Table2_Contribution of FBLN5 to Unstable Plaques in Carotid Atherosclerosis via mir128 and mir532–3p Based on Bioinformatics Prediction and Validation.XLSX

FBLN5, a member of the short fibulins in the fibulin family of extracellular matrix/matricellular proteins, is involved in interactions with components of the basement membrane and extracellular matrix proteins. It plays key roles in endothelial tissues in many vascular diseases. In this study, the relationship between FBLN5 and carotid atherosclerotic plaque stability as well as the regulatory roles of miRNAs were evaluated. Differential gene expression analyses and weighted gene co-expression network analysis (WGCNA) based on the GSE163154 dataset (including 16 samples without intraplaque hemorrhage and 27 samples with intraplaque hemorrhage) in GEO revealed that FBLN5 is related to plaque stability and is the most significantly differentially expressed gene. LASSO regression was used to evaluate genes obtained from the intersection of differentially expressed genes and clinically significant modules identified by WGCNA. A prediction model based on eight genes, including FBLN5, was constructed and showed an accuracy of 0.951 based on an ROC analysis. Low FBLN5 expression in plaque tissues was confirmed by immunohistochemistry and western blotting. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that FBLN5 acted mainly by the maintenance of the cellular matrix and reactive oxygen species production. miRNAs upstream of these eight predictive genes, including FBLN5, were identified and used to construct a network diagram. These results revealed that hsa-mir-128 and hsa-mir-532–3p were upstream regulatory factors of FBLN5, as verified by PCR assays of human plaque tissues demonstrating that both miRNAs were significantly up-regulated. Therefore, FBLN5 may play an important role in carotid atherosclerosis via hsa-mir-128 and hsa-mir-532–3p as well as become an essential target for treatment.

FBLN5是纤维蛋白家族（fibulin family）中细胞外基质/基质细胞蛋白类短纤维蛋白的成员，可参与基底膜成分与细胞外基质蛋白的相互作用。它在多种血管疾病的内皮组织中发挥关键作用。本研究评估了FBLN5与颈动脉粥样硬化斑块稳定性的关联，以及微小RNA（microRNA，miRNA）的调控功能。基于GEO数据库中GSE163154数据集（含16例无斑块内出血样本、27例伴斑块内出血样本）开展的差异基因表达分析与加权基因共表达网络分析（WGCNA）结果显示，FBLN5与斑块稳定性显著相关，且为差异表达最显著的基因。研究采用LASSO回归对差异基因与WGCNA筛选得到的临床显著模块的交集基因进行评估，构建了包含FBLN5在内的8基因预测模型，经受试者工作特征（ROC）分析验证，该模型准确率达0.951。通过免疫组织化学与蛋白质免疫印迹实验，证实FBLN5在斑块组织中呈低表达状态。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）富集分析结果表明，FBLN5主要通过维持细胞外基质稳态及活性氧生成发挥生物学功能。研究进一步鉴定出上述8个预测基因（含FBLN5）的上游miRNA，并构建了调控网络图。结果显示，hsa-mir-128与hsa-mir-532–3p为FBLN5的上游调控因子，经人类斑块组织的PCR实验验证，这两种miRNA均显著上调。综上，FBLN5可能通过hsa-mir-128与hsa-mir-532–3p介导在颈动脉粥样硬化中发挥重要作用，有望成为该疾病治疗的关键靶点。