Sohlh2 inhibited the angiogenesis of hepatocellular carcinoma through the HIF-1α/VEGFA pathway

Highlights

• •Sohlh2 functions as a tumor suppressor gene in the HCC.
• •Overexpression of Sohlh2 suppresses the proliferation of liver cancer cells, impedes the transition of cells from the G0/G1 phase to the S phase, enhances tumor cell apoptosis, and hinders angiogenesis.
• •A negative correlation between the anti-tumor properties of Sohlh2 and the HIF-1α/VEGFA signaling.
• •Overexpression of Sohlh2 leads to the downregulation of the HIF-1α/VEGFA signaling pathway, consequently demonstrating anti-tumor properties.

Abstract

Purpose

To investigate the role of Sohlh2 in hepatocellular carcinoma (HCC) and its potential mechanisms.

Methods

This study focused on Hep3B and HepG2 cells and employed lentiviral transfection to create Sohlh2 overexpression and knockdown models. Through CCK8, flow cytometry, angiogenesis and immunofluorescence experiments, the effects of Sohlh2 on tumor cell proliferation, cell cycle progression, apoptosis, and angiogenesis were investigated. Additionally, its regulatory role in the HIF-1α/VEGFA pathway was explored. The experiments were repeated after introducing the Sohlh2 agonist CoCl₂ and antagonist LW6. Additionally, a Hep3B tumor-bearing mouse model was established to evaluate the effect of Sohlh2 on liver cancer growth.

Result

Experimental results indicated that Sohlh2 was highly expressed in normal human hepatocytes but was downregulated in HCC cells. Overexpression of Sohlh2 inhibited HCC cell proliferation by blocking the transition from the G0/G1 phase to the S phase, promoted apoptosis, suppressed the expression of HIF-1α and CD31, and consequently inhibited tumor angiogenesis. Conversely, downregulation produced the opposite effects. In vivo and in vitro mechanistic studies indicated that HIF-1α agonists reversed the anti-proliferative, pro-apoptotic, and anti-angiogenic effects induced by Sohlh2 overexpression in HCC. Conversely, the antagonist LW6 significantly mitigated the effects of Sohlh2 knockdown.

Conclusion

Our research suggested that that Sohlh2 exerted a tumor-suppressing gene function in hepatocellular carcinoma by inhibiting the HIF-1α/VEGFA signaling pathway. These findings provided a potential molecular target for the development of anti-angiogenic therapies in HCC.

Introduction

Hepatocellular carcinoma (HCC) was a common malignant tumor characterized by high mortality rates, especially in East Africa, the Asia-Pacific region, and China. More than 90 % of liver cancers were HCCs. HCC was a highly malignant tumor of the digestive system characterized by high morbidity, an insidious onset, and an extremely poor prognosis [1,2]. Previous research has indicated that uncontrolled cell growth and the formation of new blood vessels significantly influenced the proliferation, migration, dissemination, and metastasis of hepatocellular carcinoma (HCC) cells [3].

Angiogenesis was a multifactorial and multimodal complex process that played a significant role in the initiation, relapse, and metastasis of HCC [[4], [5], [6]]. Angiogenesis has recently emerged as a compelling therapeutic target for the treatment of hepatocellular carcinoma [7]. For example, Wei et al. found that CLIC1 promoted angiogenesis in hepatocellular carcinoma by regulating VEGFA [8]. CD151-enriched migrasomes facilitated hepatocellular carcinoma invasion by conditioning cancer cells and enhancing angiogenesis [9]. Furthermore, Wang Xuehao’s team at Nanjing Medical University discovered that hypoxia-induced, endothelial cell-specific DGKG overexpression promotes hepatocellular carcinoma angiogenesis and immune evasion through the ZEB2/TGF-β1 axis [10]. Current anti-angiogenic therapies primarily target tyrosine kinases, vascular endothelial growth factor receptors (VEGFRs), and platelet-derived growth factor receptors (PDGFRs). These therapies are considered effective strategies for treating HCC, particularly in advanced stages [11]. Additionally, Angiogenesis-targeting medications, such as sorafenib, have been extensively studied for their diverse mechanisms of action and clinical applications. Nevertheless, their adverse effects significantly impacted the quality of life of patients [12,13]. Therefore, there is an urgent need to identify novel anti-angiogenic agents for the prevention and treatment of hepatocellular carcinoma.

Spermatogenesis and oogenesis involve Basic helix-loop-helix transcription factor 2 (Sohlh2), a member of the basic helix-loop-helix transcription factor family that played a crucial role in regulating proliferation, migration, and differentiation during these processes. It achieved this by binding to the canonical E-box sequence (CANNTG) found in the promoters of target genes [14]. Sohlh2 exhibited elevated expression levels in normal human tissues, whereas its expression was reduced in various cancers, including breast cancer [14], ovarian cancer [15], and colon cancer [16], suggesting its potential role as a tumor suppressor gene. However, the expression and mechanism of action of Sohlh2 in HCC remained poorly understood. The impact of Sohlh2 on angiogenesis in tumor tissue remained uncertain. Only one study has documented the inhibitory effect of Sohlh2 on angiogenesis in breast cancer, primarily through the downregulation of hypoxia-inducible factor 1α (HIF-1α). The results of the luciferase reporter assay and chromatin immunoprecipitation assay confirmed that Sohlh2 repressed HIF-1α transcriptional activity by directly binding to the HIF-1α promoter. The opposite effect was observed when Sohlh2 was downregulated [17].

