Liver microRNA-21 is overexpressed in non-alcoholic steatohepatitis and contributes to the disease in experimental models by inhibiting PPARα expression

Abstract

Objective: Previous studies suggested that microRNA-21 may be upregulated in the liver in non-alcoholic steatohepatitis (NASH), but its role in the development of this disease remains unknown. This study aimed to determine the role of microRNA-21 in NASH.

Design: We inhibited or suppressed microRNA-21 in different mouse models of NASH: (a) low-density lipoprotein receptor-deficient (Ldlr^-/-) mice fed a high-fat diet and treated with antagomir-21 or antagomir control; (b) microRNA-21-deficient and wild-type mice fed a methionine-choline-deficient (MCD) diet; (c) peroxisome proliferation-activator receptor α (PPARα)-deficient mice fed an MCD diet and treated with antagomir-21 or antagomir control. We assessed features of NASH and determined liver microRNA-21 levels and cell localisation. MicroRNA-21 levels were also quantified in the liver of patients with NASH, bland steatosis or normal liver and localisation was determined.

Results: Inhibiting or suppressing liver microRNA-21 expression reduced liver cell injury, inflammation and fibrogenesis without affecting liver lipid accumulation in Ldlr^-/- fed a high-fat diet and in wild-type mice fed an MCD diet. Liver microRNA-21 was overexpressed, primarily in biliary and inflammatory cells, in mouse models as well as in patients with NASH, but not in patients with bland steatosis. PPARα, a known microRNA-21 target, implicated in NASH, was decreased in the liver of mice with NASH and restored following microRNA-21 inhibition or suppression. The effect of antagomir-21 was lost in PPARα-deficient mice.

Conclusions: MicroRNA-21 inhibition or suppression decreases liver injury, inflammation and fibrosis, by restoring PPARα expression. Antagomir-21 might be a future therapeutic strategy for NASH.

Keywords: FATTY LIVER; INFLAMMATION; NONALCOHOLIC STEATOHEPATITIS.

Figure 1

MicroRNA-21 (miR-21) was overexpressed in the liver of low-density lipoprotein receptor-deficient (Ldlr^−/−) mice fed a high-fat diet (HFD), primarily in inflammatory and biliary cells. (A) Mouse study design. (B) miR-21 expression in the liver of wild-type (WT) mice fed a chow diet (CD) and of Ldlr^−/− mice fed an HFD treated with the vehicle (phosphate buffer saline (PBS)), antagomir control (AC) or antagomir-21 (A-21). **p<0.01 versus WT mice fed a CD; §§p<0.01 versus Ldlr^−/− mice treated with PBS; ##, p<0.01 versus Ldlr^−/− mice treated with AC; (C) in situ hybridisation. miR-21 was detected in liver inflammatory (arrow) and biliary cells (arrow head) from Ldlr^−/− mice fed an HFD. Original magnification ×200. (D) miR-21 expression in sorted liver inflammatory (CD45⁺) and non-inflammatory (CD45⁻) cells from Ldlr^−/− mice fed an HFD. (E) miR-21 expression in subpopulations of liver inflammatory (CD45⁺) cells from Ldlr^−/− mice fed an HFD. *p<0.05; **p<0.01. Data are given as median (horizontal bar) and IQR (error bar). PV, portal vein.

Figure 2

Antagomir-21 reduced liver injury and liver inflammation in the liver of low-density lipoprotein receptor-deficient (Ldlr^−/−) mice fed a high-fat diet (HFD). Antagomir-21 (A-21) reduced serum aspartate aminotransferase (AST) (A) and alanine aminotransferase (ALT) (B) levels, liver monocyte chemoattractant protein-1 (MCP1) (C and D) and tumour necrosis factor (TNF)-α (E and F) gene and protein expressions, as well as CD3⁺ (G, quantification; H, representative images, original magnification ×200) and CD68⁺ (I, quantification; J, representative images, original magnification ×400) cell infiltrates. *p<0.05, **p<0.01 versus wild-type (WT) mice fed a chow diet (CD); §p<0.05, §§p<0.01 versus Ldlr^−/− mice fed an HFD treated with phosphate buffer saline (PBS); #p<0.05, ##p<0.01 versus Ldlr^−/− mice fed an HFD treated with antagomir control (AC). Data are given as median (horizontal bar) and IQR (error bar). Samples were randomly selected in each group of mice.

Figure 3

Antagomir-21 (A-21) reduced liver fibrosis and expression of fibrogenic-related genes in the liver of low-density lipoprotein receptor-deficient (Ldlr^−/−) mice fed a high-fat diet (HFD). A-21 reduced liver collagen staining (Sirius red; A, quantification; B representative images, original magnification ×200), collagen deposition (C), as well as liver normalised Tgfβ (D), and collagen-1α2 (E) mRNA expression. *p<0.05, **p<0.01, ***p<0.001 versus wild-type (WT) mice fed a chow diet (CD); §p<0.05 versus Ldlr^−/− mice fed an HFD treated with phosphate buffer saline (PBS); #p<0.05, ##p<0.01 versus Ldlr^−/− mice fed a HFD treated with antagomir control (AC). Data are given as median (horizontal bar) and IQR (error bar). Samples were randomly selected in each group of mice.

Figure 4

MicroRNA-21 (miR-21) was overexpressed in the liver of wild-type (WT) mice fed a methionine-choline-deficient (MCD) diet. (A) Mouse study design. (B) miR-21 expression in the liver of WT mice fed a chow diet (CD) or an MCD diet as well as in miR-21^−/− mice fed an MCD diet. ***p<0.001 versus WT mice fed a chow diet (CD); ###p<0.001 versus WT mice fed an MCD diet. Data are given as median (horizontal bar) and IQR (error bar). (C) In situ hybridisation. Arrows point to liver inflammatory cells and arrow heads point biliary cells. Original magnification ×200. PV, portal vein.

