Targeting ESM1 via SOX4 promotes the progression of infantile hemangioma through the PI3K/AKT signaling pathway

doi:10.21203/rs.3.rs-4169901/v1

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Targeting ESM1 via SOX4 promotes the progression of infantile hemangioma through the PI3K/AKT signaling pathway

https://doi.org/10.21203/rs.3.rs-4169901/v1

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Background

Infantile hemangioma (IH) is the most prevalent benign vascular tumour in children, yet its pathogenesis remains incompletely understood. Research has established a strong association between SOX4 and tumour blood vessel formation. However, the specific role of SOX4 in IH progression has not been clearly defined. The objective of this study was to investigate the function and underlying mechanism of SOX4 in IH development, with the aim of identifying novel therapeutic targets and facilitating drug development.

Methods

The transcription factor SOX4, which is associated with IH, was identified through RNA-seq screening of IH microtumours and validated in IH tissue samples. Cell experiments were conducted to investigate the impact of SOX4 on the biological behavior of CD31+ HemECs and the PI3K/AKT pathway. Furthermore, RNA-seq analysis was performed on CD31+ HemECs exhibiting low levels of SOX4, leading to the identification of the downstream gene ESM1, which is regulated by SOX4. The targeting relationship between SOX4 and ESM1 was confirmed through database predictions and ChIP-PCR assays. Finally, the influence of the SOX4-ESM1 signaling axis on tumour growth was further elucidated through 3D microtumour models and tumour formation experiments in nude mice.

Results

During the proliferating phase, SOX4 was highly expressed in IH tissue samples and was shown to enhance the proliferation, migration, and angiogenesis of CD31+ HemECs in vitro. SOX4 was observed to bind to the promoter of ESM1, thereby transcriptionally upregulating the ESM1-mediated PI3K/AKT signaling pathway and ultimately promoting the progression of IH. The pro-proliferative effect of SOX4 on CD31+ HemECs was dependent on the expression of ESM1. Through IH 3D microtumour and in vitro animal experiments, it was demonstrated that both SOX4 and ESM1 are tumourigenic genes that independently promote tumour progression and that tumour growth could be partially reversed by knocking down SOX4 and overexpressing ESM1.

Conclusions

SOX4 plays a crucial role in the progression of IH, and the SOX4/ESM1 axis may serve as a novel biomarker and potential therapeutic target for IH.

Infantile hemangioma

CD31 + HemECs

SOX4

ESM1

Proliferation

Angiogenesis

Infantile hemangioma (IH) is the most common benign vascular tumour in children, with an incidence of approximately 4%-10% [1, 2]. The prevalence is greater in premature infants and low birth weight infants, with a male-to-female incidence ratio of approximately 1:3 [3, 4]. While 90% of IH cases resolve spontaneously, a small percentage may result in permanent pigmentation, fibrous tissue accumulation, and scarring, impacting the aesthetic appearance of children [5]. Some IH cases may be associated with bleeding, pain, infection, and ulcers, which significantly affect the quality of life of affected children [6, 7]. IH occurring in specific locations can lead to severe complications such as organ failure, visual impairment, restricted joint movement, breathing difficulties, and even death [8–10]. Furthermore, IH can have adverse effects on the mental well-being of children and their families [11, 12].

There are notable structural and physiological differences between 2D cell cultures and in vivo cells. Moreover, cell suspension implantated lesions often regress quickly in nude mice, limiting the understanding of IH pathophysiology and progression [13]. Therefore, establishing stable, dependable, and standardized models is crucial for elucidating the detailed mechanisms underlying IH onset and development, identifying new therapeutic targets, and developing novel treatments. In our previous research, we utilized decellularized extracellular matrix from porcine aortas to create a decellularized extracellular matrix (dECM) micropattern chip for constructing a three-dimensional (3D) microtumour model of CD31 + hemangioma-derived endothelial cells (HemECs). This approach allowed precise control of the size and arrangement of cell clusters, enhancing the authenticity and stability of drug screening and mechanistic exploration [14]. Using this model, we conducted RNA-seq analysis of 3D microtumours and 2D planar CD31 + HemECs. We then performed a joint analysis of the upregulated differentially expressed genes in these cellular models, as well as those identified in our previous RNA-seq results from IH tissues during the proliferating and involuting phase [15]. Through this analysis, we identified the gene SOX4, which may be closely associated with the progression of IH.