During tumor angiogenesis, the activation of HIF-1α triggered the production of abnormal vascular endothelial growth factor (VEGF), resulting in the formation of disorganized and permeable tumor vasculature [18]. Accumulating evidence has demonstrated that the suppression of HIF-1α inhibited angiogenesis in solid tumors and may serve as a potential prognostic indicator of poor outcomes in patients [[19], [20], [21]]. A recent study demonstrated that Sohlh2 reduced the malignancy of ovarian cancer cells under hypoxic conditions by negatively regulating the HIF-1α/CA9 axis [22]. Furthermore, Sohlh2 has been shown to reduce the expression of HIF-1α in breast cancer cells [14]. HIF-1α played a pivotal role as a transcription factor in regulating angiogenesis within the tumor microenvironment. The regulation of angiogenesis in HCC by Sohlh2 through HIF-1α has not been previously documented.

Hence, we conducted both in vitro and in vivo studies to elucidate the role of Sohlh2 in angiogenesis in hepatocellular carcinoma and to determine whether Sohlh2 regulated angiogenesis via the HIF-1α/VEGFA pathway.

Materials and methods

Cell culture

Human normal hepatocytes QSG7701 (C6746, Beyotime, CHN), human hepatocellular carcinoma cell lines SMMC-7721 (C6865, Beyotime, CHN) and Bel-7402 (STM-CL-5381, STEMCELL, CAN) were cultured in RPMI-1640 medium supplemented with 10 % fetal bovine serum (FBS). The human hepatocellular carcinoma cell line Hep3B (BNCC360312) was purchased from BeNa Culture Collection (Beijing, CHN) and cultured in complete EMEM medium (SNM-009D, SUNNCELL, CHN) supplemented with 10 % FBS. The human hepatocellular carcinoma cell line HepG2 (CL-0103) was purchased from Procell System and cultured in MEM medium containing non-essential amino acids (NEAA), supplemented with 10 % FBS and 1 % penicillin/streptomycin (P/S). The human hepatocellular carcinoma cell line Huh-7 (SNL-085) was purchased from SUNNCELL (CHN) and cultured in DMEM supplemented with 10 % FBS and 1 % P/S. HUVEC cells (STM-CL-5186, STEMCELL, CAN) were cultured in a specialized endothelial cell medium (CM-0122, Procell System, CHN). All cells were incubated in a 5 % CO₂ atmosphere at 37 °C.

Cellular transfection

Sohlh2-OE lentivirus, OE-NC lentivirus, Sohlh2-KD lentivirus, and KD-NC lentivirus were supplied by Shanghai GeneChem Co., Ltd. HEK293T cells were seeded in 10 cm culture dishes and maintained at 37 °C in a 5 % CO₂ atmosphere until reaching 70–80 % confluency prior to transfection. According to the RNAi Mate transfection reagent guidelines, HEK293T cells were transfected and then replenished with fresh complete culture medium 6 h after transfection. After an additional 48 h of cultivation, the cell supernatant containing lentiviral particles was harvested and then centrifuged at 200 × g for 30 min to concentrate and collect the lentivirus. Cells were cultured in a 24-well plate at a density of 1.0 × 10⁵ cells per well. Upon reaching a cell density of 70 % to 80 %, lentiviruses were added according to the desired multiplicity of infection. After 12 h of incubation, the cells were replenished with fresh, complete culture medium. After an additional incubation period of 72 h, Western blot and RT-qPCR were used to validate transfection efficiency.

Cellular group

To investigate the effects of Sohlh2 on HCC cell proliferation, cell cycle distribution, apoptosis, and angiogenesis, cells were divided into five groups: control group, Sohlh2 overexpression negative control group (OE-NC), Sohlh2 overexpression group (Sohlh2-OE), Sohlh2 knockdown negative control group (KD-NC), and Sohlh2 knockdown group (Sohlh2-KD). Hep3B and HepG2 cells were infected with lentiviruses at a multiplicity of infection (MOI) of 10. To preliminarily assess the effects of Sohlh2 expression levels (overexpression or knockdown) on the biological behaviors of HCC cells, corresponding negative control groups were established to exclude potential nonspecific effects of the lentiviral vector and transfection procedures.

To further investigate the molecular mechanism by which Sohlh2 regulated hepatocellular carcinoma proliferation, the HIF-1α/VEGFA pathway agonist CoCl₂ and the inhibitor LW6 were utilized. Cells were grouped as follows: KD-NC group, Sohlh2-KD group, Sohlh2-KD+LW6 group, OE-NC group, Sohlh2-OE group, and Sohlh2-OE+CoCl₂ group. Following lentiviral transfection, HepG2 and Hep3B cells in the Sohlh2-OE+CoCl₂ group were treated with 50 μM [23] CoCl₂ for 24 h, while cells in the Sohlh2-KD+LW6 group were treated with 80 μM [24] LW6 for 24 h. Cells or supernatants were collected for subsequent assay measurements.

