Comprehensive analysis of LncRNAs expression profiles in an in vitro model of steatosis treated with Exendin-4

Abstract

Background and aims The hallmark of non-alcoholic fatty liver disease (NAFLD) is the excessive hepatic lipid accumulation. Currently, no pharmacotherapy exists for NAFLD. However, the glucagon-like peptide-1 receptor agonists have recently emerged as potential therapeutics. Here, we sought to identify the long non-coding RNAs (LncRNAs) associated with the steatosis improvement induced by the GLP-1R agonist Exendin-4 (Ex-4) in vitro. Methods Steatosis was induced in HepG2 cells with oleic acid. The transcriptomic profiling was performed using total RNA extracted from untreated, steatotic, and Ex-4-treated steatotic cells. We validated a subset of differentially expressed LncRNAs with qRT-PCR and identified the most significantly enriched cellular functions associated with the relevant LncRNAs. Results We confirm that Ex-4 improves steatosis in HepG2 cells. We found 379 and 180 differentially expressed LncRNAs between untreated and steatotic cells and between steatotic and Ex-4-treated steatotic cells, respectively. Interestingly, 22 upregulated LncRNAs in steatotic cells became downregulated with Ex-4 exposure, while 50 downregulated LncRNAs in steatotic cells became upregulated in the presence of Ex-4. Although some LncRNAs, such as MALAT1, H19, and NEAT1, were previously associated with NAFLD, the association of others with steatosis and the positive effect of Ex-4 is being reported for the first time. Functional enrichment analysis identified many critical pathways, including fatty acid and pyruvate metabolism, and insulin, PPAR, Wnt, TGF-β, mTOR, VEGF, NOD-like, and Toll-like receptors signaling pathways. Conclusion Our results suggest that LncRNAs may play essential roles in the mechanisms underlying steatosis improvement in response to GLP-1R agonists and warrant further functional studies.

Regulation of lipid droplet (LD) formation in hepatocytes via regulation of SREBP1c by non-coding RNAs

“…On the other hand, H19 was downregulated ( Figure 3B ). This is in line with the finding of Khaoula et al who showed a decreased expression of lncRNA H19 in an HepG2 steatotic model ( 43 ). However, our findings oppose the result of Liu et al which reported an induction of H19 upon FA treatment.…”

Investigation of the Effect of Exendin-4 on Oleic Acid-Induced Steatosis in HepG2 Cells Using Fourier Transform Infrared Spectroscopy

“…We have previously shown that the incubation of HepG2 cells with 400 µM OA after 6 h of starvation induces steatosis, as reflected by the presence of lipid droplets [ 39 , 40 ]. We have further shown that treatment of the steatotic HepG2 cells with 200 nM Ex-4 for 3 h resulted in a significant reduction in steatosis [ 40 ], indicating the protective effect of this drug on steatosis.…”

“…Gupta and colleagues [ 38 ] were the first to propose that the activation of the hepatic GLP-1R plays a direct role in reducing hepatic steatosis in vitro through modulation of the elements of the insulin signaling pathway. Recently, we and others [ 39 , 40 , 41 ] have shown that the GLP-1R agonist Exendin-4 (Ex-4) reduces the fat content in an in vitro cell model of steatosis by inhibiting hepatic lipogenesis through activation of β-catenin signaling and modulation of the expression of several lipogenesis genes. In addition, β-catenin might also mediate the effect of Ex-4 on ameliorating hepatic steatosis induced by a high fructose diet in rats [ 42 ].…”

Exendin-4 alleviates steatosis in an in vitro cell model by lowering FABP1 and FOXA1 expression via the Wnt/-catenin signaling pathway

“…We further labeled the nuclei by incubating the cells with 1 μM DAPI for 1 min. After a final wash with PBS, we mounted the coverslips on microscope slides used for imaging on a Zeiss LSM 870 confocal microscope, as we reported recently 45 . To analyze the images, we used ImageJ software (version 1.8.0, NIH, USA).…”