miR-33 deletion in hepatocytes attenuates MASLD-MASH-HCC progression

Abstract

The complexity of the mechanisms underlying metabolic dysfunction-associated steatotic liver disease (MASLD) progression remains a significant challenge for the development of effective therapeutics. miRNAs have shown great promise as regulators of biological processes and as therapeutic targets for complex diseases. Here, we study the role of hepatic miR-33, an important regulator of lipid metabolism, during the progression of MASLD and the development of hepatocellular carcinoma (HCC). We report that miR-33 was elevated in the livers of humans and mice with MASLD and that its deletion in hepatocytes (miR-33 HKO) improved multiple aspects of the disease, including steatosis and inflammation, limiting the progression to metabolic dysfunction-associated steatotic hepatitis (MASH), fibrosis, and HCC. Mechanistically, hepatic miR-33 deletion reduced lipid synthesis and promoted mitochondrial fatty acid oxidation, reducing lipid burden. Additionally, absence of miR-33 altered the expression of several known miR-33 target genes involved in metabolism and resulted in improved mitochondrial function and reduced oxidative stress. The reduction in lipid accumulation and liver injury resulted in decreased YAP/TAZ pathway activation, which may be involved in the reduced HCC progression in HKO livers. Together, these results suggest suppressing hepatic miR-33 may be an effective therapeutic approach to temper the development of MASLD, MASH, and HCC in obesity.

Keywords: Fatty acid oxidation; Hepatology; Liver cancer; Metabolism; Noncoding RNAs.

Figure 1. Hepatic miR-33 deficiency improves systemic metabolism in MASL/MASH.

(A) qPCR analysis of miR-33 expression in WT and HKO hepatocytes fed a control diet and CD-HFD for 3 (MASL) and 6 months (MASH) (n = 3). (B) qPCR analysis of miR-33a and miR-33b expression in livers from healthy, MASL, and MASH human patients (n = 8). (C and D) Body weight (C) and body composition (D) analysis of WT and HKO mice during MASL/MASH time course (n = 18 WT and 16 HKO). (E–G) Levels of total cholesterol (E), HDL-C (F), and TAGs (G) in plasma of WT and HKO mice (n = 8 WT and 8 HKO chow; 6 WT and 6 HKO; MASL; 6 WT and 6 HKO MASH). (H) Cholesterol content of fast protein liquid chromatography–fractionated lipoproteins from pooled plasma of 6 WT and 6 HKO mice fed a CD-HFD for 3 (MASL) and 6 months (MASH). (I–L) GTT (n = 13, MASL; n = 12 WT and 11 HKO MASH) (I and K) and ITT (n = 8; n = 12 WT and 11 HKO MASH) (J and L) in WT and HKO mice with areas under the curve. Data represent the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ^###P < 0.001 comparing WT and HKO compared with WT animals, 2-way ANOVA followed by multiple comparison (A, B, and D–G) and unpaired 2-sided Student’s t test for 2-group comparisons (I–L).

Figure 2. miR-33 deficiency in hepatocytes reduces liver steatosis through regulation of FA synthesis and FAO.

(A) Representative images of H&E- and Oil Red O–stained livers from WT and HKO mice and (B) quantification of ORO staining (n = 3 WT and 3 HKO chow; 6 WT and 6 HKO; MASL; 7 WT and 6 HKO MASH). Liver weight (n = 3 WT and 3 HKO chow; 6 WT and 6 HKO; MASL; 7 WT and 6 HKO MASH) (C) and liver TAG (n = 7 WT and 7 HKO chow; 6 WT and 6 HKO MASL; 7 WT and 6 HKO MASH (D) in WT and HKO mice fed with a chow diet or CD-HFD. Data represent the mean ± SEM. *P ≤ 0.05, ***P ≤ 0.001 compared with WT animals, 2-way ANOVA followed by multiple comparison. FA, fatty acid.

Figure 3. miR-33 deficiency in hepatocytes increases FAO synthesis and decreases FA synthesis.

