miR-21: A therapeutic target for delaying severe liver disease and hepatocellular carcinoma in high-fat-diet-fed mice

Abstract

Liver disease, including hepatocellular carcinoma (HCC), is a major global health concern, claiming approximately 2 million lives worldwide annually, yet curative treatments remain elusive. In this study, we aimed to investigate the role of microRNA-21-5p (miR-21) in metabolic dysfunction-associated steatotic liver disease (previously NAFLD), metabolic-associated steatohepatitis (previously NASH), and HCC within the context of a Western high-fat diet, without additional choline (HFD) and offering potential therapeutic insights. We found that reduced miR-21 levels correlated with liver disease progression in WT mice fed on HFD, while miR-21 knockout mice showed exacerbated metabolic dysfunction, including obesity, hepatomegaly, hyperglycemia, insulin resistance, steatosis, fibrosis, and HCC. Our study reveals that miR-21 plays a protective role in metabolic syndrome and in the progression of liver disease to cancer. MiR-21 directly targets Transforming growth factor beta-induced (Tgfbi), a gene also known to be significantly upregulated and a potential oncogene in HCC. Further, our study showed that intervention with the administration of a miR-21 mimic in WT livers effectively improves insulin sensitivity, steatosis, fibrosis, Tgfbi expression and tumor burden in HFD conditions. These findings indicate that miR-21 could serve as an effective strategy to delay or prevent liver disease in high-fat-diet environments.

Keywords: MASLD (NAFLD); fibrosis; hepatocellular carcinoma; high-fat diet; liver; miR-21; protective.

Figure 1:. WT mice fed on HFD develop obesity, liver injury, hyperglycemia, insulin resistance, fatty liver, steatohepatitis, advanced fibrosis, and HCC

(A) Experimental timeline. B6/129S mice were injected with 25mg/kg of Diethylnitrosamine (DEN) at three weeks of age and were fed with either a chow diet (Chow) or high-fat diet, without additional choline (HFD) for up to 32 weeks, (B) change in body weight over time, (C) Liver to body weight ratio, (D) Serum ALT levels,(E) Blood glucose levels, and (F) Insulin tolerance test (ITT) for week-16 and week-24. Histology scores for (G) Steatosis,(H) Fibrosis, (I) Hypertrophy, and (J) Lobular inflammation. (K) Representative liver images at week 32, and (L) Tumor burden at weeks 24 and 32. Data are expressed as the mean±SD for six mice per group for ITT assay, at least six mice per group for body weight measurements, histology and tumor burden analysis. *p<0.05, **p<0.01, ***p<0.001.

Figure 2:. miR-21 KO mice on HFD show exacerbated signs of liver injury, hyperglycemia, and insulin resistance

(A) miR-21 levels in livers during liver disease progression in WT mice (B) Body weight of miR-21 KO mice compared to WT on Chow and HFD group. (C) Serum ALT levels at weeks 4 and 16, (D) Liver to body weight ratio (E) Blood glucose measurements, (F) Insulin tolerance test, and (G) qRT-PCR expression of genes involved in insulin resistance. N= 6–8 mice/group. ns=not significant, *p<0.05, **p<0.01, ***<0.001.

Figure 3:. miR-21 KO mice on HFD show exacerbated signs of fatty liver, steatohepatitis, and advanced fibrosis

(A) Representative images of livers at weeks 16, 24, and 32 for Hematoxylin-eosin (H&E), Sirius red, and Oil Red O staining. Histology scores for (B) Hypertrophy, (C) Lobular inflammation, (D) Steatosis, and (E) Fibrosis. ImageJ quantification of (F) Oil Red O and (G) Sirius Red staining at week 16. Quantitative real-time analysis of genes involved in (H) Inflammation (I) Fibrosis, and (J) Fatty acid uptake in Week-16, 24, and 32 liver samples. Serum levels of (K) Cholesterol and (L)Triglycerides at week 24. N= 4–8 mice/group. ns=not significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 4:. miR-21 KO mice develop hepatocellular carcinoma

(A) Tumor burden of mice at weeks 24 and 32 (B) Representative hepatic tumors (C) H&E and immunostaining of hepatic-tumor sections at weeks 24 and 32, (D) Comparative lesion burden and (E) Types of lesions in various groups, (F) Gene count of differentially expressed genes with fold change >2 in tumors from miR-21 KO vs WT fed on HFD, (G) Cancermine analysis for the nature of genes. (H) IPA analysis of canonical pathways, and diseases and functions, (I) Heatmap representing differentially expressed genes related to the advanced malignant tumor. N= 4–8 mice/group.

Fig. 5:. miR-21 directly regulates Tgfbi

(A) Venn diagram showing overlap of differentially expressed genes in various comparative datasets. (B) The in-silico binding of miR-21 on 3’UTR of Tgfbi gene, (C) Quantitative real-time analysis of Tgfbi mRNA expression in week 16, 24, and 32 liver samples. (D) TGFBI gene expression levels in the livers of HCC patients. Data is expressed as at least 4 mice/group. ns=not significant, *p<0.05, **p<0.01, ***p<0.001.

Figure 6:. Supplementation of miR-21 mimic slows down the progression of liver disease in WT mice

(A) Timeline of the experiment (B) Quantitative real-time analysis for the levels of miR-21 target gene-Tgfbi in the liver (C) Body weight over time (D) Glucose levels in mices’ serum after 5-hours of starvation, Insulin tolerance test for week-18 and 22 (E) Histology of liver sections and staining with H&E, Oil Red O and Sirius red stain, (F) Histology scoring of Hypertrophy, Lobular inflammation, Steatosis and Fibrosis (G) Representative images of negative control (NC) and miR-21 mimic administered livers and (H) Tumor burden on week-22. ns= not significant. *p<0.05.