ABSTRACT
Atherosclerotic plaques belong to the common vascular disease in the aged, which rupture will lead to acute thromboembolic diseases, the leading cause of fatal cardiovascular events. Accumulating evidence indicates that the lncRNAs-miRNAs-mRNA regulatory network plays a critical role in atherosclerosis. Based on RNA sequencing (GSE207252), we constructed expression profiles of lncRNAs, microRNAs, and mRNA in the carotid plaque of atherosclerosis patients and analyzed differentially expressed genes (DEGs). We identified three candidate lncRNAs using two algorithms (LASSO and SVM-RFE): lnc_GLRX3, lnc_FGF7-5, and DISC1FP1). LNCipedia, TargetScan, and miRDB databases were used to predict target miRNAs of lncRNAs and target genes of miRNAs. Gene ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA) analysis of DEGs was carried out using the R package clusterProfiler. A PPI network was constructed using the STRING website and visualized by Cytoscape. According to the "MCC" method of the plug-in cytoHubba in Cytoscape, ERCC4 was the top hub gene of the PPI network. We constructed a lncRNA_FGF7-5/lncRNA_GLRX3-miR-2681-5p-ERCC4 regulatory network for carotid plaque using lncRNA-miRNA and miRNA-mRNA pairs. Next, lncRNA_FGF7-5 and lncRNA_GLRX3 targeted miR-2681-5p directly to upregulate ERCC4 expression. Silencing of lncRNA_FGF7-5 and lncRNA_GLRX3 promoted apoptosis and TP53 expression in HUVECs treated with ox-LDL; however, these effects were reversed by ERCC4-overexpression. Taken together, these findings indicated that lncRNA_FGF7-5 and lncRNA_GLRX3 together reduced atherosclerosis-induced apoptosis of HUVECs via targeting miR-2681-5p to increase ERCC4 expression, thereby preventing the formation of carotid plaque and finally inhibiting atherosclerosis progression.
Subject(s)
Atherosclerosis , MicroRNAs , Plaque, Atherosclerotic , RNA, Long Noncoding , Humans , Aged , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Plaque, Atherosclerotic/genetics , Gene Regulatory Networks , MicroRNAs/genetics , MicroRNAs/metabolism , Atherosclerosis/genetics , RNA, Messenger/genetics , Carrier Proteins/genetics , Fibroblast Growth Factor 7/genetics , Fibroblast Growth Factor 7/metabolismABSTRACT
OBJECTIVE: To study the effects of octadecadienoic acid (ODA) on the proliferation and apoptosis of glioma cells and its mechanisms. METHODS: Cultured human glioma cells (cell density 2×106 cells/L) were divided into solvent control group (DMSO, 30 µl/L), 5-FU group (10 mg/L) and octadecadienic acid groups (0.3, 0.6 and 1.2 mg/L groups). The toxicity of ODA on glioma cells was detected by trypan blue and thiazolium blue (MTT). The expression levels of P53, PI3K, P21, PKB/Akt and Caspase-9 in glioma cells were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: â Cell count under optical microscope showed that the inhibition rate of cell proliferation in ODA low, medium and high dose groups and 5-FU group was significantly higher than that in the solvent control group (Pï¼0.01), but there was no statistical significance compared with the 5-FU group (Pï¼0.05). â¡ MTT assay showed that the inhibition rate of cell proliferation was increased significantly in ODA low, medium and high dose groups and 5-FU groups (Pï¼0.01), compared with the solvent control group. Compared with 5-FU group, the inhibition rate of cell proliferation was increased significantly only in ODA high dose group (Pï¼0.01). ⢠The number of G0/G1 phase cells in ODA low, medium and high dose groups and 5-FU group were increased significantly (Pï¼0.05, Pï¼0.01), the number of G2/M phase cells were decreased significantly (Pï¼0.01), and the apoptosis rate was increased significantly (Pï¼0.01),compared with the solvent control group. Compared with the 5-FU group, the number of cells in G2/M phase was decreased significantly (Pï¼0.01) and the apoptosis rate was increased significantly (Pï¼0.01) in ODA high dose group. ⣠ELISA test results showed that the protein expression levels of P53, PI3K and PKB/Akt in ODA low , medium and high dose groups and 5-FU group were significantly lower than those in solvent control group (all Pï¼0.01), but the protein expression levels in ODA high dose group were significantly lower than those in 5-FU group (Pï¼0.01). The protein expression levels of P21 and caspase-9 in ODA low , medium and high dose groups and 5-FU group were significantly higher than those in solvent control group (Pï¼0.05, Pï¼0.01), but the protein expression levels in ODA high dose group were significantly higher than those in 5-Fu group (Pï¼0.01). CONCLUSION: ODA can significantly inhibit the proliferation and promote apoptosis of glioma cells. The mechanisms are related to up-regulating the levels of P21 and caspase-9 to promote apoptosis, down-regulating the levels of P53, PI3K and PKB/Akt to inhibit the cell division cycle, and reducing the activity of PI3K-Akt signal transduction pathway.
