THSR Mediated MiR374b Targeting C/EBP β/FOXO1 to Accelerate Thyroid Stimulating Hormone-Induced Hepatic Steatosis

Introduction

In China, non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease, surpassing both viral hepatitis and alcoholic liver disease.¹ Current estimates suggest a prevalence of NAFLD in the general population ranging from 15% to 33%, with rates as high as 23.3% to 59.6% among obese children and adolescents.²

Subclinical hypothyroidism (SCH), characterized by raised thyroid stimulating hormone (TSH) levels, is a prevalent endocrine metabolic disorder and an independent risk factor for NAFLD.³ SCH affects approximately 4–10% of adults, increasing to 20% among women over 60 years old, and is becoming more widespread.⁴ This condition is a significant contributor to secondary dyslipidemia and hypertension.^5,6 TSHR is one of the genes most closely related to GD, and currently K1-70 has been used as a targeted TSHR inhibitor for the treatment of GD eye disease.⁷ Activation of TSHR in cultured orbital fibroblasts promotes enhanced HA and fat production.⁸ Despite the established association between increasing serum TSHR levels and the incidence of SCH,⁹ the mechanisms by which TSHR regulates NAFLD progression remain poorly understood.

MicroRNAs (miRNAs) are known to bind specifically to the 3′ untranslated region (UTR) of target gene mRNAs, forming a complex that suppresses gene translation. Increasing evidence indicates that miRNAs play a critical role in regulating the expression of genes associated with NAFLD, with about 100 miRNAs showing significant expression differences in NAFLD patients.^10,11 These miRNAs impact various aspects of NAFLD, including glucose and lipid metabolism,¹² oxidative stress,¹³ cell apoptosis,¹⁴ inflammation, endoplasmic reticulum stress,¹⁵ and insulin resistance.¹⁶ For example, inhibition of miR-33 in the liver significantly slows NAFLD progression,¹⁷ while upregulation of miR-122 correlates directly with the severity of liver fibrosis in NAFLD, making it a potential biomarker and therapeutic target.¹⁸ MiR-34a influences liver lipid metabolism and steatosis by targeting the transcription factor SIRT1.¹⁹ Upregulated levels of miR-378 in the liver may enhance transcription activating factor LXR α, inhibiting key enzymes and effectors in liver lipid metabolism pathways, thereby exacerbating NAFLD.²⁰ Furthermore, extracellular vesicles containing miR-223 from neutrophils and other cells can decrease the expression of the pro-fibrotic gene TAZ in liver cells.²¹ However, the specific targets of miR374b in SCH remain to be clarified.

Recent research indicates that miR374b targets CCAAT/enhancer-binding protein-b (C/EBP β) and Recombinant Forkhead Box Protein O1 (FOXO1) in adipose tissue, reducing their transcriptional stability and translational output.²² This interaction significantly suppresses the PPAR γ signaling pathway, playing a vital role in lipid metabolism and insulin resistance. It is hypothesized that miR374b could be a critical miRNA in the development and progression of fatty liver disease.

Material and Methods

Animal Model

C57BL/6J mice, purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. (Hangzhou, China), underwent a 7-day acclimatization period. After that, 20 male mice were randomized into two groups: 10 mice in the control group and 10 in the model group. The model group was fed a high-fat diet for eight weeks to induce an NAFLD model. High fat feed raw material ratio, maintaining basic feed, 0.435; Lard, 0.175; Sucrose, 0.12; Whole milk powder, 0.1; Casein, 0.13; Experimental animal premix, 0.02; Calcium hydrogen phosphate, 0.02. (XTHF60, Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd, China). The Shandong Provincial Hospital Animal Ethics Committee approved animal experiments (Ethical approval No.2021–497) in this study. Experimental animals follow the Guidelines for Ethical Review of Experimental Animal Welfare. The standard number of this guide is GB/T 35892–2018.

Cell Culture

HepG2 cells were purchased from Wuhan Pricella Biotechnology Co., Ltd. (Wuhan, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; BIOIND Israel) in a 5% CO₂ atmosphere at 37°C.