RT-qPCR

Analysis of Sohlh2 mRNA expression in Hep3B and HepG2 cell lines was conducted using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Total RNA was extracted from the cells using TRIzol® reagent (9108Q, Takara, JPN) following the manufacturer's instructions. The ratio of absorbance at 260 nm to that at 280 nm (A260/A280) was maintained within the range of 1.8 to 2.0. For cDNA synthesis, reverse transcription was performed using PrimeScript RT Master Mix (RR036Q, Takara, JPN) following the manufacturer's instructions. qPCR analysis was performed using an ABI 7900HT system (ThermoFisher Scientific, USA) and SYBR® Green PCR Master Mix (RR820Q, Takara, JPN) according to the manufacturer's instructions. The thermocycling protocol consisted of an initial denaturation at 95 °C for 30 s for one cycle, followed by 40 cycles of denaturation at 94 °C for 5 s and annealing at 60 °C for 34 s. The final step included an extension at 72 °C for 10 min. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the normalization control, and the relative expression levels of Sohlh2 mRNA were determined using the 2^-ΔΔCT method. The primer amplification efficiency was determined using the standard curve method. The average amplification efficiencies for the Sohlh2 and GAPDH primers were 95 % and 98 %, respectively, both within the acceptable range of 95 % to 105 %. All experiments complied with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. The primer sequences utilized in the study were as follows: Sohlh2 forward, 5′-GCTTCCTCAATTATCTGCCAGG-3′ and reverse, 5′-GCACTTAGTGAAGAAGGCACC-3′; and GAPDH forward, 5′-GGAGCGAGATCCCTCCAAA AT-3′ and reverse, 5′-GGCTGTTGTCATAC TTCTCATGG-3′.

CCK8

To evaluate the effects of lentiviral transfection on tumor cells, CCK8 assays were conducted to assess the viability of Hep3B and HepG2 cells 48 h after transfection. Briefly, cultured cells were seeded at a density of 5,000 cells per well in a 96-well plate. After 48 h of culture, 10 μL of CCK8 reagent (CK04, Dojindo, JPN) was added to each well, followed by incubation at 37 °C for 2 h. Optical density (OD) at 450 nm was measured using a microplate reader (1681130, Bio-Rad, USA).

Flow cytometry

Apoptosis and cell cycle distribution were evaluated using flow cytometry. For the assessment of cell apoptosis, a total of 2 × 10⁵ cells were seeded per well in a 6-well plate and incubated for 48 h. Subsequently, the cells were suspended in binding buffer and stained with Annexin V-FITC and propidium iodide (PI) (P-CA-201, Pricella, CHN) under dark conditions. Apoptotic cells were assessed using a flow cytometer (FACSDiscoverA8, BD Biosciences, USA). For cell cycle analysis, the cell cycle detection kit (CCS012, Multi sciences, CHN) was used. The cell suspension was harvested and centrifuged to remove the supernatant. The cells were fixed overnight at 4 °C in 500 μL of cold 70 % ethanol. The fixed solution was washed with phosphate-buffered saline (PBS) before being incubated with 500 μL PI/RNase A staining solution. Subsequently, it was incubated in darkness at room temperature for 60 min. Cell cycle distribution was analyzed using flow cytometry with an excitation wavelength of 488 nm.

Tube formation assay

To assess angiogenic capability, HUVECs were incubated in conditioned medium for the tube formation assay. Matrigel (356234, BD Biosciences, USA) was thawed overnight at 4 °C, and then 50 μL of diluted Matrigel was dispensed into each well of a 96-well plate. The plate was then placed in a 37 °C incubator for 30 min. Subsequently, HUVECs (3 × 10⁵ cells/well) were seeded and cultured with the supernatant from Hep3B and HepG2 cells in each group. The cells were then incubated for 4 h, followed by observation and photography of HUVEC angiogenesis using a light microscope.

ELISA assay

The levels of vascular endothelial growth factor A (VEGFA, PV963, Beyotime, CHN) and angiopoietin-1 (Ang-1, PA033, Beyotime, CHN) in the cell culture supernatant were quantified using ELISA kits according to the manufacturers' instructions.

Cellular immunofluorescence

Immunofluorescence analysis was performed to assess the expression of HIF-1α in Hep3B and HepG2 cells. Cells were fixed with a 4 % paraformaldehyde solution for 10 min, permeabilized with 0.5 % Triton X-100 for 15 min, and blocked with 5 % BSA for 1 h. They were then incubated with an anti-HIF-1α antibody (1:100, ab308433, Abcam, UK, RRID: AB_2941086) overnight at 4 °C, followed by incubation with Cy3-labeled goat anti-rabbit IgG (H+L) (1:500, A0516, Beyotime, CHN, RRID: AB_2893015) for 1 h at room temperature in the dark. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; C1002, Beyotime, CHN) for 5 min. Images were captured using an IX71 fluorescence microscope (Olympus, JPN).

Construction of a xenograft tumor model

A subcutaneous xenograft tumor model was established in male nude mice aged 4-6 weeks, obtained from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (License Number: SCXK(Lu) 20220006). Mice were maintained in a controlled environment free of specific pathogens (SPF conditions). All animal care protocols and experiments were conducted in strict accordance with the guidelines established by the China Council on Animal Care and Use. Additionally, these procedures were approved by the ethics committee of Yantaishan Hospital (Ethic number: No.2024001).

Male nude mice were randomly assigned to five groups using a random number table method, with five mice per group to ensure balanced baseline characteristics: the Model group, Sohlh2-KD group, Sohlh2-OE group, Sohlh2-KD+LW6 group (HIF-1α antagonist), and Sohlh2-OE+CoCl₂ group (HIF-1α agonist). Hep3B cells (0.2 mL, 2 × 10⁷ cells/mL) were subcutaneously inoculated into the right flank of mice to establish the tumor model. 20 mg/kg [24] LW6 and 60 mg/kg [25] CoCl₂ were into injected i.p. into the mice every three days. The tumor volume was measured weekly and computed using the following formula. After one month, the nude mice were anesthetized using 3 % pentobarbital sodium (45 mg/kg) and euthanized through cervical dislocation. Tumor tissues were excised and their weight was measured. The tumors were either fixed in 4 % paraformaldehyde or stored in a -80 °C freezer. During tumor volume measurement, weighing, and tissue analysis, the researchers were blinded to group assignments to prevent subjective bias.