Figure 5

MiR-21^−/− mice fed a methionine-choline-deficient (MCD) diet had reduced liver injury and liver inflammation. When compared with wild-type (WT) mice, miR-21^−/− mice had reduced serum aspartate aminotransferase (AST) (A) and alanine aminotransferase (ALT) (B) levels, liver monocyte chemoattractant protein-1 (MCP-1) (C, D) and tumour necrosis factor (TNF)-α (E, F) gene and protein expressions, as well as lower CD3⁺ (G, not significant) and CD11b⁺ (H) cell infiltrates. *p<0.05, **p<0.01, ***p<0.001 versus WT mice fed a chow diet (CD). #p<0.05, ##p<0.01, ###p<0.001 versus WT mice fed an MCD diet. Data are given as median (horizontal bar) and IQR (error bar).

Figure 6

MiR-21^−/− mice fed a methionine-choline-deficient (MCD) diet had reduced expression of fibrogenic-related genes. When compared with wild-type (WT) mice, miR-21^−/− mice had similar liver collagen staining (Sirius red; A, quantification; B representative images, original magnification ×200) and collagen deposition (C), but reduced liver normalised Tgfβ (D) and collagen-1α2 (E) mRNA expression. *p<0.05, **p<0.01, ***p<0.001 versus WT mice fed a chow diet (CD). ##p<0.01 versus WT mice fed an MCD diet. Data are given as median (horizontal bar) and IQR (error bar).

Figure 7

MicroRNA-21 (miR-21) inhibition or suppression restored liver peroxisome proliferation-activator receptor α (PPARα) expression. (A–C) Antagomir-21 (A-21) restored liver PPARα gene (A) and protein (B and C) expression. *p<0.05, **p<0.01 versus wild-type (WT) mice fed a chow diet (CD); §p<0.05; §§p<0.01 versus low-density lipoprotein receptor-deficient (Ldlr^−/−) mice fed a high-fat diet (HFD) treated with phosphate buffer saline (PBS); ##p<0.01 versus Ldlr^−/− mice fed an HFD treated with antagomir control (AC). (D–F) MiR-21^−/− mice fed a methionine-choline-deficient (MCD) diet had restored liver PPARα gene (D) and protein (E and F) expression. *p<0.05, **p<0.01, versus WT mice fed a chow diet (CD). ###p<0.001 versus WT mice fed an MCD diet. Data are given as median (horizontal bar) and IQR (error bar). Liver samples randomly selected in each group of mice.

Figure 8

Antagomir-21 (A-21) did not improve liver cell injury or liver inflammation in Pparα^−/− mice fed a methionine-choline-deficient (MCD) diet. (A) Mouse study design. (B) Liver microRNA-21 (miR-21) expression. (C) miR-21 expression in sorted liver inflammatory (CD45⁺) and non-inflammatory (CD45⁻) cells from wild-type (WT) mice fed an MCD diet for 5 weeks. (D) miR-21 expression in subpopulations of liver inflammatory (CD45⁺) cells and in biliary cells (Epcam+) from WT mice fed an MCD diet for 5 weeks. A-21 did neither reduce serum aspartate aminotransferase (AST) (E) or alanine aminotransferase (ALT) (F) levels, nor liver Mcp1 gene expression (G) or CD3⁺ (H) cell infiltrates in Pparα^−/− mice. *p<0.05, **p<0.01, ***p<0.001 versus WT mice fed a chow diet (CD). #p<0.05, ##p<0.01, ###p<0.001 versus WT mice fed an MCD diet and treated with antagomir control (AC). Data are given as median (horizontal bar) and IQR (error bar).

Figure 9

Antagomir-21 (A-21) did not improve liver fibrosis in Pparα^−/− mice fed a methionine-choline-deficient (MCD) diet. A-21 did not reduce liver collagen staining (A; Sirius red, representative images, original magnification ×200), collagen deposition (B) or liver normalised Tgfβ (C) and collagen-1α2 (D) mRNA expression in Pparα^−/− mice fed an MCD diet. *p<0.05, **p<0.01, ***p<0.001 versus wild type (WT) mice fed a chow diet (CD). #p<0.05, ###p<0.001 versus WT mice fed an MCD diet and treated with antagomir-21. Data are given as median (horizontal bar) and IQR (error bar).

Figure 10

MicroRNA-21 (miR-21) was overexpressed in the liver of patients with non-alcoholic steatohepatitis (NASH), primarily in inflammatory and biliary cells. (A) miR-21 expression normalised to U6 snRNA in the liver of patients with no or mild abnormalities at histological examination (control) or with bland steatosis or with NASH. *p<0.05, **p<0.01. (B) In situ hybridisation. miR-21 was detected in inflammatory (arrow) and biliary cells (arrow head). U6 probe is the positive control and no probe the negative control. Original magnification ×200. (C and D) Fluorescence in situ hybridisation (FISH). Combined FISH was performed for miR-21 and CD3 (lymphocyte marker) or CK19 (biliary cells marker) in liver sections from controls or patients with NASH. miR-21 expression (red) was detected in CD3-positive cells (C, green) from patients with NASH and in CK19-positive cells from patients with NASH and from controls (D, green). Original magnification ×630. Representative of three experiments. PV, portal vein.