MicroRNA-379-5p regulates free cholesterol accumulation and relieves diet induced-liver damage in db/db mice via STAT1/HMGCS1 axis

Abstract

Lipotoxicity induced by the overload of lipid in the liver, especially excess free cholesterol (FC), has been recognized as one of driving factors in the transition from non-alcoholic fatty liver (NAFL) to non-alcoholic steatohepatitis (NASH). MicroRNA (miR)-379-5p has been reported to play regulatory roles in hepatic triglyceride homeostasis, but the relationship of miR-379-5p and hepatic cholesterol homeostasis has never been touched. In the current study, we found that hepatic miR-379-5p levels were decreased obviously in NAFLD patients and model mice compared with their controls. Moreover, miR-379-5p was discovered to be able to inhibit intracellular FC accumulation and alleviate mitochondrial damage induced by palmitic acid (PA) in vitro. Furthermore, overexpression of miR-379-5p in HFHC-fed db/db mice could reduce the level of hepatic total cholesterol (TC) and FC, and ameliorate hepatic injury reflected by the lower serum alanine aminotransferase (ALT) and aspartate transaminase (AST). Subsequently, by combining spectrometry (MS) and luciferase assay, we identified miR-379-5p suppressed STAT1 through transcriptional and translational regulation. Finally, we confirmed that STAT1 was a transcriptional factor of HMGCS1. In conclusion, miR-379-5p inhibits STAT1 expression and regulates cholesterol metabolism through the STAT1/HMGCS1 axis, suggesting miR-379-5p might be applied to improve lipotoxicity in the future.

Keywords: Cholesterol metabolism; HMGCS1; MiR-379-5p; Non-alcoholic fatty liver disease; STAT1.

Fig. 1

Expression of miR-379-5p is decreased in vitro/in vivo/in clinic. a Hepatic miR-379 levels between healthy control (HC, n = 18), simple steatosis (SS, n = 17) and NASH patients (n = 19). Data were derived from GEO database (GSE89632). In HC group, 6 healthy living liver donors with fibrosis symptoms were excluded. b db/db mice and their lean littermates (wild type (WT)) were fed with a NCD or HFHC diet for 20 weeks respectively, and miR-379-5p in the liver was examined by RT-PCR. n = 5 in each group. c MiR-379-5p was detected by RT-PCR in the livers of C57BL/6 J mice fed with a NCD or HFHFrHC diet for 20 weeks. n = 9–10 in each group. d Primary mouse hepatocytes (PMH) and Huh7 cells were stimulated with 0.5 mM PA for 24 h, and the expression of miR-379-5p in the cells was detected by RT-PCR. For a, c and d, two-tailed student’s t-test was used to calculate statistical significance, while for b, One-Way ANOVA test was used (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Overexpression of miR-379-5p inhibits PA-stimulated FC accumulation and mitochondrial dysfunction in vitro. a FC contents in Huh7 cells treated with 0.5 mM PA for 24 h. b Representative images of TMRM (red) and Hoechst (blue) in Huh7 cells, Scale bar: 100 μm. c Quantitative analysis of TMRM fluorescence intensity in Huh7 cells treated with 0.5 mM PA for 24 h. d Seahorse XFe96 analysis of cell maximal respiration in Huh7 cells followed by treatment with 0.5 mM PA or BSA for 24 h. e Seahorse XFe96 analysis of cell OCR in Huh7 cells followed by transfected with NC (30 nM) for 48 h and treatment with 0.5 mM PA or BSA for another 24 h. f Seahorse XFe96 analysis of cell OCR in Huh7 cells followed by transfected with miR-379-5p mimic (30 nM) for 48 h and treatment with 0.5 mM PA or BSA for another 24 h. One-Way ANOVA test was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001. n = 3 independent experiments

