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

Fig. 1. Exendin-4 reduces Oleic acid-induced lipid accumulation in HepG2 cells. In all experiments, HepG2 cells were starved for 6 h and then treated with 400 μM Oleic acid for 16 h. Afterwards, cells were treated for 3 h with a fresh DMEM solution containing 400 µM OA in the presence or absence of 200 nM Ex-4. a Confocal imaging of lipid droplets after staining with 0.2 mM BODIPY 493/503 (green) and DAPI (blue). The white dots in the bright field images indicate lipid droplets. b Quantification of the lipid content with the BODIPY/DAPI fluorescence ratio in steatotic cells relative to untreated cells, and in Ex-4-treated steatotic cells relative to steatotic cells. For each treatment condition (untreated, steatotic, and Ex-4-treated steatotic cells), two independent researchers analyzed 200 individual cells from three separate experiments. Values are expressed as the mean ± SE (n = 3). *p < 0.05

Fig. 2. Hierarchical Clustered Heatmaps of the top 50 LncRNAs showing differential expression between untreated, steatotic, and Ex-4-treated steatotic cells. Each row represents one LncRNA, and each column represents one sample. The expression level of each transcript in a single sample is depicted according to the color scale. Red and blue indicate up-regulation and down-regulation, respectively. The experiment was performed in triplicate

Fig. 3. LncRNA transcriptional portrait between untreated, steatotic, and Ex-4-treated steatotic cells. A Venn Diagram of differentially expressed LncRNAs. The 22 LncRNAs at the intersection are upregulated in steatotic cells and downregulated in Ex-4 treated steatotic cells (a). The 50 LncRNAs at the intersection are downregulated in steatotic cells and upregulated in Ex-4 treated steatotic cells (b). B Fold change of 22 LncRNAs upregulated in steatotic HepG2 cells and downregulated in Ex-4-treated steatotic Cells (a) and fold change of 50 LncRNAs downregulated in steatotic HepG2 cells and upregulated in Ex-4-treated steatotic Cells (b). The experiment was performed in triplicate

Fig. 4. Validation of the differential expression of a set of LncRNAs with qRT-PCR. Comparison of the expression of LncRNAs ABALON, NEAT1, TP73-AS1, H19, MALAT1, and HOXA10-AS between steatotic cells and Ex-4-treated steatotic cells using qRT-PCR and RNA sequencing. The primers used for each LncRNA are shown in Table 1. The experiment was performed in triplicate

Fig. 5. Functional enrichment analysis of differentially expressed LncRNAs. a Signalling pathways and biological processes associated with the 22 LncRNAs that are upregulated in steatotic cells and downregulated in Ex-4-treated steatotic cells. b Signalling pathways and biological processes associated with the 50 LncRNAs that are downregulated in in steatotic cells and upregulated in Ex-4-treated steatotic cells. The experiment was performed in triplicate