SOX4, a member of the SOX gene family, is a crucial and highly conserved transcription factor that encodes an approximately 47 kDa protein. It features a highly conserved high mobility box DNA-binding domain with evolutionary conservation, playing a significant role in stable stem cell differentiation, cartilage differentiation, and progenitor cell development [16]. SOX4 is frequently implicated as a retrovirus integration site, leading to mRNA elevation [17]. Research has shown that SOX4 is involved in the differentiation, proliferation, migration, apoptosis, and epithelial-mesenchymal transition (EMT) of tumour cells [18]. Recent studies have highlighted the elevated expression of SOX4 in various malignant tumours, such as glioblastoma, prostate cancer, bladder cancer, liver cancer, lung cancer, and breast cancer, suggesting its potential role in promoting intratumoral neovascularization and tumour progression [19–24]. Numerous studies have demonstrated that SOX4 can modulate several developmental signaling pathways, including the PI3K/AKT, Wnt, and TGF-β pathways, among others, thereby influencing disease progression [25–29]. This finding implies that SOX4 may play a pivotal role in tumour angiogenesis. Despite extensive research on SOX4 in various cancers, studies focusing on SOX4 in IH are limited. The mechanism of action and the transcriptional regulatory network of SOX4 in IH remain unclear. Further exploration is needed to identify the downstream target genes regulated by SOX4 and the signaling pathways involved. Therefore, our study aimed to investigate the impact of SOX4 on IH progression through experiments involving IH tissue samples, 2D planar cells, 3D microtumour models, and animal tumour formation. By elucidating the downstream molecular mechanisms of SOX4 in regulating IH development, we aimed to provide novel targets and insights for the treatment of IH.

Patients and samples

This study was conducted with the approval of the Ethics Committee of West China Hospital, Sichuan University. Informed consent was obtained from the parents or guardians of all patients. Tissue specimens were collected from 16 children who were diagnosed with IH and who underwent surgical removal at the Pediatric Surgery Department of West China Hospital of Sichuan University between January 2021 and December 2023. The experimental group consisted of proliferating phase IH tissue (n = 10), while the control group consisted of involuting phase IH tissue (n = 6). All specimens were confirmed to be IH through pathological examination via hematoxylin-eosin (HE) staining and glucose transport albumin 1 (GLUT-1) immunohistochemistry (IHC) staining (Additional file 1: Fig. S1). The clinical data of the children with IHs were obtained surgically at our hospital (Additional file 2: Table S1).

Antibodies and reagents

Endothelial basal medium (EBM-2) supplemented with growth factors and antibodies, as well as other reagents, was obtained from Lonza (Walkersville, MD, USA). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin, and streptomycin were purchased from Gibco (New York, USA). SOX4 and ESM1 antibodies were obtained from Abcam (Cambridge, UK).

Culture and treatment of 2D cells and 3D microtumours

The methods for the separation, extraction, and screening of CD31 + HemECs have been described in previous literature reports [30, 31]. CD31 + HemECs were cultured in endothelial basal medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml). The cells used in this study were isolated from the tissues (from the proliferating phase) of 3 different children with IH. The cells were maintained in a 37°C cell incubator with 5% carbon dioxide and 95% humidity. Upon reaching 90% confluence, the cells were dissociated using 0.25% trypsin for subsequent experiments. Cells from passages 3 to 8 were used in the experiments. Additionally, the culture method for the 3D microtumour model has been described in previous reports [32].

Construction of siRNAs and overexpression plasmids

To construct the SOX4/ESM1 overexpression plasmid, the coding sequence of SOX4/ESM1 was searched in the Pubmed database. The restriction endonuclease sites within SOX4/ESM1 were analyzed, and primers containing these restriction sites were designed (Additional file 3: Table S2). The target gene fragment was amplified by PCR using cDNA as a template and then treated with the corresponding restriction enzyme to linearize the pcDNA3.4/pcDNA3.1 vector(oe-SOX4/oe-ESM1). The empty vector was used as a control (oe-NC).The linearized vector was then ligated to the amplified fragment and transformed into DH5α competent cells. Positive clones were identified through methods such as PCR and sequencing. Additionally, siRNAs were purchased from Beijing Qingke Biological Co., Ltd., and transfected using Lipofectamine 8000TM (Beyotime, China).

