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, 25 (5), 857-872

miRNA-21 Ablation Protects Against Liver Injury and Necroptosis in Cholestasis

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miRNA-21 Ablation Protects Against Liver Injury and Necroptosis in Cholestasis

Marta B Afonso et al. Cell Death Differ.

Abstract

Inhibition of microRNA-21 (miR-21) prevents necroptosis in the mouse pancreas. Necroptosis contributes to hepatic necro-inflammation in the common bile duct ligation (BDL) murine model. We aimed to evaluate the role of miR-21 in mediating deleterious processes associated with cholestasis. Mechanistic studies established a functional link between miR-21 and necroptosis through cyclin-dependent kinase 2-associated protein 1 (CDK2AP1). miR-21 expression increased in the liver of primary biliary cholangitis (PBC) patients and BDL wild-type (WT) mice at both 3 and 14 days. Notably, under BDL, miR-21 -/- mice displayed decreased liver injury markers in serum compared with WT mice, accompanied by reduced hepatocellular degeneration, oxidative stress and fibrosis. Hallmarks of necroptosis were decreased in the liver of BDL miR-21 -/- mice, via relieved repression of CDK2AP1. Further, miR-21 -/- mice displayed improved adaptive response of bile acid homeostasis. In conclusion, miR-21 ablation ameliorates liver damage and necroptosis in BDL mice. Inhibition of miR-21 should arise as a promising approach to treat cholestasis.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1. miR-21 modulates necroptosis in primary mouse hepatocytes.
Primary mouse hepatocytes isolated from WT, Rip3 −/−, and miR-21 −/− C57BL/6 mice were treated with TNF-α (10 ng/mL)/CHX (0.5 µg/mL)/zVAD-fmk (50 µM) or vehicle control. After 36 h, cells were harvested for cell death assays and protein extraction. a. General cell death in primary mouse hepatocytes as assessed by the percentage of LDH release assay (top) or bis-AAF-R110 fluorescence (bottom). Immunoblotting and densitometry of p-MLKL and MLKL in whole cells extracts (b) and in insoluble and soluble protein fraction (c). Blots of p-MLKL were normalized to MLKL. Representative immunoblots are shown. d. WT primary mouse hepatocytes were transfected with a miR-21 precursor (pre-miR-21) or control (pre-miR-Control) for 16 h as described in Materials and Methods, and then loaded with TNF-α/CHX/zVAD-fmk or vehicle control for 28 h. qRT-PCR analysis of miR-21. e. Percentage of general cell death as assessed by LDH release. f. Immunoblotting and densitometry of p-MLKL and MLKL in whole cell extracts (left), insoluble protein fraction (middle) and soluble protein fraction (right). Blots of p-MLKL were normalized to MLKL. Representative immunoblots are shown. Results are expressed as mean ± S.E.M. fold change or percentage from 3–5 independent cultures from each genotype. § p < 0.05 and *p < 0.01 from control; p < 0.05 and p < 0.01 from respective control
Fig. 2
Fig. 2. miR-21 modulates necroptosis in L929 cells.
a. L929 cells were transfected with a miR-21 specific inhibitor (anti-miR-21) or control (anti-miR-Control) for 24 h as described in Materials and Methods, and then loaded with TNF-α (10 ng/mL) or vehicle control for 7 h. qRT-PCR analysis of miR-21 (top). Percentage of general cell death as assessed by AK release (bottom). b. L929 cells were transfected with a miR-21 precursor (pre-miR-21) or control (pre-miR-Control) for 24 h as described in Materials and Methods, and then loaded with TNF-α (10 ng/mL) or vehicle control for 7 h. qRT-PCR analysis of miR-21 (top). Percentage of general cell death as assessed by AK release (bottom)
Fig. 3
Fig. 3. Deletion of miR-21 protects primary mouse hepatocytes from necroptosis via CDK2AP1.
Primary hepatocytes isolated from WT and miR-21 −/− mice were transfected for 16 h with a siRNA targeting either PTEN (siPTEN), PDCD4 (siPDCD4), CDK2AP1 (siCDK2AP1) or a scrambled control (siControl), as described in Materials and Methods, and then loaded with TNF-α/CHX/zVAD-fmk or vehicle control for 28 h. a. Representative immunoblots of PTEN and PDCD4. β-actin was used as a loading control. b. Percentage of cell death as assessed by LDH release in hepatocytes incubated with TNF-α/CHX/zVAD-fmk. c. Representative immunoblots of CDK2AP1. β-actin was used as a loading control (top). qRT-PCR analysis of CDK2AP1 (bottom). d. Percentage of cell death as assessed by LDH release. e. Immunoblotting and densitometry of p-MLKL and MLKL in whole cells extracts. Blots of p-MLKL were normalized to MLKL. Representative immunoblots are shown. f. Hepatocytes isolated from WT mice were co-transfected with a CDK2AP1 luciferase reporter vector, or negative control, and pre-miR-21/pre-miR-Control; CDK2AP1/Control siRNAs; or anti-miR-21/anti-miR-Control for 24 h. Renilla luciferase signal was used as an internal standard control. Results are expressed as mean ± S.E.M. fold change or percentage of 3-5 independent cultures from each genotype. § p < 0.05 and *p < 0.01 from control; p < 0.05 and p < 0.01 from respective control