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Immunofluorescence staining of tissues

Tumor tissue was dehydrated, embedded in paraffin, and sectioned into 3 μm slices. The paraffin sections underwent dewaxing, rehydration, antigen retrieval, and were subsequently sealed with a blocking solution. Subsequently, the samples were incubated overnight with the primary antibody at 4 °C, followed by a 1 h incubation with the Cy3-labeled goat anti-rabbit IgG (H+L) (1:200, A0516, Beyotime, CHN, RRID: AB_2893015). Subsequently, the tissue slices were stained with DAPI and sealed using neutral resin. Finally, the specimens were observed and photographed using a confocal microscope at 200 × magnification. The primary antibodies used in the study included CD31 (28083-1-AP, Proteintech, 1:200, RRID: AB_2881055) and HIF-1α (1:200, AF1009, Affinity, UK, RRID: AB_2835328).

Western blot

Cells and tissues were lysed using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (P0013C, Beyotime, CHN). The protein concentrations were assessed utilizing a bicinchoninic acid kit (P0009, Beyotime, China). Equal amounts of protein samples were separated by 10 % SDS-PAGE, transferred onto polyvinylidene difluoride membranes (HVLP14250, Millipore, USA), and subsequently obstructed using 5 % nonfat dried milk. The membranes were subjected to overnight incubation at 4 °C with the respective primary antibodies: Sohlh2 antibody (1: 500, ab101402, Abcam, UK, RRID: AB_10672266), HIF-1α (1:1000, ab308433, Abcam, UK, RRID: AB_2941086), anti-VEGFA (1:2000, 19003-1-AP, Proteintech, USA, RRID: AB_2212657), and GAPDH (1:3000, AF7021, Affinity, CHA, RRID: AB_2839421). On the subsequent day, the membranes underwent incubation with HRP-labeled Goat Anti-Rabbit IgG(H+L) (1:1000, A0208, Beyotime, CHN, RRID: AB_2892644). Finally, the blots were visualized utilizing enhanced chemiluminescence reagents (P0018S, Beyotime, CHN) and a Tanon 5200 multi-imager (Tanon Science & Technology, CHN). Quantify gray values using ImageJ software and perform band normalization. Using the gray value of the GAPDH band as a reference, normalize the gray value of the target protein band by dividing it by the corresponding gray value of the GAPDH band. This process eliminated the effects of variations in sample loading amounts.

Statistical analysis

Statistical analyses were performed utilizing SPSS version 20.0 (IBM, Armonk, USA) for the Windows operating system. Statistical comparisons among the groups were conducted utilizing one-way analysis of variance (ANOVA). Multiple comparisons were conducted utilizing the Tukey test. All data are presented as mean ± standard deviation (SD), and statistical significance was considered at a level of p<0.05. All experiments were conducted three times to ensure reproducibility.

Result

Differential expression of Sohlh2 and successful transfection with lentivirus

In this study, we first examined the expression of Sohlh2 in normal hepatocytes (QSG 7701) and in five human hepatocellular carcinoma cell lines. Western blot results (Fig. 1A) revealed that Sohlh2 was highly expressed in QSG 7701 cells but showed lower expression in hepatocellular carcinomas. Considering subsequent knockdown experiments, HepG2 and Hep3B cells, which exhibited relatively higher Sohlh2 expression, were selected. HepG2 and Hep3B cells were transfected with lentiviruses to either overexpress or knock down Sohlh2. Western blot (Fig. 1B-C) and RT-qPCR (Fig. 1D-E) results demonstrated that Sohlh2 expression was significantly upregulated following transfection with the Sohlh2-overexpressing lentivirus. Conversely, Sohlh2 expression was significantly downregulated following transfection with the Sohlh2 knockdown lentivirus. These findings confirm the successful transfection of both cell lines with Sohlh2-OE and Sohlh2-KD lentiviruses.

Fig. 1.

Sohlh2 expression differences in normal cells and liver cancer cells, as well as lentiviral transfection identification. (A) The differential expression of Sohlh2 in normal human cells QSG 7701 and human HCC cells Hep3B, HepG2, Huh-7, SMMC-7721, and Bel7402. (B-C) Western blot and (D-E) RT-qPCR were used to determine the expression of Sohlh2 in HepG2 and Hep3B cells after lentivirus transfection. Data were presented as the mean ± standard deviation, and differences between groups were analyzed using one-way analysis of variance, N = 3.

Overexpression of Sohlh2 inhibited the proliferation and promoted the apoptosis of HCC cells

The impact of Sohlh2 on the proliferation of HCC cell lines HepG2 (Fig. 2A) and Hep3B (Fig. 2B) was analyzed using CCK-8 assays. The results indicated that overexpression of Sohlh2 significantly reduced the proliferation rate of cells compared to the control and OE-NC group, whereas knockdown of Sohlh2 significantly increased the proliferation rate compared to the control and KD-NC group. These findings suggested that Sohlh2 overexpression inhibited the proliferation of HepG2 and Hep3B cells. The cell cycle experiment results demonstrated that Sohlh2 overexpression inhibited the transition of HepG2 (Fig. 2C and E) and Hep3B (Fig. 2D and F) cells from the G0/G1 phase to the S phase, whereas Sohlh2 knockdown increased the percentage of cells in the S phase. Apoptosis in HepG2 (Fig. 2G) and Hep3B (Fig. 2H) cells was assessed using flow cytometry. Sohlh2 overexpression increased apoptosis rates in both cell types, whereas Sohlh2 knockdown decreased apoptosis rates in both.