Oil Red O Staining

HepG2 cells were washed thrice with PBS and fixed with 4% formaldehyde at room temperature for 30 minutes, followed by three additional PBS washes of 5 minutes each. Cells were then stained using an Oil Red O stain kit (Sigma Aldrich, Shanghai, China) for 1 hour after diluting the stock solution with water (3:2 ratio). After staining, cells were washed thrice with PBS (5 minutes per wash) and imaged under a microscope.

TSH/TC/TG Assay

According to the manufacturer’s instructions, measure Thyroid Stimulating Hormone using a mouse/human thyroid stimulating hormone detection kit (Solarbio, China).Total cholesterol and triglyceride levels were measured using the total cholesterol/triglyceride assay kit (Solarbio, China) according to the manufacturer’s instructions.

Dual-Luciferase Reporter Assay

FOXO1/C/EBPβ 3′ UTR-WT-psicheck2 and FOXO1/C/EBPβ 3′ UTR-Mut-psicheck2 plasmids (Limibio Co., Ltd.,China) were transfected into HepG2 cells alongside hsa-miR-374a-5p mimics or miR-NC in 24-well plates. After 48 hours, cells were lysed, and luciferase activities were measured. Firefly luciferase activity was assessed using 20µL of cracking mixture followed by 100 µL of Luc reaction solution. Renilla luciferase activity was then measured using 100µL of termination solution. The ratio of Renilla to firefly luciferase activities was calculated to determine relative luciferase activity.

Quantitative Real-Time PCR

Total RNA was extracted from HepG2 cells and tissues using TRIzol Reagent (Biosharp, China). RNA concentration was measured with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). Reverse transcription was performed using Hifair III first strand cDNA Synthesis SuperMix (Yeasen Biotechnology Co., Ltd., China). qPCR was conducted using Hieff UNICON® Universal Blue qPCR SYBR Master Mix (Yeasen Biotechnology Co. Ltd., China) on a LightCycler480 Real-Time fluorescence quantitative PCR instrument (Roche, Switzerland). The gene primers used in the experiment are shown in Table 1.The thermal cycling conditions were: 1 cycle at 95°C for 2 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 20 seconds, then 95°C for 15 seconds and 60°C for 60 seconds. β-actin was used as an internal control, and each sample was tested in triplicate.

Table 1 Specific Primer Sequences for qRT-PCR

Western Blot

Protein extraction was conducted using a total protein extraction kit (Solarbio, China), and protein concentrations were determined with a BCA protein assay kit (Solarbio, China). For electrophoresis, 20 µg of protein per sample was separated on a 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature, incubated overnight at 4°C with primary antibodies, followed by a 1.5-hour room temperature incubation with corresponding secondary antibodies. Immunoblots were scanned in grayscale, using β-actin as an internal reference protein.Information on the use of the antibodies is provided in Table 2.

Table 2 Information on the Use of the Antibodies

Statistical Analysis

Statistical analyses were performed using SPSS version 21.0 (IBM, USA). Data were analyzed using the t-test and expressed as mean ± standard deviation (SD). Differences were considered statistically significant at P<0.05 and marked accordingly in the results.

Results

Effect of TSH on Adipose Degeneration in HepG2 Cells

In order to understand the role of TSH in the occurrence and development of NAFLD, ELISA was used to detect the concentration of TSH in the serum of NAFLD mouse models. The results showed that TSH was significantly overexpressed in this group (Figure 1A). In vitro, HepG2 cells were treated with varying concentrations of TSH, and lipid accumulation was assessed using oil red staining. As depicted in Figure 1B, lipid levels were significantly higher in the TSH-treated group than in controls, increasing progressively with rising TSH concentrations. At the same time, levels of total cholesterol (TC) and triglycerides (TG) were elevated in cells treated with TSH (Figure 1C). While TC levels at a 1μM concentration of TSH did not differ significantly from controls, TG levels significantly increased, particularly at a 4μM concentration. Importantly, miR374b expression was found to correlate positively with TSH concentration (Figure 1D), suggesting that TSH induces miR374b expression in HepG2 cells.

Figure 1 Effect of TSH on adipose degeneration in HepG2 cells. (A) Comparison of serum TSH levels between NAFLD mouse model and control group (n=3, * P < 0.05). (B) Oil red O staining of HepG2 cells treated with TSH (0 μM, 1 μM, 2 μM) (n=3), Scale bar = 50 μm. (C) Changes of serum TC and TG levels in each group treated with TSH (0 μM, 1 μM, 2 μM) (n=3, one-way ANOVA and Tukey’s multiple comparison test; ns, nonsignificant; * P < 0.05 versus 0 μM). (D) The expression of miR-374b-5p in each group compared with control (n=3, *** P < 0.001 versus 0 μM, ****P < 0.0001 versus 0 μM).