Fig. 3

Overexpression of miR-379-5p attenuates the overnutrition-induced cholesterol metabolism disorder and liver damage in db/db mice. a Hepatic miR-379-5p levels after 9 weeks of AAV injection in mcherry and mcherry-miR-379-5p group of db/db mice with HFHC diet. b The serum TC of mcherry and mcherry-miR-379-5p group with HFHC diet. c & d The hepatic FC and TC of mcherry and mcherry-miR-379-5p group with HFHC diet. e The proportion of liver calculated with following formula: Liver weight / body weight × 100% of mcherry and mcherry-miR-379-5p group with HFHC diet. f The serum ALT and AST of mcherry and mcherry-miR-379-5p group with HFHC diet. g Representative H&E liver sections of mcherry and mcherry-miR-379-5p group with HFHC diet. Scale bar, 400 μm. h Histological scores of mcherry and mcherry-miR-379-5p group with HFHC diet. Two-tailed student’s t-test was used to calculate statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001, n = 7–8 in each group

Fig. 4

Regulatory effect of miR-379-5p on HMGCS1. a The mRNA expression of cholesterol metabolism-related genes (SREBF2, HMGCR, HMGCS1, SCARB1, CEH, Cyp7a1, Cyp27a1, ABCA1 and ABCG1) in the livers of HFHC mice, n = 7–8 in each group. b HMGCS1 protein levels in the livers of HFHC mice, n = 7–8 in each group. c FC contents in Huh7 cells that were transfected with miR-379-5p mimics (30 nM) or siHMGCS1 (30 nM) for 24 h and then cultured with 0.5 mM PA for another 24 h. d & e HMGCS1 protein and mRNA levels in Huh7 cells transfected with miR-379-5p mimics (30 nM) or NC (30 nM) for 48 h and then cultured with 0.5 mM PA for another 24 h. f The relative activities of HMGCS1 3’UTR presented by relative luciferase activity of HEK293 cells co-transfected with related plasmids and miR-379-5p mimics (30 nM) or NC(30 nM) for 24 h. For a, c, e-f, two-tailed student’s t-test was used to calculate statistical significance, while for b, One-Way ANOVA test was used. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3 independent experiments

Fig. 5

Regulatory effect of miR-379-5p on STAT1. a Heatmap for 18 down-regulated proteins in the nucleus of miR-379-5p (30 nM)-treated Huh7 cells, compared to NC (30 nM)-treated Huh7 cells. b Schematic diagram of predicted transcription factors of HMGCS1. c Relative STAT1 protein level between miR-379-5p- and NC-transfected cells obtained from the data of MS. d & e STAT1 protein and mRNA levels in the livers of HFHC mice, n = 7–8 in each group. f & g STAT1 mRNA and protein levels in Huh7 cells transfected with different concentrations of miR-379-5p mimics or NC for 72 h. h STAT1 3’UTR activity in miR-379-5p (30 nM)- and NC (30 nM)-transfected HEK293 cells. i Five putative binding sequence between miR-379-5p and STAT1 3’UTR and their related mutants. j The activity of WT or mutants of STAT1 3’UTR in miR-379-5p (30 nM)- and NC (30 nM)-transfected HEK293 cells. k STAT1 promoter activity in miR-379-5p (30 nM)- and NC (30 nM)-transfected HEK293 cells. l Four putative binding sites between miR-379-5p and STAT1 promoter and their related mutants. m The activity of WT or mutants of STAT1 promoter in miR-379-5p (30 nM)- and NC (30 nM)-transfected HEK293 cells. For c, f and i, two-tailed student’s t-test was used to calculate statistical significance, while for d, h and k, One-Way ANOVA test was used. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3 independent experiments

Fig. 6

Regulatory effect of STAT1 on HMGCS1. a & b STAT1 and HMGCS1 mRNA and protein levels in Huh7 cells transfected with siSTAT1 (30 nM) or siNC (30 nM) for 72 h. c The protein levels of STAT1 and HMGCS1 in Huh7 cells treated with different concentrations of Fludarabine for 24 h. d Relative activity of HMGCS1 promoter in HEK293 cells under the condition of siSTAT1 (30 nM) transfection or Fludarabine (10 μM) treatment. e Five binding sequences between STAT1 and HMGCS1 promoter predicted by the JASPAR. f Schematic diagram of HMGCS1 promoter (WT) and related truncations (H1 and H2). g Relative luciferase activity of HMGCS1 promoter WT, H1 and H2 in HEK293 cells transfected with siSTAT1 (30 nM) or treated with Fludarabine (10 μM) for 24 h. Two-tailed student’s t-test was used to calculate statistical significance. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3 independent experiments