Identification of a novel human long non-coding RNA that regulates hepatic lipid metabolism by inhibiting SREBP-1c

Figures

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  Figure 1. HCV-related lncRNA regulates SREBP-1c promoter activity. (A) Immunofluorescent assay confirmed HCV infection of Huh7 cells according to antibody staining against viral core protein. DAPI staining (blue) indicates nuclei. Scale bars, 30 μm. (B) Representative circos plot demonstrates differentially expressed lncRNAs (outer track) and mRNAs (inner track) in chromosomes. Red indicates up-regulated genes; blue is down-regulated genes. (C) LncHR1 was quantified in Huh7 cells in response to HCV infection. PBS treatment was used as a negative control. SREBP-1c promoter reporter gene was co-transfected with either lncHR1 overexpression (D) or knockdown (E) constructs in Huh7 cells, and luciferase reporter expression was measured and normalized. (F) HCV infection in 2-3+ replicon cells was cured with the NS5A inhibitor PD790052 (left), and lncHR1 expression was measured with qRT-PCR (right). Data are means ± SD. *p < 0.05, **p < 0.01.

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  Figure 2. Biological characteristics of lncHR1. (A) Structural diagram of lncHR1 gene. (B) Endogenous lncHR1 expression was measured in 11 cell lines using absolute qRT-PCR. (C) Intracellular localization of lncHR1 was visualized in Huh7 cells with RNA-FISH assays (blue for nucleus and green for lncHR1). Scale bars, 13 μm.

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  Figure 3. LncHR1 negatively regulates endogenous SREBP-1c and FAS. (A) LncHR1 overexpression plasmid was transfected into Huh7 cells in a dose dependent manner and intracellular lncHR1 was quantified by qRT-PCR. An empty vector was a negative control. The data were normalized to relative fold changes. (B) Post-transfection of lncHR1 overexpression plasmid for 48 h, endogenous SREBP-1c mRNA (up) and protein (down) were measured by qRT-PCR and Western blot, respectively. (C) LncHR1 knockdown plasmid (shRNA) was transfected into Huh7 cells in a dose dependent manner and intracellular lncHR1 lever was quantified by qRT-PCR. (D) Forty-eight hours post transfection of lncHR1 knockdown plasmid, endogenous SREBP-1c mRNA (up) and protein (down) levels were measured by qRT-PCR and Western blot, respectively. (E) HCV was inoculated into Huh7 cells 24 h before transfection with either control vector or lncHR1 overexpression plasmid and 48 h post-transfection, endogenous SREBP-1c protein was measured with Western blot. Actin was used as an equal loading control. (F) Endogenous FAS protein expression was measured in Huh7 cells with overexpressed (up) or decreased (down) lncHR1. Empty vectors were…

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  Figure 4. LncHR1 suppressed TG accumulation and LD formation in steatotic hepatocytes. Huh7 cells were transfected with lncHR1 overexpression (A) or knockdown (B) plasmids, and then treated with OA at 24 h post-transfection. An empty vector was used as negative control. Endogenous SREBP-1c protein was measured with Western blot (A, B) and intracellular TG was quantified (C, D). Intracellular LD formation in Huh7 cells was visualized with oil red O staining. The nuclei were indicated by DAPI staining. Scale bars, 30 μm. (E, F). Volume of LD in each treatment was determined with Image J software (G, H). Data are means ± SD. *p < 0.05, **p < 0.01, *** p < 0.001.

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  Figure 5. LncHR1 regulated lipid metabolism in HFD-induced transgenic mice. (A) LncHR1 expression in the lncHR1TG group was ~9-fold greater than the WT group (n= 6/group). (B) In liver tissues, SREBP-1c, FAS and ACCα level in both groups were measured with Western blot. β-actin was used as an equal loading control. (C-E) Serum TG, VLDL and LDL were measured. (F) Liver TG were measured in the lncHR1TG group and WT group (n=6/group). (G) At week 9 after HFD induction, the ratio of liver to body weight in transgenic mice was calculated in both groups (n=6/group). (H) Weight gain of two groups was measured weekly up to the 9th week. Data are means ± SD. *p < 0.05, **p < 0.01.