TSH-Induced Hepatic Steatosis in HepG2 Cells Dependent on TSHR

To determine if TSH expression depends on TSHR, three siRNAs targeting the TSHR gene in HepG2 cells were designed. Effective knockdown was achieved with sequences TSHR-Si-385 (CGGAAUACCAGGAACUUAATT) and TSHR-Si-656 (GGACAAAGCUGGAUGUGUGUGUGUGUGUTT), which reduced TSHR expression by 70% and 50%, respectively (Figure 2A). TSHR-Si-385 was used for further experiments to knock down TSHR, and recombinant TSH protein was employed for intervention treatments. The Oil Red O staining results, as shown in Figure 2B, showed no significant lipid accumulation in the OE-NC and si NC groups compared to the Control group. The OE-TSHR group showed a significant increase in lipids compared to the OE-NC group, while the si TSHR group showed a decrease in lipid content compared to the si NC group; After TSH treatment of cells, the lipid content of OE-NC+TSH and si-NC+TSH significantly increased; The OE-TSHR+TSH group had the highest lipid content, significantly higher than the lipid content of cells treated with a single treatment; The lipid content of the si TSHR+TSH group was significantly increased compared to the si TSHR group, indicating that TSH promotes liver lipid accumulation in a TSHR dependent manner. Additionally, levels of TC and TG correlated with TSHR expression, increasing in the TSHR overexpression group (Figure 2C). Real-time quantitative PCR analysis demonstrated that both TSH and TSHR overexpression could upregulate miR-374b expression. Notably, even after TSHR knockdown, TSH overexpression still elevated miR-374b levels (Figure 2D), indicating that miR-374b expression is primarily regulated by TSH, with TSHR exerting a regulatory effect.

Figure 2 TSH-induced hepatic steatosis in HepG2 cells dependent on TSHR. (A) Comparison of TSHR mRNA expression levels between the interference and overexpression TSHR groups and the control group (n=3,* P < 0.05; ** P < 0.01; *** P < 0.01). (B) Oil red O staining of HepG2 cells treated with TSHR combined with TSH (2 μM) (n=3,Red arrow lipid accumulation site,Scale bar = 50 μm. (C) Changes of serum TC and TG levels in each group treated with TSHR-siRNA and TSH (2 μM) (n=3;*** P < 0.001). (D) The expression of miR-374b-5p in each group checked by Realtime-PCR (n=3; **P < 0.01; ***P < 0.001).

Effect of miR374b on the Regulation of the Transcription Factor C/EBP β and FOXO1 Expression

To delineate the gene regulatory function of miR374b in NAFLD, gene manipulation was conducted on miR374b, with both mimics and inhibitors synthesized and transfected into HepG2 cells. C/EBP β, FoxO1, PPAR γ, SREBP2, and SREBP1c expression levels were assessed using real-time qPCR. Results showed a decrease in the expression of C/EBP β, PPAR γ, and FoxO1 following miR374b upregulation, while miR-33, SREBP2, and SREBP1c expression significantly increased (Figure 3A). In contrast, miR374b knockdown elevated C/EBP β, PPAR γ, and FoxO1 expression and reduced miR-33, SREBP2, and SREBP1c. Western blot analysis corroborated the qPCR findings, confirming the regulatory effects at both protein and mRNA levels (Figure 3B), thus indicating miR374b’s role in modulating the expression of these key metabolic regulators.

Figure 3 Effect of miR374b on the regulation of the transcription factor C/EBP β and FOXO1 expression. (A) The mRNA expression of C/EBP β, FoxO1, PPAR γ, SREBP2, SREBP1c, and miR33a in each group compared with the normal control (n=3,* P < 0.05). (B) Western blot showing the expression of TSHR, C/EBPβ, FOXO1, PPARγ, SREBP2, and SREBP1c in each group compared with normal control (n=3,* P < 0.05).