IHC/immunofluorescence staining

The 4 µm paraffin sections were cut, dewaxed in water, and subjected to antigen retrieval using sodium citrate solution under high pressure for 3 minutes. After cooling to room temperature, the sections were treated with 3% H₂O₂ at 37°C for 10 minutes to inactivate endogenous peroxidase activity. Subsequently, the antigen was blocked with sheep serum for 30 minutes following three washes with PBS. Primary antibody (1:200, Abcam) was added, and the sections were refrigerated at 4°C overnight. The following day, the slices were removed, rinsed with PBS three times, and then incubated with a secondary antibody (1:1000 dilution) (fluorescent secondary antibody) at 37°C for 30 minutes, followed by another three washes with PBS. After the sections were restained with hematoxylin, they were subjected to diaminobenzidine (DAB) color development, dehydration, transparency, and sealing, and images were acquired using an optical microscope (Leica AG, Germany).

Western blot

Total protein was extracted from IH tissue in liquid nitrogen using RIPA lysis buffer. The protein concentration was measured using the bicinchoninic acid (BCA) method. Protein samples were then quantitatively diluted, and 20 µl of each sample was transferred onto a polyvinylidene fluoride (PVDF) membrane. Polyclonal monoclonal antibody (1:1000 dilution) and β-actin (1:2000 dilution) were added overnight at 4°C. The following day, after washing with Tris-buffered saline with tween (TBST), a secondary antibody (1:10,000 dilution) was added, and the membrane was incubated at room temperature for 1 hour. The membrane was washed with TBST, and color development was performed.

qRT-PCR

The target gene sequence was designed using Primer-BLAST software based on the full gene sequence provided by NCBI GenBank, with GAPDH serving as the internal reference. The gene primers used were synthesized by Beijing Qingke Biological Company (Additional file 4: Table S3). Total RNA was extracted from tissues and cells using the TRIzol method, and its concentration and quality were determined using a spectrophotometer. Reverse transcription was performed using 2 µg of RNA as the template following the instructions of the reverse transcription kit. After obtaining cDNA, PCR was conducted using a SYBR Green 10 µl assay, and the results were analyzed using the 2-ΔΔCt method.

CCK-8 assay

The cell suspensions were inoculated into 96-well plates, 100 µl of medium was added to each well, and the plates were then placed in a cell incubator for 24 hours. Then, plasmids or siRNAs were added to each well for 24, 48, 72, or 96 hours, and the cell proliferation capacity was assessed. At each time point, 10 µl of CCK-8 reagent was added to each well, and the plates were further incubated in the cell incubator for 2 hours. The absorbance at a wavelength of 450 nm was then measured.

5-Ethynyl-29-deoxyuridine (EdU) assay

The cells were cultured in a Petri dish, and the appropriate concentration of EdU solution was added to the medium 2 hours prior to allowing the cells to fully interact with the EdU. After 2 hours, the cells were fixed with 4% paraformaldehyde for 15 minutes. To enhance stain penetration, the cells were permeated with 0.5% Triton X-100 for 20 minutes. Following the instructions provided with the kit, an appropriate amount of EdU stain solution was added to covalently bind to the DNA of the cells. The cell nuclei were then stained with DAPI for 15 minutes and analyzed using flow cytometry.

Scratch assay

The cells were inoculated into a 6-well plate and cultured for 24 hours, after which they were lightly marked with the tip of a 200 µl pipette. Shed cells were then washed away with PBS, and serum-free medium was added. The plate was observed and photographed, and after 24 hours, the serum-free medium was replaced, and a picture was taken again at the same position. The area between cells and the scratch mobility were calculated using ImageJ software.

Transwell assay

A migrating cell with an 8 µm pore size was utilized for the migration test. The lower chamber was filled with medium containing 10% fetal bovine serum, while cell suspensions were inoculated into the upper chamber and incubated for 24 hours. After incubation, the cells in the upper compartment were gently scraped using a cotton swab and then fixed by staining with Rehsen-Giemsa reagent. Finally, the number of cells that migrated through the chamber was counted under a microscope.

