调控棕色脂肪产热关键基因的新筛选方法

Aü novel screening approach for identifying key genes involved in the regulation of brown adipose tissue thermogenesis

摘要:

目的]通过多组学整合分析,系统解析棕色脂肪组织(BAT)产热功能的分子调控网络,发现新型产热调控基因,为代谢性疾病的治疗提供新靶点。 [方法]构建筛选棕色脂肪产热关键基因的新方法:首先,对小鼠棕色脂肪Bulk RNA-seq数据进行差异表达分析,以ABS(log₂FoldChange)＞1且Padj＜0.05为条件筛选差异基因,并分别对上调和下调基因取交集。然后,根据所得候选基因的表达量对scRNA-seq数据中棕色脂肪细胞进行分组,对高表达组和低表达组进行差异分析和差异基因的基因集富集分析(GSEA),以分析候选基因和产热功能的相关性。通过实时荧光定量PCR和蛋白质印迹对部分候选基因进行实验验证。 [结果]生物信息学分析共鉴定出产热正相关基因65个和负相关基因7个,实时荧光定量PCR分析发现候选基因Mfsd2a、Me1、Slc25a34、Pfkp、Ankrd9、Hsd17b12、Aldoa、Ctsz和Pcyt2上调倍数超过5倍,Pid1、Angpt1下调超过50%(均P＜0.01),可进行进一步的产热相关功能学和分子机制研究。 [结论]本研究创建了“计算筛选→模拟敲除→实验验证”的靶点发现范式,系统揭示了参与BAT产热调控的分子网络,发现了11个核心基因,可能在BAT产热激活过程中发挥关键调控作用,有望为改善能量代谢、治疗肥胖及其并发症提供新的药物干预靶点。

Abstract:

Aim To systematically elucidate the molecular regulatory network of thermogenic function in brown adipose tissue (BAT) through multi-omics integrative analysis, to discover novel thermogenic regulatory genes and provide novel therapeutic targets for metabolic disorders. Methods A novel methodology for screening key genes regulating thermogenesis in BAT was constructed:First, differential expression analysis was performed on bulk RNA-seq data from murine BAT. Genes meeting the thresholds of ABS(log₂FoldChange)＞1 and Padj＜0.05 were identified as differentially expressed genes. Intersectional analysis was then applied to obtain consensus upregulated and downregulated gene sets.Subsequently, scRNA-seq data of brown adipocytes were partitioned into high-expression group and low-expression group based on the expression levels of candidate genes. Differential analysis and gene set enrichment analysis (GSEA) were conducted between these groups to assess the correlation between candidate genes and thermogenic function. Finally, experimental validation of selected candidate genes was performed using quantitative real-time PCR and Western blot. Results Bioinformatics analysis identified 65 thermogenesis-positive correlated genes and 7 thermogenesis-negative correlated genes. Subsequent quantitative PCR validation demonstrated that candidate genes Mfsd2a, Me1, Slc25a34, Pfkp, Ankrd9, Hsd17b12, Aldoa, Ctsz and Pcyt2 exhibited upregulation exceeding 5-fold, while Pid1 and Angpt1 showed downregulation over 50%. All observed expression changes demonstrated statistical significance (P＜0.01) through rigorous hypothesis testing. These findings highlight the potential involvement of these genes in thermogenic regulation, warranting further functional investigations to elucidate their molecular mechanisms in energy metabolism pathways. ConclusionsThis study established a novel “computational screening → in silico knockout → experimental validation” paradigm for target discovery, systematically unveiling the molecular network involved in BAT thermogenic regulation. This methodology is equally applicable for identifying key regulatory genes in other physiological or pathological processes. The study identified 11 core genes that may play pivotal regulatory roles during BAT thermogenic activation, which could potentially offer novel pharmacological intervention targets to improve energy metabolism and treat obesity-related complications.