Fig. 4
Fig. 4. miR-21 is overexpressed in the liver of PBC patients and BDL mice and ablation of miR-21 ameliorates liver injury in BDL mice.
a. qRT-PCR analysis of miR-21 in the liver of 10 healthy controls and 5 PBC patients. Results are expressed as mean ± S.E.M. fold change. b. C57BL/6 WT and miR-21 −/− mice were subjected to sham or BDL surgical procedures and euthanized at days 3 and 14. qRT-PCR analysis of miR-21. miR-21 expression was not detected in miR-21 −/− mice. c. Serum ALT and AP levels. d. Representative images of H&E or Masson’s Trichrome stained liver sections. n, necrosis; h, hemorrhage; black arrowhead, hydropic degeneration; white arrowhead, cytoplasmic changes characterized by increased cytoplasmic granularity, cell swelling, and eosinophilia. e. Hepatocellular degeneration, scored as described in Materials and Methods. f. qRT-PCR analysis of Mip-2, Il-1β and Tlr4 in mouse liver. Results are expressed as mean ± S.E.M. arbitrary units or fold change of 4–7 individual mice. § p < 0.05 and *p < 0.01 from sham-operated mice; p < 0.05 and p < 0.01 from BDL WT mice at respective time-point
Fig. 5
Fig. 5. miR-21 ablation decreases hepatic fibrogenic and apoptosis markers in response to BDL.
C57BL/6 WT and miR-21 −/− mice were subjected to sham or BDL surgical procedures and euthanized at days 3 and 14. a. qRT-PCR analysis of Tgf-β, α-Sma and collagen1a1 in mouse liver. b. Liver hydroxyproline content. c. Immunoblotting and densitometry of caspase-3 in whole liver extracts. Blots were normalized to endogenous β-actin. Representative immunoblots are shown. d. Caspase-3/7 and -8 enzymatic activities. e. Immunoblotting and densitometry of p-JNK and JNK in whole liver extracts. Blots of p-JNK were normalized to JNK. Representative immunoblots are shown. f. TUNEL staining of liver tissue sections (red). Nuclei were counterstained with Hoechst 33258 (blue). Scale bar, 50 μm. Histogram shows the quantification of TUNEL-positive cells/mm2. Results are expressed as mean ± SEM fold change of 5–7 individual mice. § p < 0.05 and *p < 0.01 from sham-operated mice; p < 0.05 from BDL WT mice at respective time-point. Casp-3/-7/-8, caspase-3/-7/-8; Cl Casp-3, cleaved caspase-3
Fig. 6
Fig. 6. Ablation of miR-21 prevents necroptosis in the BDL mice liver.
C57BL/6 WT and miR-21 −/− mice were subjected to sham or BDL surgical procedures and euthanized at days 3 and 14. a. qRT-PCR analysis of RIP3 and MLKL in mouse liver. Immunoblotting and densitometry of RIP3, p-MLKL, MLKL, and CDK2AP1 in whole liver extracts (b) and RIP3, p-MLKL, and MLKL in insoluble and soluble protein fractions (c). Blots of RIP3 and CDK2AP1 were normalized to endogenous β-actin, whereas p-MLKL was normalized to MLKL. Representative immunoblots are shown. Results are expressed as mean ± S.E.M. fold change of 5–7 individual mice. § p < 0.05 and *p < 0.01 from sham-operated mice; p < 0.05 and p < 0.01 from BDL WT mice at respective time-point
Fig. 7
Fig. 7. Absence of miR-21 ameliorates oxidative stress during the acute phase of BDL.
C57BL/6 WT and miR-21 −/− mice were subjected to sham or BDL surgical procedures and euthanized at days 3 and 14. a. qRT-PCR analysis of HO-1 in mouse liver. Labile iron content (b), lipid peroxidation and ROS generation (c) were measured as described in Materials and Methods. Results are expressed as mean ± SEM arbitrary units or fold change of 5–7 individual mice. § p < 0.05 and *p < 0.01 from sham-operated mice; p < 0.05 from BDL WT mice at respective time-point
Fig. 8
Fig. 8. miR-21 ablation provides an adaptive response in the expression of bile acid homeostasis-associated genes in response to BDL.
C57BL/6 WT and miR-21 −/− mice were subjected to sham or BDL surgical procedures and euthanized at days 3 and 14. qRT-PCR analysis of Fxr, Pxr and Lxr (a); Cyp7a1 and Sult2a1 (b); Oatp and Ntcp (c); and Mrp3 and Bsep in mouse liver (d). e. Serum bile acid levels. Results are expressed as mean ± SEM arbitrary units or fold change of 5–7 individual mice. § p < 0.05 and *p < 0.01 from sham-operated mice; p < 0.05 and p < 0.01 from BDL WT mice at respective time-point
Fig. 9
Fig. 9
Schematic model depicting the deleterious effects of increased miR-21 expression during cholestasis

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