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

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.