(A) Ex vivo analysis of FAO in WT and HKO livers (n = 5 WT and 4 HKO chow; 6 WT and 6 HKO; MASL; 6 WT and 6 HKO MASH). (B and C) Mitochondrial respiratory analysis inferred from oxygen consumption rate measurements of primary mouse hepatocytes isolated from WT and miR-33 HKO livers (n = 4 WT and 3 HKO MASL; 5 WT and 5 HKO MASH). (D) Western blot and densitometric analysis of CROT, CPT1α, phosphorylated acetyl-CoA carboxylase (p-ACC) (Ser79), total ACC, p-AMPKα (T172), total AMPKα, and housekeeping standard VINCULIN in WT and HKO livers. Data represent the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with WT animals, unpaired 2-sided Student’s t test for 2-group (B) comparisons and 2-way ANOVA followed by multiple comparison (A and D). CPM, counts per million.

Figure 4. RNA-Seq in MASL livers reveals global changes in gene expression regulated by miR-33.

(A, B, and D) Heatmaps of pathways relevant to MASLD progression in livers from WT and HKO mice. Cutoff values were settled as fold-change > log₂1.5 and Padj < 0.05. (n = 4.) (C) Comparison of liver BAs and BA distribution of the 12α-hydroxy/non–12α-hydroxy ratio in WT and HKO livers. (n = 4–5.) (E) Venn diagram depicting the overlap between miR-33 predicted targets and genes upregulated in HKO livers versus WT livers in mice fed a chow diet and CD-HFD. Data represent the mean ± SEM (*P ≤ 0.05 compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons and 2-way ANOVA followed by multiple comparison).

Figure 5. Loss of hepatic miR-33 attenuates liver fibrosis and MASH.

(A) Representative images of Sirius red–stained livers from WT and HKO mice. Indicated quantification on the right (n = 3 chow; 7 WT and 6 HKO MASH). (B) Graphical quantification of macrovesicular fat and hepatocyte ballooning from WT and HKO livers normalized per high-magnification field area (HMF) (n = 7 WT and 6 HKO). (C) Western blot and densitometric analysis of FN1, COL1A1, and housekeeping standard VINCULIN in WT and HKO livers. (D) Hydroxyproline content in MASH WT and HKO livers (n = 3 chow; 7 MASH). (E) Serum ALT in WT and HKO mice (n = 3 chow; 6 MASH). Data represent the mean ± SEM (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons and 2-way ANOVA followed by multiple comparison).

Figure 6. Hepatic loss of miR-33 attenuates liver injury through the crosstalk of different cell types.

Uniform manifold approximation and projection (UMAP) (A) and heatmap (B) representation of cell clusters identified from scRNA-Seq analysis. (C) UMAP of single-cell profiles from WT (gray) and HKO (red) mouse hepatocytes identified from scRNA-Seq analysis. (D) Canonical pathways represented by z score among differentially expressed genes in scRNA-Seq analysis of hepatocytes from WT and HKO mice. Red bars indicate pathways in which genes are upregulated in HKO, and gray indicates downregulated pathways on the predicted z score. All represented pathways were significantly changed with a –log P > 1.5. (E–I) Violin plots representing the top upregulated and downregulated genes significantly altered in the indicated pathways in hepatocytes. Violin plot data display single-cell distribution of the indicated experimental groups. Orig.Ident, original identity.

Figure 7. miR-33 deficiency attenuates liver fibrosis without regulating miR-33 target genes in nonhepatocyte liver cells.

(A and B) Heatmap showing HSC activation (A) and macrophage inflammatory/noninflammatory markers (B) identified from scRNA-Seq from WT and HKO mice livers. Color codes referred to z score. (C–F) Violin plots showing expression changes of miR-33 target genes in nonhepatocyte cells, including HSCs (C), endostellate cells (D), macrophages (E), and cholangiocytes (F). Violin plot data display single-cell distribution of the indicated experimental groups. ***Padj < 0.001 using default statistical test (Wilcox test) from Seurat package in R studio.

Figure 8. Hepatic miR-33 deficiency improves mitochondrial function and homeostasis.

