Table1_Association of the LEP gene with immune infiltration as a diagnostic biomarker in preeclampsia.docx

Objective: Preeclampsia (PE) is a serious condition in pregnant women and hence an important topic in obstetrics. The current research aimed to recognize the potential and significant immune-related diagnostic biomarkers for PE.
Methods: From the Gene Expression Omnibus (GEO) data sets, three public gene expression profiles (GSE24129, GSE54618, and GSE60438) from the placental samples of PE and normotensive pregnancy were downloaded. Differentially expressed genes (DEGs) were selected and determined among 73 PE and 85 normotensive control pregnancy samples. The DEGs were used for Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO) enrichment analysis, and Gene Set Enrichment Analysis (GSEA). The candidate biomarkers were identified by the least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) analysis. The receiver operating characteristic curve (ROC) was applied to evaluate diagnostic ability. For further confirmation, the expression levels and diagnostic value of biomarkers in PE were verified in the GSE75010 data set (80 PE and 77 controls) and validated by qRT-RCR, Western blot, and immunohistochemistry (IHC). The CIBERSORT algorithm was used to calculate the compositional patterns of 22 types of immune cells in PE.
Results: In total, 15 DEGs were recognized. The GO and KEGG analyses revealed that the DEGs were enriched in the steroid metabolic process, receptor ligand activity, GnRH secretion, and neuroactive ligand−receptor interaction. The recognized DEGs were primarily implicated in cell-type benign neoplasm, kidney failure, infertility, and PE. Gene sets related to hormone activity, glycosylation, multicellular organism process, and response to BMP were activated in PE. The LEP gene was distinguished as a diagnostic biomarker of PE (AUC = 0.712) and further certified in the GSE75010 data set (AUC = 0.850). The high expression of LEP was associated with PE in clinical samples. In addition, the analysis of the immune microenvironment showed that gamma delta T cells, memory B cells, M0 macrophages, and regulatory T cells were positively correlated with LEP expression (P < 0.05).
Conclusion:LEP expression can be considered to be a diagnostic biomarker of PE and can offer a novel perspective for future studies regarding the occurrence and molecular mechanisms of PE.

研究目的：子痫前期（Preeclampsia, PE）是妊娠期女性的严重病症，亦是产科学领域的重要研究议题。本研究旨在识别子痫前期潜在且具有重要价值的免疫相关诊断生物标志物。
研究方法：从基因表达综合数据库（Gene Expression Omnibus, GEO）中下载3组公开的基因表达谱数据集（GSE24129、GSE54618及GSE60438），其样本均来自子痫前期胎盘组织与正常妊娠胎盘组织。本研究共纳入73例子痫前期患者样本与85例正常妊娠对照样本，筛选并鉴定其中的差异表达基因（Differentially expressed genes, DEGs）。利用所得DEGs开展基因本体论（Gene Ontology, GO）、京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）、疾病本体论（Disease Ontology, DO）富集分析及基因集富集分析（Gene Set Enrichment Analysis, GSEA）。通过最小绝对收缩和选择算子（least absolute shrinkage and selection operator, LASSO）与支持向量机递归特征消除（support vector machine recursive feature elimination, SVM-RFE）分析筛选候选生物标志物，并采用受试者工作特征曲线（receiver operating characteristic curve, ROC）评估其诊断效能。为进一步验证，本研究在GSE75010数据集（包含80例子痫前期样本与77例对照样本）中验证候选生物标志物在子痫前期中的表达水平与诊断价值，并通过qRT-RCR、蛋白质印迹法及免疫组化（immunohistochemistry, IHC）进行验证。采用CIBERSORT算法计算子痫前期样本中22种免疫细胞的组成模式。
研究结果：本研究共鉴定得到15个DEGs。GO与KEGG富集分析结果显示，DEGs主要富集于类固醇代谢过程、受体配体活性、促性腺激素释放激素分泌及神经活性配体-受体相互作用通路。鉴定得到的DEGs主要与细胞型良性肿瘤、肾衰竭、不孕症及子痫前期相关。在子痫前期样本中，与激素活性、糖基化、多细胞生物过程及骨形态发生蛋白应答相关的基因集被激活。本研究筛选出LEP基因作为子痫前期的诊断生物标志物（曲线下面积AUC=0.712），并在GSE75010数据集中得到进一步验证（AUC=0.850）。临床样本分析显示，LEP的高表达与子痫前期显著相关。此外，免疫微环境分析结果表明，γδ T细胞、记忆B细胞、M0型巨噬细胞及调节性T细胞的浸润水平与LEP的表达呈正相关（P<0.05）。
研究结论：LEP的表达可作为子痫前期的诊断生物标志物，为未来子痫前期发生机制及分子通路的相关研究提供全新视角。