Long Non-Coding RNAs Involved in Progression of Non-Alcoholic Fatty Liver Disease to Steatohepatitis

Figures

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  Figure 1. Study overview of HepG2 stimulation. Stimulation of HepG2 hepatocytes with free fatty acids (FFA) to mimic liver steatosis and tumor necrosis factor alpha (TNFα) to mimic liver inflammation in NASH. BSA (Bovine Serum Albumin).

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  Figure 2. Differentially expressed genes upon FFA and TNFα stimulation. (A) Heatmap of differentially expressed (DE) genes in each condition at FDR < 0.1. Fold change of gene expression Z-scores is shown. (B) Number and Venn diagrams of DE genes at FDR < 0.1 in each condition for coding (left) and non-coding genes (right). (C) Pathway analysis for DE coding genes including gene ontology (GO) terms and false discovery rate (FDR) for each condition.

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  Figure 3. lncTNF expression in FFA- and TNFα-exposed HepG2 cells and correlation with NASH phenotypes in human livers. (A) Correlation plot representing the correlation coefficients (Spearman) between 18 NASH-associated lncRNAs that showed the same direction of dysregulation in the HepG2 model of NASH. Positive correlations are shown in yellow. Negative correlations are shown in blue. LncTNF, corresponding to RP11-91KP.1, is marked with a red arrow. Rows represent genes and columns represent NASH phenotypes. (B) Correlation between normalized lncTNF expression (y-axis) and NASH grade (x-axis) in human liver samples. (C) Normalized lncTNF expression (y-axis) in HepG2 cells upon exposure to free fatty acids (FFA, green line), tumor necrosis factor alpha (TNFα, blue line), or control conditions (BSA: bovine serum albumin, red line). Mean values for three replicates are shown. x-axis represents exposure time in hours. (D) Gene ontology (GO) terms (y-axis) and FDR corrected p-value (x-axis) as defined by DAVID. Genes co-expressed with lncTNF from the human liver data (FDR < 0.05) were used as input for this analysis.

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  Figure 4. LncTNF structure and function. (A) Chromosomal location of lncTNF and read distribution based on the RNA sequencing in control (BSA-treated, all time points) and TNFα-stimulated cells (all time points). qPCR primers are located in exon 1 (forward primer) and exon 2 (reverse primer). shRNAs used for knock-down experiments are located in exon 1 (shRNA1) and exon 2 (shRNA2). (B) Gene expression of lncTNF relative to the Bactin (y-axis) measured by qPCR in HepG2 cells transduced with adenovirus containing IκBα dominant negative construct (Ad5IκB; IκBα-DN) and cells transfected with Cre adenovirus used as control (x-axis). Three time points were analyzed (0 h or no stimulation, 3 h, and 5 h of TNFα stimulation). (C) Gene expression of lncTNF relative to Bactin (y-axis) upon lncTNF knock-down using two different shRNAs (shRNA1 and 2) and three time points (x-axis). Scrambled shRNA sequence was used as control (mock). (D) Activity of NF-κB measured by luciferase/renilla ratio (y-axis) in lncTNF-KD cells (shRNA1) compared to mock control cells (x-axis). Transfected cells were stimulated with TNFα for 6, 12, and 24 h or not stimulated (0 h). (E) Gene expression level of A20 and IKBA…