Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct;70(4):1150-1167.
doi: 10.1002/hep.30645. Epub 2019 Jun 5.

MicroRNA-223 Ameliorates Nonalcoholic Steatohepatitis and Cancer by Targeting Multiple Inflammatory and Oncogenic Genes in Hepatocytes

Affiliations

MicroRNA-223 Ameliorates Nonalcoholic Steatohepatitis and Cancer by Targeting Multiple Inflammatory and Oncogenic Genes in Hepatocytes

Yong He et al. Hepatology. 2019 Oct.

Abstract

Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of diseases ranging from simple steatosis to more severe forms of liver injury including nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC). In humans, only 20%-40% of patients with fatty liver progress to NASH, and mice fed a high-fat diet (HFD) develop fatty liver but are resistant to NASH development. To understand how simple steatosis progresses to NASH, we examined hepatic expression of anti-inflammatory microRNA-223 (miR-223) and found that this miRNA was highly elevated in hepatocytes in HFD-fed mice and in human NASH samples. Genetic deletion of miR-223 induced a full spectrum of NAFLD in long-term HFD-fed mice including steatosis, inflammation, fibrosis, and HCC. Furthermore, microarray analyses revealed that, compared to wild-type mice, HFD-fed miR-223 knockout (miR-223KO) mice had greater hepatic expression of many inflammatory genes and cancer-related genes, including (C-X-C motif) chemokine 10 (Cxcl10) and transcriptional coactivator with PDZ-binding motif (Taz), two well-known factors that promote NASH development. In vitro experiments demonstrated that Cxcl10 and Taz are two downstream targets of miR-223 and that overexpression of miR-223 reduced their expression in cultured hepatocytes. Hepatic levels of miR-223, CXCL10, and TAZ mRNA were elevated in human NASH samples, which positively correlated with hepatic levels of several miR-223 targeted genes as well as several proinflammatory, cancer-related, and fibrogenic genes. Conclusion: HFD-fed miR-223KO mice develop a full spectrum of NAFLD, representing a clinically relevant mouse NAFLD model; miR-223 plays a key role in controlling steatosis-to-NASH progression by inhibiting hepatic Cxcl10 and Taz expression and may be a therapeutic target for the treatment of NASH.

