitotic clonal expansion (MCE) at the early stage of adipocyte differentiation. Exposing 3T3-L1 cells to EGCG reduced the expression of fat mass and obesity-associated (FTO) protein, an mA demethylase, which led to increased overall levels of RNA mA methylation. Cyclin A2 (CCNA2) and cyclin dependent kinase 2 (CDK2) play vital roles in MCE. The mA levels of CCNA2 and CDK2 mRNA were dramatically enhanced by EGCG. Interestingly, EGCG increased the expression of YTH N-methyladenosine RNA binding protein 2 (YTHDF2), which recognized and decayed methylated mRNAs, resulting in decreased protein levels of CCNA2 and CDK2. As a result, MCE was blocked and adipogenesis was inhibited. FTO overexpression and YTHDF2 knockdown in 3T3-L1 cells significantly increased CCNA2 and CDK2 protein levels and ameliorated the EGCG-induced adipogenesis inhibition. Thus, mA-dependent CCNA2 and CDK2 expressions mediated by FTO and YTHDF2 contributed to EGCG-induced adipogenesis inhibition.
CONCLUSION
Our findings provide mechanistic insights into how mA is involved in the EGCG regulation of adipogenesis and shed light on its anti-obesity effect.
背景/目的：mRNA 中的 N6-甲基腺苷（mA）修饰在调节脂肪生成中发挥作用。然而，其潜在机制在很大程度上仍不清楚。表没食子儿茶素没食子酸酯（EGCG）是绿茶中含量最丰富的儿茶素，在抗肥胖和抗脂肪生成中起着关键作用。
方法
采用高效液相色谱-串联三重四极杆质谱法（HPLC-QqQ-MS/MS）测定 3T3-L1 前脂肪细胞中的 mA 水平。通过甲基化 RNA 免疫沉淀结合定量实时 PCR（meRIP-qPCR）测定 EGCG 对特定基因中 mA 水平的影响。通过定量实时 PCR（qPCR）和蛋白质印迹法分析几种脂肪生成标志物和细胞周期基因。通过油红 O 染色评估脂质积累。所有测量均至少进行了三次。
结果
在这里，我们表明 EGCG 通过阻断脂肪细胞分化早期的有丝分裂克隆扩张（MCE）来抑制脂肪生成。暴露于 EGCG 的 3T3-L1 细胞降低了脂肪质量和肥胖相关（FTO）蛋白的表达，这是一种 mA 去甲基化酶，导致 RNA mA 甲基化的整体水平增加。细胞周期蛋白 A2（CCNA2）和细胞周期蛋白依赖性激酶 2（CDK2）在 MCE 中发挥重要作用。CCNA2 和 CDK2 mRNA 的 mA 水平被 EGCG 显著增强。有趣的是，EGCG 增加了 YTH N6-甲基腺苷 RNA 结合蛋白 2（YTHDF2）的表达，该蛋白识别并降解甲基化的 mRNAs，导致 CCNA2 和 CDK2 蛋白水平降低。结果，MCE 被阻断，脂肪生成受到抑制。在 3T3-L1 细胞中转染 FTO 过表达和 YTHDF2 敲低显著增加了 CCNA2 和 CDK2 蛋白水平，并改善了 EGCG 诱导的脂肪生成抑制。因此，FTO 和 YTHDF2 介导的 mA 依赖性 CCNA2 和 CDK2 表达有助于 EGCG 诱导的脂肪生成抑制。
结论
我们的研究结果提供了关于 mA 如何参与 EGCG 调节脂肪生成的机制见解，并揭示了其抗肥胖作用。
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