Long Noncoding RNA lnc-HC Regulates PPARγ-Mediated Hepatic Lipid Metabolism through miR-130b-3p

Figure 1. lnc-HC Expression Is Linked to Hepatocytic Fatty Acid Metabolism in CBRH-7919 Cells (A) Oil-red O staining of CBRH-7919 cells treated with vehicle or FFAs for 24 h. (B) Quantitative real-time PCR detection of Fasn, Cpt-1α, Dgat1, and Dgat2 expression in the CBRH-7919 cell model. (C) Quantitative real-time PCR analysis of PPARα, PPARγ, FXR, LXRα, and Srebp1c expression in the CBRH-7919 cell model. (D) Quantitative real-time PCR analysis of lnc-HC and Slc25a15 expression in the CBRH-7919 cell model overtime (0, 3, 6, 12, 24, and 48 h). Data are expressed as means ± SEM. An unpaired t test was performed to determine the statistical significance. *p < 0.05; **p < 0.01; ***p < 0.001, as compared with, respectively, the vehicle group, the 0 h time point, or the NC group.

Figure 2. lnc-HC Controls PPARγ Expression and Its Signaling Pathway (A and B) Quantitative real-time PCR analysis of gene expression, including lnc-HC, Slc25a15, PPARα, PPARγ, FXR, LXRα, and Srebp1, in lnc-HC stably overexpressed CBRH-7919 (A) and lnc-HC stable-knockdown CBRH-7919 cells (B). (C) Western blotting detection of PPARγ expression (left) and quantification histograms (right) in lnc-HC-overexpressed/stable-knockdown CBRH-7919. β-Actin was the loading control in the western blotting assay. Western blotting shows one representative result and quantitative data from 4 independent experiments. (D) Quantitative real-time PCR analysis of PPARγ target gene expressions in indicated cells. (E and F) Quantitative real-time PCR (E) and Western blotting detection (F) of PPARγ expression in lnc-HC stable-knockdown cells with siNC or siPPARγ transfection for 24 h. β-Actin was the loading control in the western blotting assay. Each western blot shows one representative result and quantitative data from three independent experiments. (G) Quantitative real-time PCR analysis of PPARγ target gene expression in lnc-HC stable-knockdown cells with siNC or siPPARγ transfection for 24 h. Data are…

Figure 3. lnc-HC Modulates miR-130b-3p Expression (A) Quantitative real-time PCR analysis of PPARγ mRNA stability overtime (0, 6, 12, and 24 h) after α-amanitin treatment (50 μM) in lnc-HC stably overexpressed/knockdown cells. Data are expressed as means ± SEM. *p < 0.05, NC shR versus lnc-HC shR, two-way ANOVA. (B) RIP detection of the interaction between hnRNPA2B1 protein and lnc-HC or indicated mRNAs, including PPARα, PPARγ, Cyp7a1, and U6, by using anti-hnRNPA2B1 antibody (5 μg), with IgG (5 μg) used as antibody isotype control. (C) RNA pull-down assay to check the interaction among lnc-HC, nucleoprotein (NP), and indicated mRNAs, including PPARα, PPARγ, Cyp7a1, and U6. (D) RIP detection of the interaction between lnc-HC and mRNAs, including PPARγ and U6, by using anti-GFP antibody (5 μg), using IgG (5 μg) as the antibody isotype control and lnc-HC antisense (AS) as the negative control. (E) Bioinformatics prediction results of the candidate miRNAs targeting PPARγ 3′UTR and the binding sequence by using miRanda and TargetScan software. (F) Quantitative real-time PCR analysis of indicated miRNA expression in lnc-HC stably interfered CBRH-7919. (G) Quantitative real-time PCR analysis of…

Figure 4. lnc-HC Controls PPARγ Expression through miR-130b-3p (A) Quantitative real-time PCR analysis of PPARγ expression in lnc-HC stably interfered CBRH-7919, with transient transfection of NC mimics (mNC, 50 nM) or miR-130b-3p mimics (50 nM). (B) Western blotting measurement (left panel) and quantification histograms (right panel) of PPARγ expression under the same conditions as in (A). Quantitative data are from 4 independent experiments. (C) Quantitative real-time PCR analysis of PPARγ expression with transfection of NC inhibitor (iNC, 100 nM) or indicated miRNA inhibitors (100 nM). (D) Western blotting detection (left panel) of PPARγ expression in CBRH-7919 cells with transfection of iNC (100 nM) or miR-130b-3p inhibitor (100 nM) and quantification histograms (right panel). Western blotting and quantitative data are from 6 independent experiments. (E) Schematic diagram of wild-type (Wt) and mutated (Mut) binding sites of miR-130b-3p targeting PPARγ 3′ UTR. (F) Dual-luciferase activity of PPARγ-3′UTR-wild and PPARγ-3′UTR-mut in CBRH-7919 cells with transfection of mNC/miR-130b-3p mimics (50 nM) or iNC/miR-130b-3p inhibitor (100 nM). Data are expressed as means ± SEM. *p < 0.05; **p <…

Figure 5. lnc-HC Negatively Regulates Hepatocytic Lipid Accumulation (A and B) CBRH-7919, NCshRCBRH, and Lnc-HCshRCBRH cells were treated with FFAs for 12 h, and the cellular lipid droplets in each group were (A) analyzed by oil red O staining and (B) measured by color density scanning using ImageJ. (C and D) The concentration of (C) intracellular FFAs and (D) TGs were detected. (E) Quantitative real-time PCR analysis of lnc-HC, PPARγ, and indicated miRNA expression in lnc-HC stably knocked down cells with vehicle or FFA treatment for 12 h. (F) overLnc-HCCBRH cells were transfected with NC inhibitor (iNC, 100 nM) or miR-130b-3p inhibitor (100 nM) for 24 h and then treated with FFAs for 12 h. Oil red O staining was used to detect the lipid droplet formation in each group. (G) Quantification histograms of the relative lipid droplet content in the indicated cells by using ImageJ. (H) The lipid droplet formation was measured in Lnc-HCshRCBRH cells under the transfection with NC mimics (mNC, 50 nM) or miR-130b-3p mimics (50 nM) for 24 h and then treated with FFAs for 12 h. (I) Quantification histograms of the relative lipid droplet content in indicated cells. Data are expressed as means ± SEM.…

Figure 6. lnc-HC/miR-130b-3p Pathway Regulates Liver Lipid Metabolism (A) H&E-stained liver sections with the HCD-induced lipid metabolism disorder E3 rat model. (B) At the end of the HCD-induced hyperlipidemia model (E3 rats, 8-week induction), serum concentrations of TG, HDL-C, and LDL-C were measured. (C and D) The liver/body weight ratio (C) and body weight gain (D) at the 8th week. (E) The hepatic TG concentration. (F and G) Quantitative real-time PCR analysis of lnc-HC expression (F) and miR-130b-3p and miR-130a-3p expression (G) in rat livers. (H and I) Quantitative real-time PCR analysis (H) and western blotting detection (I) of PPARγ expression in model livers. Data are expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. An unpaired t test was performed to determine the statistical significance.