MiR374b Regulates NAFLD by Targeting C/EBPβ and FOXO1

To elucidate the role of miR374b in NAFLD, we regulated miR374b expression in a TSH-induced HepG2 cell model by constructing miR374b mimics and inhibitors. Functional validation via oil red staining demonstrated that overexpression of miR374b enhanced lipid accumulation while its knockdown decreased lipid levels (Figure 4A). This effect was exacerbated with TSH intervention, though fat accumulation was significantly reduced in the miR374b knockdown group compared to controls, suggesting that miR374b operates independently of TSH in influencing NAFLD. Biochemical assays confirmed that miR374b overexpression increased TC and TG, which were reduced upon miR374b knockdown (Figure 4B). C/EBP β and FOXO1 were identified as target genes of miR374b, both containing binding sites within their 3′UTR regions, confirmed via dual-luciferase reporter assays (Figure 4C and D). Real-time qPCR analysis revealed that upregulation of miR374b decreased the expression of C/EBP β, FOXO1, and PPAR γ, while miR-33, SREBP2, and SREBP1c expressions were elevated. Conversely, miR374b knockdown led to increased expression of C/EBP β and slight changes in FOXO1 and PPAR γ (Figure 4E). Western blot analysis corroborated these findings (Figure 4F), underscoring miR374b’s role in targeting and suppressing C/EBP β and FOXO1 expression, impacting lipid metabolism in NAFLD.

Figure 4 MiR374b regulates NAFLD by targeting C/EBP β and FOXO 1. (A) Oil red O staining of HepG2 cells transfected with miR374b mimics and inhibitor (n=3), Scale bar = 50 μm. (B) Changes of serum TC and TG levels in each group treated with miR374b mimics and inhibitors combined with TSH (2 μM) (n=3,* P < 0.05 versus control). (C) Predicted binding sites with C/EBP β and FOXO1. (D) The dual luciferase reporter gene assay confirmed the binding of miR374b to C/EBP β and FOXO1 genes; Error bars indicate the mean ± SD; ** P < 0.01, (E) The mRNA expression of C/EBP β, FoxO1, PPAR γ, SREBP2, SREBP1c, and miR33a in each group compared with normal control (n=3,* P < 0.05). (F) Western blot showed the expression of TSHR, C/EBPβ, FOXO1, PPARγ, SREBP2, and SREBP1c in each group compared with normal control (n=3, * P < 0.05).

Effect of miR374b on the Regulation of NAFLD in vivo

An NAFLD mouse model was replicated, and fatty lesions in the liver tissues of the model mice were identified via HE pathological staining (Figure 5A). Oil red O staining indicated a significant accumulation of adipocytes in the liver tissues of NAFLD mice (Figure 5B). Quantitative analyses of TC and TG in serum and liver tissues showed elevated levels in the model group compared to the control group. TSH levels were significantly increased in the model group, as illustrated in Figure 5C. qPCR analysis detected upregulated expression of TSHR and miR-374b in the model group (Figure 5D), indicating that TSHR promotes the expression of miR-374b. As a target gene of miR-374b, C/EBP β expression was reduced, leading to downregulation of the transcription factor FOXO1 and its downstream target genes. Western blot analyses corroborated the qPCR findings (Figure 5E). In line with prior studies, an increase in miR-33-3p expression was observed, which may be associated with the regulation of SREBP1. Collectively, these findings suggest that TSH upregulates miR374b expression via TSHR in liver cells, which then influences C/EBP β and modulates critical molecules in lipid metabolism, thereby promoting lipid accumulation in liver cells and inducing hepatic steatosis.

Figure 5 Effect of miR374b on the regulation of NAFLD in vivo. (A) Liver HE staining in NAFLD mouse model and control group (n=3,Red arrow lipid vacuoles, Scale bar = 50 μm). (B) Oil red O staining in NAFLD mouse model and control group (n=3,Red arrow lipid accumulation site, Scale bar = 50 μm). (C) Changes of serum and liver tissues TC and TG levels in NAFLD mouse model compared with negative control (n=6, *P < 0.05). (D) The mRNA expression of C/EBP β, FoxO1, PPAR γ, SREBP2, SREBP1c, and miR33a in each group compared with normal control (n=3, *P < 0.05). (E) Western blot showed the expression of TSHR, C/EBPβ, FOXO1, PPARγ, SREBP2, and SREBP1c in each group compared with normal control (n=3, * P < 0.05).