PubMed Disclaimer

Conflict of interest statement

Potential conflict of interest: Nothing to report

Figures

Figure 1.
Figure 1.. miR-223 is highly elevated in hepatocytes in HFD-fed mice and NASH patients.
(A-C) C57BL/6J mice were fed an HFD or control diet (CD) for three months. Liver and serum samples were collected and subjected to the measurement of miR-223 by RT-qPCR (panel A). Frozen liver tissue sections were also subjected to miR-223 in situ hybridization along with immunofluorescence staining of hepatocyte marker HNF-4α. Representative images of miR-223 expression (green), HNF-4α (red) and nuclei (blue) are shown (panel B). MiR-223+ hepatocytes were counted (panel C). (C-D) Formalin-fixed human liver tissue sections were subjected to miR-223 in situ hybridization and albumin staining for hepatocytes. Albumin+MiR-223+ hepatocytes were counted (panel C). Representative images of miR-223 (green) and nuclei (blue) are shown (Panel D). Arrows indicate miR-223+ hepatocytes. Values represent means ± SEM (n=8–12). *P< 0.05, **P< 0.01, ***P<0.001.
Figure 2.
Figure 2.. miR-223KO mice are more susceptible to 3m-HFD-induced liver injury, steatosis and inflammation.
WT and miR-223KO mice were fed an HFD or CD for three months. Serum and liver tissue samples were collected. (A) Serum ALT levels were measured. (B) Liver body ratio was measured. Lipids was stained with Oil Red and the quantitation of Oil Red+ area per field was determined. Representative images are shown in supporting Fig. S2D. (C) Liver tissues were also subjected to MPO and F4/80 immunostaining and the quantitation of MPO+ and F4/80+ cells per field was determined. Representative images are shown in Supporting Fig. S3A–B. (D, E) RT-qPCR analyses of liver Ly6g and F4/80 mRNA, and other inflammatory mediators. (F) Quantification of 4-hydroxynonenal (HNE)+ and malonaldehyde (MDA)+ area per field. Values represent means ± SEM (n=8–12). *P< 0.05, **P< 0.01, ***P<0.001 in comparison with corresponding WT groups; #P<0.05, ##P<0.01, ###P<0.001 in comparison with WT CD group.
Figure 3.
Figure 3.. miR-223KO mice are more susceptible to HFD-induced fibrosis.
WT and miR-223KO mice were fed an HFD or CD for three months. Liver tissue samples were collected. (A) Representative images of Sirius red staining, Masson staining, α-SMA staining and P62 staining of liver tissue sections are shown. (B) Quantification of fibrotic area per field. (C) RT-qPCR analyses of hepatic fibrogenesis genes. Values represent means ± SEM (n=8–12). *P< 0.05, **P< 0.01, ***P< 0.001 in comparison with WT HFD groups; #P<0.05, ##P<0.01, ###P<0.001 in comparison with WT CD groups.
Figure 4.
Figure 4.. Upregulation of hepatocarcinogenesis-related genes in miR-223KO mice versus WT mice after 3m-HFD feeding.
WT and miR-223KO mice were fed an HFD or CD for three months. Liver tissue samples were collected, and then subjected to microarray analysis. (A) Heat map of gene expression (>2-fold changes) from microarray analysis is shown. (B) Heat map of 172 liver cancer-related genes expression from microarray analysis is shown. (C) Ingenuity Pathway Analysis of liver cancer, inflammation, and cirrhosis in WT and miR-223KO mice. (D) RT-qPCR analyses of hepatic proliferation markers. (E) RT-qPCR analyses of HCC markers. Values represent means ± SEM (n=4 in panels A-C; n=8 in panels D-E). *P< 0.05, **P< 0.01 in comparison with WT HFD groups; #P<0.05, ##P<0.01 in comparison with WT CD groups.
Figure 5.
Figure 5.. miR-223KO mice show higher tumor incidence and greater degree of liver fibrosis compared with WT mice after HFD feeding for one year.
WT (n=21) and miR-223KO mice (n=19) were fed an HFD or CD for one year. Liver tissue samples were collected. (A) Representative photographs of liver tumors that developed in WT and miR-223KO mice are shown. Arrows indicate liver tumor. (B) Liver tumor incidence in WT and miR-223KO mice was analyzed. (C) RT-qPCR analysis of hepatic miR-223 levels after HFD feeding for one year. (D) Representative Sirius red staining and α-SMA staining of liver tissue sections are shown. (E) Quantification of fibrotic area per field. (F) RT-qPCR analyses of hepatic fibrogenesis genes. Values represent means ± SEM (n=6–21). *P< 0.05, ***P< 0.001 in comparison with WT HFD groups; #P<0.05, ##P<0.01, ###P<0.001 in comparison with WT CD groups.
Figure 6.
Figure 6.. A greater number of hepatocytes strongly expressed CXCL10 and TAZ in HFD-fed miR-223KO mice versus WT mice.
