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

Lin Zheng,Honglin Dong,Guoping Zheng,Ruijing Zhang,Bojin Zhang,Minhui Li,Ruihan Chen,Xinyang Yue,Jie Hu

doi:10.3389/fgene.2022.821650

Abstract

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.

Highlights

McGeachie et al (2009) constructed a Bayesian model to predict atherosclerosis based on 13 genes, including FBLN5, and 5 clinical variables and obtained a prediction accuracy of 85%; the mechanism by which the genes contribute to atherosclerosis was not explored
Colige et al (2019) found that FBLN5 is connected to FBLN1 via LTBP4, and ADAMTST degrades LTBP4 in cardiovascular diseases, resulting in extracellular matrix degradation and disease development
To test whether FBLN5 plays a role in carotid atherosclerotic plaque stability, we download an expression matrix from a Gene Expression Omnibus (GEO) dataset including 27 samples with intraplaque hemorrhage (IPH) and 16 samples without IPH

Summary

Introduction

Carotid atherosclerosis is responsible for more than a third of strokes. Little is known about the factors that cause atherosclerotic plaque instability, rupture, and embolism. It is critical to clarify the molecular mechanism underlying the development and progression of intraplaque hemorrhage as well as to identify new molecular targets for pharmacological therapy to decrease plaque instability and avoid ischemic events. McGeachie et al (2009) constructed a Bayesian model to predict atherosclerosis based on 13 genes, including FBLN5, and 5 clinical variables and obtained a prediction accuracy of 85%; the mechanism by which the genes contribute to atherosclerosis was not explored. Nox may negatively regulate FBLN5 to degrade the arterial middle layer (Hu et al, 2019). Carotid atherosclerosis involves changes in the middle layer of the artery

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Results

Conclusion