Discussion

TSH, a key protein in SCH lesions, is involved in inducing NAFLD through its interaction with the TSH receptor (TSHR).²³ TSH regulates thyroid activity and comprehensively activates thyroid metabolism,²⁴ including the synthesis of thyroid-specific proteins such as thyroglobulin (Tg), thyroid peroxidase (TPO), and the sodium-iodide symporter (NIS). Thyroid hormone receptors (TRs), TRα and TRβ, bind with Thyroid hormone (3,5,3′-triiodothyronine, T3) to regulate gene expression associated with TSH.²⁵ Beyond its thyroid-specific roles, TSH influences various hepatic metabolic activities, promoting lipid output, oxidation, and de novo adipogenesis.²⁶ The TSH/TSHR signaling pathway is crucial for the morphogenesis and differentiation of embryonic glands in mice,²⁷ affecting lipid metabolism through modulation of AMPK and PPARα signal transduction.^28,29 However, the relationship between TSH and miRNAs has not yet been elucidated.

This study elucidates the novel role of miR374b in the pathogenesis of NAFLD mediated by TSH. The results demonstrate that miR374b significantly influences lipid metabolism and regulates FOXO1 and its downstream target genes in HepG2 cells. MiR374b is involved in the complex regulation of NAFLD lipid metabolism. Notably, miR374b activity is significantly elevated in a NAFLD mouse model; TSH promotes its expression via the TSHR. Moreover, miR374b directly targets C/EBP β and its downstream target genes, playing a critical role in regulating lipid metabolism. Overexpression of miR374b leads to increased fat accumulation, while its downregulation has the opposite effect, highlighting its essential role in maintaining intracellular lipid balance.

The discovery of miR374b’s targeted regulation of C/EBP β, a key factor in triglyceride metabolism and NAFLD pathogenesis,^30,31 is particularly significant. C/EBP β as a transcription factor that controls energy metabolism³² can improve lipid accumulation and metabolism in zebrafish larvae,³³ while FOXO1 is an important regulatory factor for energy balance in dormant embryos and is crucial for lipid metabolism in animals.³⁴ Increased miR374b levels lead to elevated C/EBP β expression, whereas knocking down miR374b reduces its expression. This study also confirms that miR374b regulates the expression of FOXO1 and its downstream genes. These findings suggest that TSH contributes to NAFLD by influencing miR374b’s targeting of C/EBP β and its downstream gene regulation, presenting a novel therapeutic target for NAFLD management by modulating miR374b activity.

Additionally, this research uncovered that miR374b-induced lipid accumulation correlates negatively with lipid metabolism, thereby exacerbating NAFLD. It also examined the role of the TSHR in NAFLD pathogenesis. Findings demonstrated that TSHR is involved in TSH-induced NAFLD, where TSH binding to TSHR enhances miR374b transcription, subsequently activating C/EBP β to induce lipid production in adipose tissue, highlighting the therapeutic potential of targeting TSHR to mitigate NAFLD onset.The function of TSHR in NAFLD.TSH binds to TSHR, which activates the expression of miR374b. Resulting in a decrease in the expression levels of miR374b target genes C/EBP β and FOXO 1, thereby regulating the expression of downstream lipid metabolism pathway proteins PPAR γ, SREBP2, and SREBP1c.

Despite these insights, the study has limitations that warrant further investigation. Primarily, it relies on the HepG2 cell line, which might not fully mimic the complex cellular dynamics of human lipid metabolism. Future studies should incorporate primary human liver cells to validate these findings. Additionally, while the findings offer valuable in vitro insights, they require validation in clinical settings to confirm their relevance to NAFLD pathogenesis. Moreover, although it was established that miR374b targets C/EBP β and FOXO1, impacting lipid metabolism in NAFLD, the study did not thoroughly explore the detailed molecular mechanisms and downstream lipid metabolism processes. Further investigations are necessary to elucidate the specific signaling pathways and molecular interactions involved.

Conclusion

These findings suggest that TSHR-induced upregulation of miR374b accelerates NAFLD progression by modulating lipid metabolism pathways through C/EBP β and FOXO1, positioning miR374b as a potential therapeutic target for NAFLD.