WT and miR-223KO mice were fed an HFD or CD for three months or one year. (A) Liver tissues were collected for RT-qPCR analyses of Cxcl10 and Taz. (B) Liver tissues were also subjected to immunohistochemistry analyses. Representative CXCL10 staining and TAZ staining of liver tissue sections. Quantification of CXCL10+ hepatocytes or CXCL10+ area and TAZ+ area per field was quantified. Values represent means ± SEM (n=8–12). *P< 0.05, **P<0.01, ***P< 0.001 in comparison with corresponding WT groups; #P<0.05, ##P<0.01, ###P<0.001 in comparison with WT CD groups.
Figure 7.
Figure 7.. miR-223 regulates Cxcl10 and Taz expression in hepatocytes.
(A) Bioinformatics approach analyses of the target prediction of miR-223. (B) Dual-Luciferase activity assay was performed to verify binding between miR-223 and CXCL10 or TAZ. AML12 cells were co-transfected with control luciferase vector, Cxcl10 3’UTR vector or Taz 3’UTR vector and miR-223 mimics or non-specific (NS)-miRNA mimics for 48h. Relative luciferase activity was determined. (C) AML12 cells were transfected with NS-miRNA mimics and miR-223 mimics for 24h, and then challenged with 0.1 mM palmitic acid (PA) for 3h. The Cxcl10 and Taz mRNA levels were measured by RT-qPCR. (D) Primary hepatocytes from WT and miR-223KO mice were treated with PA for 3h. The Cxcl10 mRNA expression was measured by RT-qPCR. (E) AML12 cells were transfected with NS-miRNA mimics and miR-223 mimics for 24h. The YAP and TAZ proteins were measured by western blotting. (F) AML12 cells were transfected with NS-miRNA mimics and miR-223 mimics for 24h, and then treated with 0.1 mM PA for 24h. The protein levels in whole hepatocyte lysates were determined by Western blot analyses. Values represent means ± SEM from three independent experiments. *P< 0.05 as indicated; #P<0.05, ###P<0.001 in comparison with control NS-miRNA mimic groups in panel C or 0h WT group in panel D.
Figure 8.
Figure 8.. Several potential targets of miR-223 positively correlate with CXCL10 and TAZ expression in NASH patients.
(A) Normal liver and NASH samples were subjected to immunohistochemistry analysis of CXCL10 and TAZ. Representative images are shown, and quantification of CXCL10+ and TAZ+ area per field was performed. Values represent means ± SEM. **P< 0.01, ***P< 0.001 as indicated. (B) The gene expression profiles in healthy control liver, fatty liver, and NASH patients were obtained from published microarray data (the accession number E-MEXP-3291 [http://www.webcitation.org/5zyojNu7T]). Positive correlation of CXCL10 or TAZ with several proven or potential miR-223 targeted genes and cancer-related genes in NASH patient liver samples. P value is indicated. (C) Gene expression profiles in the livers of normal (n=10) and NASH (n=14) patients were analyzed by RT-qPCR. Positive correlation of miR-223 with cytokine, chemokine, and fibrogenic genes, and proven or potential miR-223 targeted genes and cancer-related gene in NASH patients. P value is indicated. (D) A model depicting the critical role of miR-223 in controlling the progression of NASH. Obesity associated fatty liver upregulates miR-223, which subsequently attenuates many downstream target genes including pro-inflammatory and oncogenic genes, thereby ameliorating NASH progression. In contrast, miR-223 is markedly downregulated in HCC. Such downregulation of miR-223 accelerates HCC progression. Thus, miR-223 is a critical regulator of NASH progression and could be a novel therapeutic target for the treatment of NASH and liver cancer.

Similar articles

Cited by

References

    1. Friedman SL, Neuschwander-Tetri BA, Rinella M, Sanyal AJ. Mechanisms of NAFLD development and therapeutic strategies. Nat Med 2018;24:908–922. - PMC - PubMed
    1. Ibrahim SH, Hirsova P, Gores GJ. Non-alcoholic steatohepatitis pathogenesis: sublethal hepatocyte injury as a driver of liver inflammation. Gut 2018;67:963–972. - PMC - PubMed
    1. Machado MV, Diehl AM. Pathogenesis of Nonalcoholic Steatohepatitis. Gastroenterology 2016;150:1769–1777. - PMC - PubMed
    1. Younossi Z, Tacke F, Arrese M, Sharma BC, Mostafa I, Bugianesi E, et al. Global Perspectives on Non-alcoholic Fatty Liver Disease and Non-alcoholic Steatohepatitis. Hepatology 2018. - PubMed
    1. Estes C, Razavi H, Loomba R, Younossi Z, Sanyal AJ. Modeling the epidemic of nonalcoholic fatty liver disease demonstrates an exponential increase in burden of disease. Hepatology 2018;67:123–133. - PMC - PubMed

Publication types

MeSH terms