(A) Western blot and densitometric analysis of different mitochondrial subunits blotted with the Total OXPHOS Rodent WB Antibody Cocktail (Abcam ab110413) and housekeeping standard VINCULIN in WT and HKO livers from mice fed with CD-HFD for 6 months (n = 6). (B) qPCR analysis of mitochondrial DNA and nuclear DNA in WT and HKO livers. Data represented as mtDNA/nDNA (n = 6). (C) Activity of the ETC complex I and complex II in MASH livers. Enzyme activities are expressed as change in absorbance/min/μg protein/citrate synthase activity (n = 4–6). (D) Representative electron micrographs of mitochondria profiles in WT and HKO hepatocytes from MASH livers. (E–H) Mitochondrial coverage (E), mitochondrial density (F), cumulative distribution and mean of mitochondrial area (G), and mitochondria aspect ratio (H) from WT and HKO hepatocytes (n = 3–4). (I) Western blot of PGC1α, TFAM, MFN2, OPA1, and housekeeping standard VINCULIN or GAPDH in WT and HKO livers. Data represent the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons and 2-way ANOVA followed by multiple comparison (B, C, and I).

Figure 9. miR-33 HKO mice have reduced oxidative stress and cell death in MASH livers.

(A) Representative DHE staining and quantification in WT and HKO livers from mice fed with CD-HFD for 6 months (n = 6 WT and 6 HKO). (B) Lipid peroxidation measured by MDA assay (Invitrogen) in MASH livers (n = 3 chow; 7 WT and 6 HKO MASH). (C) Glutathione reductase activity measured in MASH livers. Data represented as change in absorbance/min/μg of protein) (n = 4 WT and 6 HKO chow; 6 MASH). (D) Western blot and densitometric analysis of 4-HNE and (E) protein carbonylation measured by OxyBlot in WT and HKO livers. Data represent the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons (A) and 2-way ANOVA followed by multiple comparison (B–E).

Figure 10. Upstream and downstream analysis of AMPKα signaling pathway.

(A and B) Western blot analysis and densitometric analysis of housekeeping standard VINCULIN and (A) p-LKB1 (Ser428), LKB1, (B) SIRT1, SIRT2, SIRT3, SIRT5, SIRT6, and SIRT7 in WT and HKO livers from mice fed with CD-HFD for 6 months (MASH). (C) NAD⁺ levels in WT and HKO MASH livers represented as pmol/mg of tissue (n = 4 chow; 5 MASH). (D) Western blot analysis and densitometric analysis of p-ULK1 (Ser555), ULK1, LC3bI/II, ATG5, and housekeeping standard VINCULIN in WT and HKO MASH livers. Data represent the mean ± SEM (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons and 2-way ANOVA followed by multiple comparison).

Figure 11. Hepatic miR-33 deficiency reduces diet-induced tumor incidence.

(A) Representative images of WT and HKO livers after 15 months of CD-HFD; dashed line used to outline tumors and relative number of mice with and without tumor. (B–D) Graphical representation of (B) total number of tumors/mouse and (C) number of tumors larger > 20 mm³/mouse. (D) Circulating AFP levels (n = 13 WT –and 12 HKO). (E) Representative images of Ki67 staining in liver tumors from WT and HKO mice after 15 months of CD-HFD. Indicated quantification on the right (n = 3). Data represent the mean ± SEM (*P ≤ 0.05, ***P ≤ 0.001 compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons and 2-way ANOVA followed by multiple comparison).

Figure 12. Hepatic miR-33 deficiency reduces Hippo signaling.

(A) Western blot and densitometric analysis of TAZ and housekeeping standard VINCULIN in WT and HKO livers fed chow diet and CD-HFD for 6 months. (B) Western blot and densitometric analysis of TAZ and housekeeping standard LAMINb in nuclear fractions of livers from WT and HKO mice after 6 months of CD-HFD. (C) qPCR analysis of mRNA expression indicated genes in WT and HKO livers after 6 months of CD-HFD (n = 6 WT and 5 HKO). (D) Liver total and free cholesterol levels after 6 months of CD-HFD measured by gas chromatography–MS (n = 6). Data represented as nmol cholesterol/mg liver protein. Data represent the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 compared with WT animals, unpaired 2-sided Student’s t test for 2-group comparisons (B–D) and 2-way ANOVA followed by multiple comparison (A).

Figure 13. Schematic representation of probable miR-33 mechanisms of action