Apoptosis assay

The cell concentration was adjusted to 1×106 cells/ml using an Annexin-V-FITC apoptosis detection kit. The cells were then resuspended in 300 µl of binding buffer, and 5 µl of YF647A-Annexin V and 10 µl of PI reagent were added sequentially. The mixture was thoroughly mixed and kept away from light for 15 minutes. Finally, the cell suspension was transferred to a flow tube, and apoptosis analysis was conducted using a BD FACSVerse.

Angiogenesis assay

Next, 50 µl of Matrigel was added to a precooled 96-well plate, which was then allowed to solidify for 60 minutes. Then, 3×104 cells/ml of cell suspension were added to each well. After culturing for 4 hours, the formation of blood vessels was observed, and photos were taken. Five fields were randomly selected under the microscope to observe the number of cell tubes, and ImageJ software was used to calculate the number of nodes and the length of the main tube.

Microtumour RNA-seq

We extracted RNA from CD31 + HemEC 3D microtumours and 2D planar cells (three in each group). Following quality inspection, a library was constructed, and RNA-seq was conducted to identify downstream targeted molecules and signaling pathways associated with SOX4. An Illumina NovaSeq 6000 was used for sequencing, quantification, and enrichment of products ranging from 200 to 500 bp.

The database predicted the promoter binding sites of SOX4 and ESM1

The JASPAR database, which is maintained by the University of Copenhagen (https://jaspar.genereg.net/), is a comprehensive open database that regularly updates chromatin immunoprecipitation sequencing (ChIP-seq) data and literature on transcription factor-binding sites (motifs). This database allows for the prediction of transcription factor-binding sites based on provided promoter sequences [33]. To search the JASPAR database, we selected SOX4 as a potential binding transcription factor (Additional file 5: Table S4). We then obtained the ESM1 promoter sequence in Fasta format from the Scan bar and set the prediction threshold at 80%.

ChIP-PCR

After cell collection, formaldehyde is used to cross-link chromatin and proteins, the cell is dissolved, and the nucleus is extracted to obtain chromatin and related proteins. Antibodies are added to the nuclear extract, which binds to the target protein; the proteins and impurities that are not specifically bound are removed; chromatin and protein complexes are separated, cross-linked DNA is extracted; and chromatin fragments bound to the target protein are obtained. The target DNA fragments were amplified using specific primers. According to the sequencing results of the JASPAR database, the ESM1 promoter primer sequence (Additional file 6: Table S5) was designed by primer-BLAST, an online primer software, and synthesized by Beijing Qingke Company. A 10 µl SYBR Green system was used for qPCR, and the specific reaction conditions were as follows: stage 1, predenaturation (1 cycle) at 95°C for 5 min; stage 2, PCR (49 cycle) at 95°C for 15 s and 60°C for 30 s. The 2-ΔΔCt method was used for analysis.

Experiment of subcutaneous tumour formation in nude mice

Male BALB/c-nude nude mice (4–6 weeks old) were obtained from Beijing Huafukang Biological Company and raised in a specific pathogen-free (SPF) laboratory at constant temperature (22–26°C) and humidity (55 ± 5%). After 1 week of acclimatization, CD31 + HemECs with good growth status were collected, counted, centrifuged, and suspended. The nude mice were randomly assigned to four groups(five mice per group): the siRNA/plasmid no-loaded control group, low SOX4 group, overexpressing ESM1 group, and low SOX4 + overexpressing ESM1 group. The mice were anesthetized and subcutaneously injected in the back at a dose of 1×10⁷ cells/200 µl (including 100 µl of matrigel). After the nude mice were fed for 1 week, they were euthanized, and the tumour size was measured and photographed. Tissue samples were fixed with 4% paraformaldehyde for HE and CD31 IHC staining, and the microtumour vessel density(MVD) was calculated using Image J software.

Statistical analysis

The data were analyzed using SPSS version 26.0 software(SPSS, Chicago, IL, USA). The measurement data are expressed as the mean ± standard deviation. Independent sample t tests were used for comparisons between two groups, ANOVA was used for comparisons between multiple groups, and Pearson analysis was used for correlation analysis. Statistical significance was set at P values < 0.05.

