Huh-7 cell line treated with GDF-11. Homo sapiens

Growth differentiation factor 11 (GDF11) has been characterized as a key regulator of differentiation in cells that retain stemness features. Recently, it has been reported that GDF11 exerts tumor suppressive properties in hepatocellular carcinoma cells decreasing clonogenicity, proliferation, spheroid formation and cellular function, all associated to a decrement in stemness features, resulting in mesenchymal to epithelial transition and loss of aggressiveness. The aim of the present work was to figure out the mechanism associated to the tumor suppressive properties displayed by GDF11 in liver cancer cells. Hepatocellular carcinoma-derived cell lines were exposed to GDF11 (50 ng/ml), RNA-seq analysis in Huh7 cell line revealed that GDF11 exerted profound transcriptomic impact involved regulation of cholesterol metabolic process, steroid metabolic process as well as key signaling pathways resembling endoplasmic reticulum-related functions. Cholesterol and triglycerides determination in Huh7 and Hep3B cells treated with GDF11 exhibited a significant decrement in the content of these lipids. The mTOR signaling pathway was downregulated, and this was associated to a decrement in key proteins involved in mevalonate pathway. In addition, real-time metabolism assessed by Seahorse technology showed abridged glycolysis as well as glycolytic capacity, closely related to an impaired oxygen consumption rate and decrement in ATP production. Finally, transmission electron microscopy revealed mitochondria abnormalities, such as cristae disarrangement, consistent with metabolic changes. Results provide evidence that GDF11 impairs cancer cell metabolism particularly targeting cholesterol homeostasis, glycolysis and mitochondria function and morphology.

生长分化因子11（Growth differentiation factor 11，GDF11）已被证实为维持干细胞特性的细胞分化关键调控因子。近期有研究报道，GDF11在肝细胞癌细胞中发挥抑癌作用，可降低克隆形成能力、增殖活性、球体形成能力及细胞功能，上述效应均与干细胞特性的下调相关，最终诱导间质上皮转化并降低细胞侵袭性。本研究旨在阐明GDF11在肝癌细胞中发挥抑癌作用的具体分子机制。将肝细胞癌来源细胞系暴露于50 ng/ml的GDF11中，对Huh7细胞株开展RNA测序（RNA-seq）分析显示，GDF11可产生显著的转录组学影响，涉及胆固醇代谢过程、类固醇代谢过程的调控，以及与内质网相关功能高度相似的关键信号通路。对经GDF11处理的Huh7与Hep3B细胞进行胆固醇与甘油三酯含量检测，结果显示这两种脂质的水平均显著降低。mTOR信号通路被下调，且该现象与甲羟戊酸途径关键蛋白的表达下调相关。此外，通过海马生物能量代谢分析仪（Seahorse）检测的实时代谢情况显示，GDF11可削弱糖酵解水平及糖酵解能力，这与耗氧率受损及ATP生成减少密切相关。最后，透射电子显微镜观察发现线粒体出现嵴排列紊乱等异常，该结果与代谢改变的特征相符。本研究结果证实，GDF11可损害癌细胞代谢，尤其通过靶向调控胆固醇稳态、糖酵解以及线粒体功能与形态发挥抑癌效应。