Supplementary Material for: MicroRNA Expression Profiles in the Subcutaneous Adipose Tissues of Morbidly Obese Chinese Women

Introduction: Obesity is a main global health issue and an outstanding cause of morbidity and mortality. Exploring miRNA profiling may help further studies on obesity. Methods: Three morbidly obese and 5 normal-weight Chinese women were enrolled in the microarray testing group. Abdominal subcutaneous adipose tissue (SAT) samples were excised. Total RNAs including miRNAs were extracted. Affymetrix GeneChip miRNA 4.0 Array was used to compare the expression profiles of miRNAs between the 2 groups. Two algorithms, miRanda and TargetScan, were used to predict target messenger RNAs (mRNAs). Bioinformatics analysis was then done based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. The sample sizes were further expanded to 8 morbidly obese and 9 normal-weight subjects, and quantitative real-time PCR (qRT-PCR) was utilized to verify the expression of differential miRNAs and target genes. Results: As per the microarray assay, 58 miRNAs were differentially expressed in the SAT from the morbidly obese and normal-weight groups (Fold >4, p < 0.01, FDR <0.05); 54 of these were downregulated and 4 were upregulated in morbidly obese subjects. A total of 1,333 target genes were jointly predicted by miRanda and TargetScan. Further bioinformatics analysis showed that the differential miRNAs were involved in 269 significant biological functions and 89 significant signaling pathways. The validation experiment by qRT-PCR showed that the expression levels of miRNA-143-5p, miRNA-143-3p, miRNA-145-5p, and let-7a-5p were downregulated in morbidly obese subjects, consistent with the microarray detection. High-mobility group A2 (HMGA2), a target gene of the downregulated miRNA let-7a-5p, was first found to be upregulated 3.19-fold in the SAT of morbidly obese Chinese women when compared to normal-weight controls. Conclusions: MiRNA downregulation is a hallmark of intact SAT in a morbidly obese state. Transcription (DNA-dependent), small-molecule metabolic processes, the MAPK signaling pathway, and cancer-related pathways may play important roles in the occurrence and development of obesity. For the first time, we proved that HMGA2, a target gene of let-7a-5p, is upregulated in the SAT of morbidly obese Chinese women.

引言：肥胖是全球性主要公共卫生问题，亦是致病与死亡的重要诱因。探索微小核糖核酸（microRNA, miRNA）表达谱，可为肥胖相关后续研究提供重要参考。
方法：本研究的芯片检测组纳入3名病态肥胖中国女性与5名体重正常中国女性。采集受试者的腹部皮下脂肪组织（subcutaneous adipose tissue, SAT）样本，提取包含miRNA在内的总核糖核酸（ribonucleic acid, RNA）。采用Affymetrix GeneChip miRNA 4.0芯片，对比两组受试者的miRNA表达谱。采用miRanda与TargetScan两种算法，预测靶信使核糖核酸（messenger RNA, mRNA）。随后基于基因本体（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）数据库进行生物信息学分析。随后扩大样本量至8名病态肥胖者与9名体重正常者，采用实时定量聚合酶链式反应（quantitative real-time PCR, qRT-PCR）验证差异表达miRNA及靶基因的表达水平。
结果：芯片检测结果显示，病态肥胖组与正常体重组的SAT组织中共有58个miRNA存在差异表达（倍数变化>4，p<0.01，错误发现率（false discovery rate, FDR）<0.05）；其中54个在病态肥胖受试者中呈下调表达，4个呈上调表达。miRanda与TargetScan共同预测得到1333个靶基因。进一步生物信息学分析表明，差异表达miRNA参与了269个显著生物学功能及89条显著信号通路。实时定量PCR验证实验结果显示，miRNA-143-5p、miRNA-143-3p、miRNA-145-5p及let-7a-5p在病态肥胖受试者中呈下调表达，与芯片检测结果一致。下调表达miRNA let-7a-5p的靶基因高迁移率族蛋白A2（high-mobility group A2, HMGA2）首次被发现，在病态肥胖中国女性的SAT组织中较正常体重对照组上调3.19倍。
结论：miRNA下调是病态肥胖状态下完整SAT组织的特征性标志。DNA依赖的转录过程、小分子代谢过程、丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）信号通路及癌症相关通路，可能在肥胖的发生与发展中发挥重要作用。本研究首次证实，let-7a-5p的靶基因HMGA2在病态肥胖中国女性的SAT组织中呈上调表达。