Screening for the gene SOX4, which may be closely related to the development of IH

RNA-seq of CD31 + HemEC 3D microtumour and 2D planar cells (three in each group) revealed a total of 2219 DEGs, of which 1288 genes were upregulated and 931 genes were downregulated (Fig. 1A-B). In addition, we performed RNA-seq on the tissues of children with proliferating and involuting phase IH (three in each group) in our preliminary study. A total of 374 DEGs were identified, of which 115 genes were upregulated and 259 genes were downregulated (Fig. 1C-D). We analyzed the genes with upregulated expression according to the RNA-seq results in CD31 + HemECs and the genes with upregulated expression according to the RNA-seq results in IH tissues and determined the intersection of the two. The results showed that 10 genes (NOTCH3, ADAMTS4, ADAMTS12, COL4A1, SOX4, ADAM12, MY010, FAT1, and KDRID3) were simultaneously upregulated (Fig. 1E). After reviewing the literature, we found that SOX4 may be closely related to the occurrence and development of IH. Therefore, we selected the transcription factor SOX4 as the target gene for subsequent studies. Next, we verified the expression of SOX4 in CD31 + HemEC 3D microtumours and 2D cells by qRT-PCR. Compared with that in 2D cells, the expression of SOX4 mRNA in 3D microtumours was significantly greater (Fig. 1F), suggesting that SOX4 may play an important role in the pelletization process of CD31 + HemECs.

SOX4 was highly expressed in IH tissues during the proliferating phase

According to the qRT-PCR and Western blot results, the mRNA and protein expression levels of SOX4 were significantly greater in IH tissues at the proliferating phase than in IH tissues at the involuting phase (Fig. 2A-B). IHC staining demonstrated that the SOX4 protein was predominantly localized in the nucleus of IH tissues, and its positive expression was significantly greater in proliferating phase IH tissues than in involuting phase IH tissues. Proliferating phase IH tissues exhibited strong SOX4 expression, with abundant brown-yellow particles deposited in the nucleus. In contrast, involuting phase IH tissues showed weak SOX4 expression, with only a few brown-yellow particles observed in the nucleus (Fig. 2C). Immunofluorescence staining for SOX4 yielded results similar to those of IHC (Fig. 2D). Based on these findings, we speculate that SOX4 may play a role in the development of IH.

SOX4 promotes CD31 + HemEC proliferation, migration, angiogenesis and the expression of vascular endothelial function-related factors (VEGFA and MMP2)

siRNA and an overexpression plasmid targeting SOX4 were separately transfected into CD31 + HemECs, and the SOX4 knockdown and overexpression groups were successfully established (Additional file 7: Figure S2). The results of CCK-8, EdU, cell scratch, Transwell, apoptosis, and angiogenesis experiments demonstrated that SOX4 knockdown significantly inhibited the proliferation, migration, and angiogenesis of CD31 + HemECs compared to those in the control group, while it had no significant effect on apoptosis (Fig. 3A,3C,3E,3G,3I,3M). Conversely, overexpression of SOX4 promoted the aforementioned biological behaviors of CD31 + HemECs but had no significant effect on apoptosis (Fig. 3B,3D,3F,3H,3J,3N). Western blot analysis showed that SOX4 depletion reduced the protein expression of VEGF-A and MMP2 compared to that in the control group (Fig. 3K). In contrast, overexpression of SOX4 increased the protein expression of vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase 2 (MMP2) (Fig. 3L).

SOX4 may play a role by activating the PI3K/AKT signaling pathway

Previous studies have indicated that SOX4 family proteins may act as positive regulators of the PI3K/AKT signaling pathway [32]. Therefore, we hypothesized that SOX4 might be involved in the progression of IH by activating the PI3K/AKT signaling pathway. To investigate this possibility, changes in the expression of key node proteins in the PI3K/AKT pathway were examined by Western blot analysis after manipulating the expression of SOX4 in CD31 + HemECs. The results demonstrated that SOX4 knockdown significantly inhibited the protein expression of p-PI3K, p-AKT, and p-mTOR compared to that in the control group, while it had no significant effect on the total protein levels of PI3K, AKT, or mTOR (Fig. 4A). Conversely, overexpression of SOX4 significantly promoted the protein expression of p-PI3K, p-AKT, and p-mTOR without significantly altering the total protein levels of PI3K, AKT, or mTOR (Fig. 4B). These findings suggest that SOX4 may promote the progression of IH by activating the downstream PI3K/AKT signaling pathway.