Fig. 2.

The overexpression of Sohlh2 inhibited the proliferation and the proportion of cells in the S phase, promoting apoptosis in HCC cells. (A-B) CCK8 for cell proliferation. Flow cytometry was applied for cell cycle analysis in HepG2(C, E) and Hep3B (D, F). Flow cytometry was used to detect apoptosis in HepG2 (G) and Hep3B cells (H). Data were presented as the mean ± standard deviation, and differences between groups were analyzed using one-way analysis of variance, N = 3.

Overexpression of Sohlh2 inhibited the vascular formation ability of HUVEC cells

The regulatory role of Sohlh2 in angiogenesis was analyzed through a vascular formation experiment, with representative images shown in Fig. 3A and Fig. 3B. After analyzing the number of junctions and total segment length, it was found that the Sohlh2-KD group had a higher number of junctions and total segment length compared to the KD-NC group and the Control group. Conversely, the Sohlh2-OE group showed a decrease in these parameters compared to the OE-NC group and the Control group. We further analyzed the secretion of the pro-angiogenic factors VEGFA (Fig. 3C-D) and Ang-1 (Fig. 3E-F) in HepG2 and Hep3B cells. Knocking down Sohlh2 increased the levels of VEGFA and Ang-1 in the cell culture supernatant, while overexpression of Sohlh2 reduced their levels.

Fig. 3.

The overexpression of Sohlh2 inhibited the vascular formation ability of HUVEC cells. (A-B) Tube formation assay. HUVEC cells were cultured with the conditioned medium which was the supernatant of HepG2 and Hep3B cells. ELISA methods were performed to detected the VEGFA (C-D) and Ang-1 (E-F) in the supernatant of HepG2 and Hep3B cells. Magnification: 200 ×; Scale bar: 50 μm. Data were presented as the mean ± standard deviation, and differences between groups were analyzed using one-way analysis of variance, N = 3.

We also assessed the expression of HIF-1α (Fig. 4A-B) and CD31 (Fig. 4C-D) in HepG2 and Hep3B cells using immunofluorescence detection. In cells overexpressing Sohlh2, the fluorescence intensities of HIF-1α and CD31 decreased compared to those in the control and OE-NC groups. Conversely, in cells with low Sohlh2 expression, the fluorescence intensity of these factors increased significantly compared to the control and KD-NC groups. Additionally, Western blot analysis of HIF-1α and VEGFA protein expression in HepG2 (Fig. 5A) and Hep3B (Fig. 5B) cells revealed that, compared to the control and OE-NC groups, overexpression of Sohlh2 downregulated HIF-1α and VEGFA protein levels, whereas Sohlh2 knockdown produced the opposite effect. In conclusion, a significant finding emerged: overexpression of Sohlh2 inhibited the secretion of angiogenic factors in HepG2 and Hep3B cells, thereby suppressing the vascular formation capacity of HUVEC cells.

Fig. 4.

The downregulation of the HIF-1α/VEGF pathway in Sohlh2-overexpressing HepG2 and Hep3B cells. Immunofluorescence experiments detected the expression of HIF-1α (A-B) and CD31 (C-D) in HepG2 and Hep3B cells. Magnification: 200 ×; Scale bar: 50 μm. Data were presented as the mean ± standard deviation, and differences between groups were analyzed using one-way analysis of variance, N = 3.

Fig. 5.

The downregulation of the HIF-1α/VEGF pathway in Sohlh2-overexpressing HepG2 and Hep3B cells. HIF-1α and VEGFA proteins in HepG2 (A) and Hep3B (B) cells were detected by western blot. Image J software was applied to analyze the grayscale value of protein bands. Data were presented as the mean ± standard deviation, and differences between groups were analyzed using one-way analysis of variance, N = 3.

In vitro mechanism validation

The effects of Sohlh2 on proliferation and angiogenesis in HepG2 and Hep3B cells have been confirmed. Further mechanistic studies were conducted using the HIF-1α antagonist LW6 and the HIF-1α agonist CoCl₂. The proliferative capacity of HepG2 (Fig. 6A) and Hep3B (Fig. 6B) cells was evaluated. The addition of CoCl₂ and LW6 reversed the antiproliferative effect of Sohlh2 overexpression and the proliferative effect of Sohlh2-KD, respectively. Additionally, the tube-forming capacity of HUVECs was evaluated (Fig.6C-D). Conditioned media collected from HepG2 and Hep3B cells were used to culture HUVECs. It was found that incubation with Sohlh2-OE cell supernatant impaired HUVEC tube formation, whereas the addition of CoCl₂ enhanced this ability. Similarly, incubation with supernatant from Sohlh2-knockdown cells increased HUVEC tube formation, whereas LW6 inhibited this angiogenic capacity.

Fig. 6.

