Deficiency of miR-208a Exacerbates CCl4-Induced Acute Liver Injury in Mice by Activating Cell Death Pathways

Abstract

Acute liver injury (ALI) is associated with multiple cellular events such as necrosis, apoptosis, oxidative stress and inflammation, which can lead to liver failure. In this study, we demonstrate a new role of microRNA (miR)-208a in ALI. ALI was induced in wild-type (WT) and miR-208a knockout (KO) mice by CCl₄ administration. Increased alanine aminotransferase and decreased hepatic miR-208a levels were found in WT mice after acute CCl₄ treatment. Histopathological evaluations revealed increased necrosis and decreased inflammation in miR-208a KO compared with WT mice after CCl₄ treatment. CCl₄ treatment induced a higher alanine aminotransferase elevation and increased numbers of circulating extracellular vesicles (exosomes and microvesicles) in miR-208a KO compared with WT mice. We found increased CCl₄-induced nuclear factor kappa B activation and tumor necrosis factor-α induction and decreased monocyte chemoattractant protein 1 levels in miR-208a KO compared with WT mice. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay indicated aggravated hepatic apoptosis and necrosis in CCl₄ -treated miR-208a KO compared with WT mice. CCl₄ treatment induced a greater increase in cleaved caspase-8, p18, and caspase-3 in miR-208a KO compared with WT mice. p53 is involved in various cell death pathways, including necrosis and apoptosis. Our in silico analysis revealed p53 as a predicted miR-208a target, and we found enhanced p53 and cyclophilin D protein expressions in miR-208a KO mice after CCl₄ treatment. Increased liver injury in miR-208a KO mice was further associated with increased Bax (B cell lymphoma 2-associated X protein) and p21 expression. Our in vitro results indicated a role of miR-208a in cell death. We found that CCl₄-induced cytotoxicity was partially rescued by miR-208a overexpression in RAW macrophages. Altogether, our results revealed a role of miR-208a in ALI in mice and suggest a role for miR-208a in regulating cell death.

FIG. 1

Increased liver injury in miR‐208a KO mice after acute CCl₄ treatment. (A) miR‐208a levels were quantified from the livers of WT mice by quantitative PCR after 48 hours of CCl₄ treatment, and snoRNA202 was used to normalize the Cq values of each sample. (B) WT or miR‐208a KO mice received a single dose of either oil (n = 4‐5) or CCl₄ (n = 6) for 48 hours. Serum ALT levels were determined from serum samples to assess liver damage. (C) Total number of EVs, exosomes, and microvesicles were measured from serum samples using NanoSight. (D) Liver injury was evaluated by H&E staining of the liver sections. (E) Blinded histopathological evaluations of H&E‐stained liver sections were performed, and necrosis, lobular/centrivenular inflammation, regenerative changes (mitotic activity), and ballooning degeneration scoring. Chi‐square tests were used to evaluate the categorical data and studied for histopathological changes using GraphPad Prism 8 software. *P < 0.05 versus oil‐WT or oil‐KO treated mice. Abbreviations: n.d., not detected; ns, nonsignificant.

FIG. 2

Enhanced NF‐κB activation in miR‐208a KO mice after acute CCl₄ treatment. WT or miR‐208a KO mice received oil (n = 4‐5/group) or CCl₄ (n = 6/group) treatment for 48 hours. (A) Total RNA was extracted from the liver samples, and the levels of TNFα were determined by quantitative PCR. (B) NF‐κB activation was determined by EMSA from nuclear liver extracts, and representative results are shown along with density units in the bar diagram (B). MCP1 mRNA levels were quantified using quantitative PCR (C), and protein levels of MCP1 were measured from whole cell liver extracts by enzyme‐linked immunosorbent assay (D). (E) VEGF expression was determined by quantitative PCR from RNA samples (E). mRNA levels of α‐SMA (F), TIMP1 (G), MMP12 (H), MMP9 (I), and TGF (J) were determined by quantitative PCR. 18S was used to normalize the Cq values of each sample. *P < 0.05 versus oil‐WT or oil‐KO treated mice. Abbreviation: comp, complementary sequence.

