PeerJ (Jun 2020)
Integrative analysis of competitive endogenous RNA network reveals the regulatory role of non-coding RNAs in high-glucose-induced human retinal endothelial cells
Abstract
Background Increasing evidence has suggested that non-coding RNAs (ncRNAs) play critical roles in the pathogenesis of diabetic retinopathy (DR), but their underlying mechanisms remain unclear. The purpose of this study was to determine latent key genes and to structure a competing endogenous RNA (ceRNA) regulatory network to discover the potential molecular mechanisms governing the effects of high glucose on human retinal endothelial cells (HRECs). Methods We obtained microarray data for long non-coding RNA (lncRNA) and mRNA of high-glucose-induced HREC samples from NCBI GEO datasets. The ceRNA network was screened using intersecting prediction results from miRcode, TargetScan, miRTarBase and miRDB. The protein–protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes and hub genes were obtained using the cytoHubba app. The ClusterProfiler package was applied for performing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The expression of key RNAs was verified using the qRT-PCR method. A key ceRNA subnetwork was constructed based on the criticality of the genes and its binding sites were verified by luciferase reporter assay. The viability and apoptosis of HRECs were tested using the transfection of the miR-449c inhibitor. Results A total of 3,328 lncRNAs and 2,017 mRNAs were screened for differentially expressed (DE) profiles. The newly constructed ceRNA network was composed of 410 lncRNAs, 35 miRNAs and 122 mRNAs. The 10 hub genes were identified through the PPI network. GO and KEGG analysis revealed that DE mRNAs were mainly related to the positive regulation of the mRNA catabolic process, cell polarity, and the G1/S transition of mitotic and cell cycle signaling pathways. QRT-PCR was used to verify RNAs and the most important genes were screened out. A key ceRNA subnetwork OIP5-AS1/miR-449c/MYC was established. The binding site was verified by luciferase reporter assay. The expression levels of OIP5-AS1 and MYC increased after miR-449c inhibitor transfection, miR-449c decreased, HRECs activity increased, and apoptosis decreased, compared with the control group. Conclusion We successfully built the key ceRNA subnetwork, OIP5-AS1/miR-449c/MYC, by applying the GEO database for data analysis and mining. The results from the ceRNA network allow us to better understand the effect of ncRNAs on HRECs under hyperglycemic conditions and the pathogenesis of DR.
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