Overexpression Sohlh2 inhibited cell proliferation and angiogenesis of HCC through the downregulation of the HIF-1α/VEGF pathway. (A-B) CCK8 assay of HepG2 and Hep3B cells. (C-D) Angiogenesis was evaluated using tube formation assay. Immunofluorescence experiments detected the expression of HIF-1α (E-F) and CD31 (G-H) in HepG2 and Hep3B cells. Western blot was used to determine the HIF-1α and VEGFA proteins in HepG2 (I) and Hep3B (J) cells. The protein bands were analyzed by Image J software. Magnification: 200 ×; Scale bar: 50 μm. Data were presented as the mean ± standard deviation, and differences between groups were analyzed using one-way analysis of variance, N=3.

In HepG2 and Hep3B cells, the expression levels of HIF-1α (Fig. 6E–F) and CD31 (Fig. 6G–H) were analyzed using immunofluorescence staining. The results showed that, compared with the OE-NC and KD-NC group, the expression of HIF-1α and CD31 decreased in the Sohlh2-OE group, whereas their expression increased in the Sohlh2-KD group. Treatment with CoCl₂ increased HIF-1α and CD31 expression in Sohlh2-OE cells, whereas treatment with LW6 decreased their expression in Sohlh2-KD cells. This suggested that Sohlh2 overexpression suppressed HIF-1α and CD31 production, both of which was related to HIF-1α. We also conducted Western blot analysis to examine the protein expression of HIF-1α and its downstream target VEGFA in HepG2 (Fig. 6I) and Hep3B (Fig. 6J) cells in response to Sohlh2. The Western blot results were consistent with the immunofluorescence findings. Sohlh2-KD upregulated the HIF-1α/VEGFA pathway, increasing the protein expression of HIF-1α and VEGFA, whereas LW6 inhibited this upregulation. Sohlh2-OE downregulated the HIF-1α/VEGFA pathway by decreasing the protein expression levels of HIF-1α and VEGFA. Treatment with CoCl₂ reversed the downregulation induced by Sohlh2-OE.

Results of Animal Experiments

A Hep3B xenograft nude mouse model was established, and tumor images for each group are shown in Fig. 7A. Throughout the experiment, tumor volume (Fig. 7B) and the body weight of nude mice (Fig. 7C) were measured weekly. Compared to the control group, tumor volume significantly increased in the Sohlh2-KD group and significantly decreased in the Sohlh2-OE group. Compared to the Sohlh2-OE group, the Sohlh2-OE+CoCl₂ group exhibited a significantly increased tumor volume. Conversely, the Sohlh2-KD+LW6 group exhibited a significantly reduced tumor volume compared to the Sohlh2-KD group. At the end of the experiment, tumor weight was measured (Fig. 6D), and the results were consistent with the observed tumor volume trends. Results indicated that Sohlh2-KD promoted tumor growth, whereas Sohlh2 overexpression inhibited tumor growth.

Fig. 7.

Sohlh2 inhibited the growth of Hep3B tumors in xenograft nude mice. (A) Tumor images. (B) Tumor size. (C) Mice weight. (D) Tumor weight. Immunofluorescence detection of CD31 (E) and HIF-1α (F) expression in tumor tissues. (G) Some proteins (Sohlh2, HIF-1α, VEGFA) in tumor tissues were measured by Western blot. These proteins were quantified by Image J software. Magnification: 200 ×; Scale bar: 50 μm. Data were presented as the mean ± standard deviation, and differences between groups were analyzed using one-way analysis of variance, N = 5.

Next, we evaluated the expression of factors related to angiogenesis. Initially, we assessed the expression of HIF-1α (Fig. 7E) and CD31 (Fig. 7F) in tumor tissues using immunofluorescence staining. We observed a significant decrease in the fluorescence intensity of HIF-1α and CD31 in Sohlh2-overexpressing tumor tissues compared to the control group, whereas the fluorescence intensity of HIF-1α and CD31 significantly increased in Sohlh2-knockdown tumor tissues. When Sohlh2-OE mice were treated with the HIF-1α agonist CoCl₂, the fluorescence intensity of both markers significantly increased compared to untreated mice. Conversely, Sohlh2-KD mice treated with the HIF-1α antagonist LW6 exhibited a significant decrease in the fluorescence intensity of these markers relative to untreated mice.

Additionally, we quantitatively analyzed the protein expression of HIF-1α and its downstream molecule VEGFA in tumor tissues using Western blotting (Fig. 7G). The Western blot results were consistent with those obtained from immunofluorescence analysis. Sohlh2-KD upregulated the expression of HIF-1α and VEGFA proteins, whereas Sohlh2 overexpression downregulated their expression. Moreover, the levels of HIF-1α and VEGFA proteins increased and decreased, respectively, in the Sohlh2-OE+CoCl₂ group and the Sohlh2-KD+LW6 group compared to the Sohlh2-OE and Sohlh2-KD groups, showing significant differences.

We analyzed the expression of the Sohlh2 protein in tumor tissues and found that Sohlh2-OE mice exhibited the highest expression levels, whereas Sohlh2-KD mice showed the lowest. However, after treatment with CoCl₂ and LW6, significant changes were observed in the expression levels of the Sohlh2 protein in both groups, with decreased following CoCl₂ treatment and increases following LW6 treatment.