FIG. 3

Induction of cellular death in miR‐208a KO mice after acute CCl₄ treatment. WT or miR‐208a KO mice received oil (n = 4‐5/group) or CCl₄ (n = 6/group) treatment for 48 hours. (A) TUNEL assay was performed on paraffin‐embedded liver sections. The arrows indicate dark brown nuclei for TUNEL‐positive cells. The cytoplasmic staining in miR‐208a KO represents necrosis. Western blot analysis was used to determine the protein levels of procaspase‐8, caspase‐8 (B) and caspase‐3 (C), and density units for procaspase‐8 and p18, active form of caspase‐8 (B), and procaspase‐3 and caspase‐3 (C) are shown in the bar diagram (n = 4 [oil], n = 6 [CCl₄]). (D, upper panel) Predicted miR‐208a binding site at the 3′UTR region of p53. (D, lower panel) Western blot analysis for p53 protein and density units are shown in the bar diagram (n = 4 [oil], n = 6 [CCl₄]). (E) p21 mRNA levels were determined by quantitative PCR. Western blot analysis for Bcl2 (F) and Bid (G) protein levels and density units are shown in the bar diagram (n = 3 [oil], n = 3 [CCl₄]). Glyceraldehyde 3‐phosphate dehydrogenase (B,C,G) or β‐actin was used as a loading control. The mRNA levels of Bcl2 (F) and Bid (G) were determined by quantitative PCR. 18S was used to normalize the Cq values of each sample. *P < 0.05 versus oil‐treated WT mice, ^# P < 0.05 versus CCl₄‐treated WT mice. Abbreviation: GAPDH, glyceraldehyde 3‐phosphate dehydrogenase.

FIG. 4

Increased expression of genes involved in necrosis in miR‐208a KO mice after acute CCl₄ treatment. WT or miR‐208a KO mice received oil (n = 4‐5/group) or CCl₄ (n = 6/group) treatment for 48 hours. Whole‐liver cell lysates were used to determine the protein levels of Bax (A), CypD (B), PCNA (C), and cyclin D1 (D) by western blot analysis. β‐actin was used as a loading control. Density units are shown in the bar diagram from mice, Bax (n = 4 [oil], n = 6 [CCl₄]), CypD (n = 4 [oil]; n = 6 [CCl₄]), PCNA (n = 3 [oil], n = 3 [CCl₄]), and cyclin D1 (n = 3 [oil], n = 3 [CCl₄]). *P < 0.05 versus oil‐treated WT mice, ^# P < 0.05 versus CCl₄‐treated WT mice.

FIG. 5

Overexpression of miR‐208a attenuates CCl₄‐induced cytotoxicity in RAW macrophages. miR‐208a expression was determined from Kupffer cells (n = 4) and primary hepatocytes (n = 4) isolated from WT mice using quantitative PCR. (A) SnoRNA‐202 was used as an endogenous control to normalize Cq values. (B) RAW 264.7 macrophages were treated either with DMSO control (0.25%) or 0.1%, 0.2%, 0.3%, and 0.4% concentration of CCl₄ for 6 hours. Cell‐free supernatants were used to test cytotoxicity using an LDH‐cytotoxicity assay kit II according to the manufacturer’s instructions (n = 7). (C) Raw macrophages were either transfected with negative control miR mimic or miR‐208a mimic at the concentration of 2 pM for 24 hours. Cells were either treated or not with 0.3% CCl₄ for the last 6 hours of experiment. Cell‐free supernatants were used to test cytotoxicity using an LDH‐cytotoxicity assay kit II (n = 7). In vitro experiments were repeated three times, and results are shown as an average of two independent experiments. ***P < 0.001, ****P < 0.0001.