MicroRNA-451 and Genistein Ameliorate Nonalcoholic Steatohepatitis in Mice

Abstract

Effective, targeted therapy for chronic liver disease nonalcoholic steatohepatitis (NASH) is imminent. MicroRNAs (miRNAs) are a potential therapeutic target, and natural products that regulate miRNA expression may be a safe and effective treatment strategy for liver disease. Here, we investigated the functional role of miR-451 and the therapeutic effects of genistein in the NASH mouse model. MiR-451 was downregulated in various types of liver inflammation, and subsequent experiments showed that miR-451 regulates liver inflammation via IL1β. Genistein is a phytoestrogen with anti-inflammatory and anti-oxidant effects. Interestingly, we found that the anti-inflammatory effects of genistein were related to miR-451 and was partially antagonized by the miR-451 inhibitor. MiR-451 overexpression or genistein treatment inhibited IL1β expression and inflammation. Taken together, this study shows that miR-451 has a protective effect on hepatic inflammation, and genistein can be used as a natural promoter of miR-451 to ameliorate NASH.

Keywords: NASH; genistein; hepatocarcinoma; miR-451.

Figure 1

Downregulation of miR-451 in hepatic inflammation. (A,B) Expression of miR-451 in various liver diseases in the GEO database (A: GSE59492, [human]; B: GSE13840, [mouse]). Data represent means ± SEM. * p < 0.05, ** p < 0.01 compared to the control or normal groups. (C) The expression of IL-6 and TNF-α in NCTC1469 cells treated with LPS 10 μM and 20 μM (n = 3). (D) The expression of miR-451 in NCTC1469 cells treated with LPS 10 μM and 20 μM (n = 3). (E) The expression of IL-6 and TNF-α in Raw264.7 cells treated with LPS 10 μM and 20 μM (n = 3). (F) The expression of miR-451 in Raw264.7 cells treated with LPS 10 μM and 20 μM (n = 3). Data represent means ± SEM. * p < 0.05, ** p < 0.01 compared to the 0 μM LPS group.

Figure 2

IL1β is a target gene of miR-451. (A) miR-451 expression in NCTC1469 cells after transfection with the miR-451 mimic, inhibitor or negative control. (B,C) The expression of IL6, TNFα, and IL1β in NCTC1469 cells after transfection with the miR-451 mimic, inhibitor, or negative control. (D) miR-451 expression in Raw264.7 cells after transfection with the miR-451 mimic, inhibitor or negative control. (E,F) The expression of IL6, TNFα, and IL1β in Raw264.7 cells after transfection with the miR-451 mimic, inhibitor, or negative control. (G) Binding site of miR-451 and IL1β. (H) HeLa cells were co-transfected psiCHECKTM-2 vectors and the miR-451 mimic or negative control; the luciferase activity was determined. Data represent means ± SEM. * p < 0.05, ** p < 0.01, as compared to the negative control (NC).

Figure 3

miR-451 promotes the expression of inflammatory factors. (A–C) Correlation analysis of the expression of miR-451 and IL6, TNFα, and IL1β. (D,E), (F,G) IL6 and IL1β protein levels in NCTC1469 cells. Data represent means ± SEM. * p < 0.05, ** p < 0.01, as compared to the negative control (NC).

Figure 4

Genistein induces miR-451 to inhibit liver inflammation. (A) The expression of IL6 and TNFα in NCTC1469 cells after treatment with 0 μM LPS (NC), 20 μM LPS (LPS), 20 μM genistein (Gen), miR-451 inhibitor (451I), 20 μM LPS + 20 μM genistein (LPS-G), and 20 μM LPS + 20 μM genistein + miR-451 inhibitor (L-G-451I). (B) The expression of miR-451, IL1β, and Cab39 in NCTC1469 cells. Data represent means ± SEM. * p < 0.05, ** p < 0.01, compared to the NC group. # p < 0.05, ## p < 0.01, compared to the LPS group.

Figure 5

Genistein inhibits LPS-induced hepatic inflammatory responses. (A) Images of the whole livers of mice. (B) Liver weight index. (C) Chromatic aberration. (D) HE staining. The bars of the charts indicate 50 µm. (E) The expression of IL6, TNFα, and IL1β. (F) MiR-451 expression. Data represent means ± SEM. * p < 0.05, ** p < 0.01, compared to the PBS group. # p < 0.05, ## p < 0.01, compared to the LPS group.

Figure 6

Genistein inhibits high-fat diet (HFD)-induced nonalcoholic steatohepatitis (NASH). (A) Liver weight index. (B) Chromatic aberration. (C) The expression of miR-451 in mice liver after treated with HFD and genistein. (D,E) The mice liver of oil red O (D) and HE (E) staining. The bars of the charts indicate 50 µm. (F) Steatosis degree of mice liver in each group. ((G,H) The expression of Cab39 (G), TNFα, IL6, and IL1β (H) in mice liver tissues were detected by RT-qPCR. (I) Liver homogenate concentrations of TNFα, IL6, and IL1β. Data represent means ± SEM. (J) NASH activity score of mice in each group. * p < 0.05, ** p < 0.01, compared to the ND group. # p < 0.05, ## p < 0.01, compared to the HFD group.

Figure 7

Effect of miR-451 and its target genes on liver cancer. (A) miR-451 expression in healthy individuals and HCC (hepatocellular carcinoma) patients in the GEO database (GSE21362). (B) miR-451 in HCC patients with and without vascular invasion (HCC/V+ and HCC/V-, respectively) in the GEO database (GSE67139). (C,D) The overall survival rate of patients with liver cancer expressing high and low levels of IL1β and Cab39 identified in the Human Protein Atlas database. (E,F) The expression of miR-451, Cab39, and IL1β in HePG2 cells after treatment with miR-451 mimic, miR-451 inhibitor, and genistein. Data represent means ± SEM. * p < 0.05, ** p < 0.01, compared to the NC group. (G) Schematic diagram showing the theoretical mechanism by which genistein functions as a potential anti-hepatocarcinoma drug.