Discussion

With the advancement of precision medicine and molecular biology, targeted therapy has become an important approach in cancer treatment. Targeted therapy aims to block specific signaling pathways or related proteins involved in tumor growth and progression, specifically targeting molecular targets that are overexpressed or mutated in tumor tissues [26]. Currently, vascular endothelial growth factor (vascular endothelial growth factor, VEGF) [27], mammalian target of rapamycin (mammalian target of rapamycin, mTOR) [28], platelet-derived growth factor receptor (PDGFRa) [29], Transforming growth factor alpha (TGF-α) [30], epidermal growth factor receptor (EGFR) [31], among others, have been demonstrated to be effective targets for the treatment of HCC. In addition, we are currently exploring novel molecular targets to provide additional possibilities for the treatment of liver cancer.

Sohlh2 has been studied in various cancers, including breast cancer [32], renal cancer [33], colon cancer [16], ovarian cancer [34], and multiple myeloma [35]. Research has found that Sohlh2 was highly expressed in normal tissues but was expressed at low levels in tumor tissues of triple-negative breast cancer, ovarian cancer, renal cell carcinoma, and other cancers. Sohlh2 exhibited inhibitory effects on cancer metastasis, angiogenesis, and tumor cell proliferation. A study indicated that Sohlh2 suppressed the invasion and metastasis of human ovarian cancer cells by inhibiting MMP9 transcription. Furthermore, Sohlh2 can suppress the growth of breast cancer cells by inhibiting the Wnt/β-catenin signaling pathway. It was noteworthy that Sohlh2 can also inhibit breast cancer angiogenesis by downregulating HIF-1α expression. However, to date, no studies have elucidated the role of Sohlh2 in HCC. Therefore, our research focused on the expression of Sohlh2 in HCC and investigated whether Sohlh2 influenced proliferation, apoptosis, cell cycle distribution, and angiogenesis in HCC. This study found that Sohlh2 expression levels were higher in normal human hepatocytes (QSG 7701) than in human hepatocellular carcinoma cells. These findings are consistent with reports of Sohlh2 expression in various other cancer types. Subsequently, the study focused on investigating the role of Sohlh2 in the proliferation, apoptosis, and angiogenesis of HCC. Observations revealed that Sohlh2 overexpression suppressed hepatocellular carcinoma cell proliferation and angiogenesis, promoted apoptosis, and inhibited the transition from the G0/G1 phase to the S phase of the cell cycle. Conversely, underexpression of Sohlh2 produced opposite effects in hepatocellular carcinoma. These findings confirm that Sohlh2 functions as a tumor suppressor in HCC.

Blood vessels were the most extensively distributed tissues in the human body. On one hand, blood vessels transported oxygen, nutrients, and metabolic waste; on the other hand, endothelial cells (ECs) within these vessels regulated the stability of the tissue microenvironment, playing a crucial role in both normal tissue development and the onset and progression of diseases such as tumors. In tumors, angiogenesis was one of the fourteen hallmarks of cancer [36]. As tumors growed and exceeded 2 mm in diameter, internal hypoxia became more severe. This stimulated tumor cells to secrete pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which prompted endothelial cells to form new blood vessels. These vessels supplied oxygen and nutrients to the tumor while also providing pathways for invasion and metastasis [37].

Hypoxia-inducible factor (HIF) was a critical molecule that senses the hypoxic microenvironment and regulates cell proliferation, differentiation, apoptosis, and metabolic activity through downstream mechanisms. Consequently, it played a key role in embryonic development, angiogenesis, and various physiological and pathological processes [38]. In tumors, the degree of hypoxia increased with tumor growth. The hypoxic microenvironment inhibited the degradation of HIF in tumor cells, inflammatory cells, and endothelial cells via the ubiquitin-proteasome pathway, thereby maintaining HIF stability within these cells [38,39]. The elevated HIF protein entered the cell nucleus to exert transcriptional regulatory effects, upregulating a series of factors that promoted angiogenesis, such as VEGF and FGF. These cytokines bound to their corresponding receptors on ECs, promoting EC activation and the formation of new blood vessels [40,41]. Studies have shown that blocking HIF-1α in tumors can downregulate angiogenesis-related molecules such as VEGF, resulting in reduced tumor angiogenesis, slowed tumor progression, and enhanced efficacy of immunotherapy. Conversely, the accumulation of HIF in tumors can upregulate VEGF expression, thereby promoting tumor angiogenesis [42]. Research has shown that the HIF-1α protein is highly expressed in HCC and liver cirrhosis tissues, primarily influenced by hypoxic conditions. The expression of the HIF-1α protein is associated with tumor differentiation as well as intrahepatic and extrahepatic metastases [43]. Numerous studies have reported the significant role of the HIF-1α/VEGFA pathway in promoting angiogenesis in HCC. Research has shown that dexmedetomidine can upregulate the expression of HIF-1α and VEGFA, thereby increasing the number of vasculogenic mimicry (VM) channels and microvessel density (MVD) [44]. Conversely, dandelion polysaccharides downregulate the expression of VEGF and HIF-1α, thereby inhibiting angiogenesis in liver cancer [45].

Angiogenesis, a central driving force behind tumor initiation, progression, and metastasis, created a malignant feedback loop with the hypoxic microenvironment in HCC. However, current targeted therapeutic strategies often encounter the significant challenge of drug resistance. Sohlh2, a member of the bHLH transcription factor family, has been shown to possess tumor-suppressive activity in various malignancies. Specifically, it has been confirmed to exert anti-tumor effects by inhibiting angiogenesis in breast cancer models. However, the role of this molecule in regulating angiogenesis in HCC remained unclear, leaving a critical gap in the research. At the molecular level, VEGFA, as the initiating factor of angiogenesis, directly drived the proliferation and migration of endothelial cells to form primitive vascular buds. ANGPT1 was responsible for vascular maturation and stabilization, ensuring the transformation of newly formed blood vessels into functional vascular networks [46] These two molecules were functionally complementary and synergistically regulated the entire angiogenesis process. Based on this, the present study specifically focused on elucidating the regulatory role of Sohlh2 in HCC angiogenesis, selecting VEGFA and ANGPT1 as downstream effector molecules involved in critical stages of angiogenesis. This study aimed to address the research gap regarding Sohlh2 in the context of HCC angiogenesis and to provide experimental evidence elucidating its mechanism of action.

