Zhang Ruihao, Sun Qi, Huang Lixia, Li Jian
Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Cheeloo College of Medicine, Shandong University, Jinan 250012, China.
Int J Mol Sci. 2025 Jul 23;26(15):7108. doi: 10.3390/ijms26157108.
Cholesterol stress profoundly modulates cellular processes, but its underlying mechanisms remain incompletely understood. To investigate cholesterol-responsive networks, we performed integrated transcriptome (RNA-seq) and metabolome (LC-MS) analyses on HeLa cells treated with cholesterol for 6 and 24 h. Through transcriptomic analysis of cholesterol-stressed HeLa cells, we identified stage-specific responses characterized by early-phase stress responses and late-phase immune-metabolic coordination. This revealed 1340 upregulated and 976 downregulated genes after a 6 h cholesterol treatment, including induction and suppression of genes involved in cholesterol efflux and sterol biosynthesis, respectively, transitioning to Nuclear Factor kappa-B (NF-κB) activation and Peroxisome Proliferator-Activated Receptor (PPAR) pathway modulation by 24 h. Co-expression network analysis prioritized functional modules intersecting with differentially expressed genes. We also performed untargeted metabolomics using cells treated with cholesterol for 6 h, which demonstrated extensive remodeling of lipid species. Interestingly, integrated transcriptomic and metabolic analysis uncovered GFPT1-driven Uridine Diphosphate-N-Acetylglucosamine (UDP-GlcNAc) accumulation and increased taurine levels. Validation experiments confirmed upregulation and downregulation through RT-qPCR and increased O-GlcNAcylation via Western blot. Importantly, clinical datasets further supported the correlations between / expression and cholesterol levels in Non-Alcoholic Steatohepatitis (NASH) liver cancer patients. This work establishes a chronological paradigm of cholesterol sensing and identifies and as key regulators bridging glycosylation and lipid pathways, providing mechanistic insights into cholesterol-associated metabolic disorders.
胆固醇应激深刻地调节细胞过程，但其潜在机制仍未完全了解。为了研究胆固醇反应网络，我们对用胆固醇处理6小时和24小时的HeLa细胞进行了综合转录组（RNA测序）和代谢组（液相色谱-质谱联用）分析。通过对胆固醇应激的HeLa细胞进行转录组分析，我们确定了以早期应激反应和晚期免疫-代谢协调为特征的阶段特异性反应。这揭示了胆固醇处理6小时后1340个基因上调和976个基因下调，分别包括参与胆固醇流出和甾醇生物合成的基因的诱导和抑制，到24小时转变为核因子κB（NF-κB）激活和过氧化物酶体增殖物激活受体（PPAR）途径调节。共表达网络分析确定了与差异表达基因相交的功能模块。我们还对用胆固醇处理6小时的细胞进行了非靶向代谢组学分析，结果表明脂质种类发生了广泛重塑。有趣的是，综合转录组和代谢分析发现谷氨酰胺果糖-6-磷酸转氨酶1（GFPT1）驱动的尿苷二磷酸-N-乙酰葡糖胺（UDP-GlcNAc）积累和牛磺酸水平升高。验证实验通过逆转录定量聚合酶链反应（RT-qPCR）证实了基因的上调和下调，并通过蛋白质免疫印迹法证实了O-连接的N-乙酰葡糖胺糖基化（O-GlcNAcylation）增加。重要的是，临床数据集进一步支持了非酒精性脂肪性肝炎（NASH）肝癌患者中/表达与胆固醇水平之间的相关性。这项工作建立了一个按时间顺序的胆固醇感知范式，并确定了作为连接糖基化和脂质途径的关键调节因子，为胆固醇相关的代谢紊乱提供了机制性见解。