RNA-seq and enrichment analysis results of SOX4-knockdown CD31 + HemECs

To further investigate the downstream molecular genes and signaling pathways associated with SOX4, we conducted RNA-seq screening of DEGs in CD31 + HemECs with SOX4 knockdown and in the control group. The results revealed 690 DEGs in the SOX4 knockdown group, with 592 genes significantly upregulated and 98 genes significantly downregulated compared to those in the control group (Fig. 5A-C). Gene Ontology (GO) enrichment analysis of the upregulated genes revealed that they are involved in the regulation of the PI3K/AKT signaling pathway, G protein-coupled receptor binding, nervous system development, and autophagosome assembly (Fig. 5D). Additionally, GO enrichment analysis of the downregulated genes indicated enrichment in cell differentiation, mitochondrial depolarization, the PI3K/AKT signaling pathway, and mitochondrial autophagy (Fig. 5E). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis of the differentially expressed genes highlighted pathways such as animal autophagy, mitochondrial autophagy, estrogen signaling pathway, and cell adhesion (Fig. 5F). Similarly, KEGG signaling pathway enrichment analysis of the downregulated genes revealed enrichment of genes related to mitochondrial autophagy, animal autophagy, the TGF-β signaling pathway, and spliceosome action (Fig. 5G). These findings shed light on potential downstream targets and pathways influenced by SOX4 in IH progression.

Database prediction and ChIP-PCR confirmed that ESM1 was the downstream target gene of SOX4

After analyzing the RNA-seq data from the SOX4 knockdown and control groups, we identified the top 10 downregulated genes (CHRNA5, PPARGC1B, RP11-417L19.4, FAM216B, ACTR3B, DCLK1, AC092835.2, RP11-136L23.2, ESM1, and NDP) for further investigation (Additional file 8: Table S6). Among these genes, ESM1 was found to be closely associated with angiogenesis [35]. ESM1, which is secreted by vascular endothelial cells, plays a significant role in cell proliferation, differentiation, adhesion, and angiogenesis by promoting the production of inflammatory cytokines and angiogenic factors [36]. To confirm whether ESM1 is a downstream target of SOX4, we utilized the JASPAR database to predict potential binding sites for SOX4 transcription factors within five different sequences of the ESM1 promoter region (Fig. 6A). The predicted locus with the highest score was identified as -GGACAATGGA- at -248 to -257 bp. We selected this site and designed specific primers for the ESM1 promoter sequence. qRT-PCR analysis revealed significant enrichment of the ESM1 promoter in the chromatin complex pulled down by the SOX4 antibody compared with that in the IgG control group (Fig. 6B). These findings indicated that SOX4 can directly bind to the ESM1 promoter sequence. To further validate the regulatory relationship between SOX4 and ESM1, we transfected CD31 + HemECs with si-SOX4 and SOX4-overexpressing cells for 48 hours. The qRT-PCR and Western blot results demonstrated that ESM1 expression was significantly lower in the si-SOX4 group than in the control group (Fig. 6C-D), while it was notably greater in the SOX4-overexpressing group (Fig. 6E-F). These results suggest a positive correlation between SOX4 expression and ESM1 expression, supporting the hypothesis that ESM1 may indeed be a downstream positive regulatory target of SOX4.

ESM1 is highly expressed in proliferating phase IH tissues and promotes the proliferation, migration and angiogenesis of CD31 + HemECs

Following the identification of SOX4 as a transcriptional activator of ESM1, we proceeded to validate the expression of ESM1 in tissue samples from children with IH at both the proliferating and involuting phases. Through qRT-PCR and Western blot analyses, we found that ESM1 expression was significantly greater in IH tissues at the proliferating phase than at the involuting phase (Fig. 7A-B). Furthermore, Pearson correlation analysis revealed a strong correlation (r = 0.7) between the relative expression levels of SOX4 and ESM1 mRNA in 16 IH tissue samples (Fig. 7C). IHC results showed that the ESM1 protein was predominantly localized in the cytoplasm of IH tissue, with significantly greater positive expression during the proliferating phase than during the involuting phase (Fig. 7D). Similarly, ESM1 immunofluorescence staining yielded similar results (Fig. 7E). These findings suggest that ESM1 may be involved in the progression of IH, given its significantly greater expression in proliferating phase IH tissues than in subsided tissues. To further explore the role of ESM1 in IH, we transfected siRNA targeting ESM1 and an ESM1 overexpression plasmid into CD31 + HemEC cells and successfully established an ESM1 knockdown group and an overexpression group (Additional file 9: Figure S3). Functional assays, including CCK-8, EdU, cell scratch, Transwell, and angiogenesis assays, demonstrated that ESM1 knockdown significantly inhibited the proliferation, migration, and angiogenesis of CD31 + HemECs (Fig. 7F,7J,7L,7N,7P). Conversely, overexpression of ESM1 promoted these biological behaviors in CD31 + HemECs (Fig. 7G,7K,7M,7O,7Q). Western blot analysis revealed that ESM1 knockdown suppressed the protein expression of VEGF-A and MMP2(Fig. 7H), while ESM1 overexpression enhanced their expression (Fig. 7I).