A more in-depth investigation was conducted to explore the potential mechanisms underlying Sohlh2. The relationship between Sohlh2 and the HIF-1α/VEGF pathway was primarily investigated. Previous studies have indicated that Sohlh2 inhibits angiogenesis in triple-negative breast cancer by downregulating HIF-1α [17]. Therefore, it was hypothesized that Sohlh2 could suppress the progression of liver cancer by downregulating the HIF-1α/VEGF pathway. To address this, our study employed the HIF-1α agonist CoCl₂ and the inhibitor LW6 in the context of Sohlh2 overexpression or knockout. Upon treatment with CoCl₂, overexpression of Sohlh2 in HepG2 and Hep3B cells led to a reduced inhibitory effect on cell proliferation and angiogenesis. Conversely, following treatment with LW6, knockdown of Sohlh2 suppressed its promoting effects on cell proliferation and angiogenesis. Our experimental findings confirm the involvement of Sohlh2 in liver cancer via the HIF-1α/VEGF pathway. Sohlh2 inhibits liver cancer cell proliferation and cell cycle progression by downregulating the HIF-1α/VEGF pathway.

Recent single-cell RNA sequencing studies have revealed significant heterogeneity in HIF-1α expression within HCC tissues, offering new insights into the complex regulatory mechanisms of Sohlh2 [47,48]. Studies indicate that HIF-1α expression is not uniformly distributed but is specifically enriched in hypoxic tumor cells, tumor-associated endothelial cells, and macrophage subpopulations. Distinct downstream target activation patterns are observed across these different cellular subpopulations. Notably, in HCC patients with elevated cancer stem cell (CSC) levels, HIF1A regulates the increased expression of matrix metalloproteinases (MMP7, MMP9, MMP12) in SPP1⁺ macrophages, indicating a close association between CSCs and SPP1⁺ macrophages within hypoxic tumor regions. Their synergistic action is closely associated with poor patient prognosis and a suboptimal response to immunotherapy [49]. This study demonstrates that Sohlh2 broadly downregulates HIF-1α and VEGFA expression at both the cellular and tissue levels; however, the specific regulatory mechanisms of Sohlh2 on HIF-1α in different cell subpopulations remain to be elucidated. Future studies employing single-cell sequencing technology to thoroughly analyze the expression patterns of Sohlh2 across different HCC cell subpopulations and its regulatory role in the HIF-1α/VEGFA pathway will provide critical evidence for elucidating the cell-specific mechanisms by which Sohlh2 inhibits HCC angiogenesis.

Although this study has produced valuable findings, it is important to address the limitations of the experimental models and techniques employed. First, the subcutaneous xenograft model used in this study offers several advantages, including ease of operation, quantifiable tumor growth, and high reproducibility, thereby effectively validating the in vivo antitumor effects of Sohlh2. However, this model cannot replicate the liver-specific microenvironment that contributes to the development of clinical HCC. Furthermore, the blood supply pattern of subcutaneous tumors differs from that of clinical HCC, potentially causing discrepancies between the observed tumor growth inhibition and angiogenesis changes in this study and their clinical relevance. Future research should utilize patient-derived xenograft (PDX) models or transgenic HCC models to validate the specificity and stability of Sohlh2 regulation in a more clinically relevant context. Second, this study employed lentiviral shRNA to establish a Sohlh2 knockdown model. Although this technique is widely used, off-target effects remain a potential risk. shRNA may bind to non-target mRNAs through complete or partial complementarity, resulting in non-specific gene silencing. Future validation of these findings will be conducted using the more precise CRISPR-Cas9 technology to generate Sohlh2 knockout cell lines. Additionally, cirrhosis is a critical precancerous condition in the development of HCC. Due to limitations in obtaining clinical samples, this study was unable to determine Sohlh2 expression levels in cirrhotic tissues. Future research using paired samples of normal liver, cirrhotic liver, and HCC tissues will help clarify the expression pattern of Sohlh2 during HCC development and assess its potential as an early diagnostic biomarker.

In summary, this study confirms that Sohlh2 plays a crucial tumor-suppressive role by significantly inhibiting hepatocellular carcinoma cell proliferation and angiogenesis through targeted suppression of the HIF-1α/VEGFA signaling pathway. Moving forward, the research team will conduct in-depth investigations to address the limitations of the current study, thereby providing essential theoretical foundations and practical support to advance the clinical translation of Sohlh2-targeting strategies.

Funding

This study was surpported by the Yantai Science and Technology Plan Project (2022YD050).

CRediT authorship contribution statement

Qing Liu: Writing – review & editing, Writing – original draft, Software, Methodology, Investigation, Data curation. Yongming Zhang: Writing – review & editing, Validation, Methodology, Data curation. Yanfang Wu: Software, Investigation, Data curation. Sen Chen: Software, Methodology, Investigation, Data curation. Jing Wang: Software, Data curation.

Declaration of competing interest

The author declared no conflict of interest.