Identify Functional lncRNAs in Nonalcoholic Fatty Liver Disease by Constructing a ceRNA Network.

ABSTRACT

Aim: To identify functional long noncoding RNAs (lncRNAs) by constructing a NAFLD-related lncRNA-miRNA-mRNA network (NLMMN) based on the hypothesis that lncRNAs, as competitive endogenous RNAs (ceRNAs), are able to regulate mRNA functions by competitive binding to shared miRNAs. Methods: The "Limma R package" was used to identify differentially expressed lncRNAs and mRNAs (DElncRNAs and DEmRNAs). The "miRcode online tool" was used to predict the potential interactions between DElncRNAs or DEmRNAs using Perl, and "multiMiR R package" was used to predict the potential interactions between DElncRNAs and miRNAs. The NLMMN was viewed by Cytoscape. The DEmRNAs were further analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to identify functional lncRNAs in human liver tissue and FFAs-induced fat-overloading HepG2 cells. The role of functional lncRNA was explored in the HepG2 cell line. Results: A total of 336 DElncRNAs (154 upregulated and 182 downregulated, |log 2 (fold change) |>0.655 and P < 0.05) and 399 DEmRNAs (152 upregulated and 247 downregulated, |log 2 (fold change) |>0.608 and P < 0.05) were identified. A total of 142 DElncRNA-miRNA interaction pairs and 643 miRNA-DEmRNA interaction pairs were retained to construct the NLMMN, which contained 19 lncRNAs, 47 miRNAs, and 228 mRNAs. The results of GO and KEGG enrichment analyses were related to an extracellular matrix (ECM). Two upregulated lncRNAs (LINC00240 and RBMS3-AS3) and one downregulated lncRNA (ALG9-IT1) were identified by qRT-PCR in liver tissues. But only LINC00240 was significantly upregulated in fat-overloading HepG2 cells. Overexpression of LINC00240 did not affect lipid accumulation but increased the reactive oxygen species (ROS) content in HepG2 cells. Conclusion: LINC00240, RBMS3-AS3, and ALG9-IT1 might be novel functional lncRNAs that attenuate liver fibrosis in NAFLD by influencing the ECM through the ceRNA network. Among them, LINC00240 might have a key role.