ESM1 may play a role by activating the PI3K/AKT signaling pathway

Through database prediction and ChIP-PCR analysis, we discovered that ESM1 is a downstream gene directly targeted and regulated by SOX4. A literature review also indicated that ESM1 can promote various cellular behaviors, such as proliferation, migration, invasion, and angiogenesis, by activating the PI3K/AKT signaling pathway, thereby accelerating tumour progression[37]. Based on these findings, we hypothesized that ESM1 may also participate in the occurrence and development of IH through the PI3K/AKT signaling pathway. To investigate this further, we manipulated the expression of ESM1 in CD31 + HemECs and examined the expression of key proteins in the PI3K/AKT signaling pathway using Western blot analysis. ESM1 knockout significantly inhibited the protein expression of p-PI3K, p-AKT, and p-mTOR in cells compared to that in the control group. However, it had no significant effect on the total protein levels of PI3K, AKT, or mTOR (Fig. 8A). Conversely, overexpression of ESM1 promoted the protein expression of p-PI3K, p-AKT, and p-mTOR in cells without affecting the total protein levels of PI3K, AKT, or mTOR (Fig. 8B). These findings suggest that ESM1 may promote the progression of IH through activation of the PI3K/AKT signaling pathway.

SOX4-mediated transcriptional activation of ESM1 promotes CD31 + HemEC progression

In previous studies, we demonstrated that SOX4 can transcriptionally activate ESM1, leading to the promotion of proliferation, migration, and angiogenesis in CD31 + HemECs. Given this relationship, can ESM1 function as an effector of SOX4 to enhance the biological behaviors of CD31 + HemECs? To investigate this possibility, we conducted a functional experiment by simultaneously overexpressing ESM1 in CD31 + HemEC cells with SOX4 knockdown. We transfected si-SOX4 and si-SOX4 + oe-ESM1 into CD31 + HemECs for 48 hours and then conducted a cell behavior recovery experiment. The results revealed that, compared to those in the control group, the proliferation, migration, and angiogenesis of cells in the low SOX4 group were significantly reduced (Fig. 9A-E). Furthermore, compared with those in the low SOX4 group, the proliferation, migration, and angiogenesis of CD31 + HemECs in the low SOX4 + overexpressing ESM1 group were partially restored (Fig. 9A-E). Additionally, Western blot analysis demonstrated that the expression of VEGF-A, MMP2, and factors related to the PI3K/AKT pathway and vascular endothelial function significantly decreased in the SOX4 knockout group compared to the control group(Fig. 9F). However, compared with those in the SOX4-knockdown group, the levels of VEGF-A, MMP2 protein, and PI3K/AKT pathway-related factors in the SOX4-overexpressing ESM1-cotransfection group were partially rescued (Fig. 9G).

SOX4-mediated transcriptional activation of ESM1 promotes 3D microtumour proliferation

We will continue to investigate the effect of the SOX4-ESM1 signaling axis on the proliferation ability of CD31 + HemEC 3D microtumours. Ki-67 immunofluorescence staining was performed to further observe the difference in the cell proliferation ability of the microtumour in each group. Compared with those in the negative control group, the proliferation ability of cells in the low SOX4 group was significantly reduced, while the proliferation ability of cells in the ESM1 overexpression group was significantly enhanced(Fig. 10). Compared with that in the low SOX4 group, the proliferation of cells in the low SOX4 + overexpressing ESM1 cotransfection group was partially restored (Fig. 10). According to the above results, SOX4 can promote the progression of IH by affecting the proliferation of 3D microtumour cells, but this effect can be partially inhibited by ESM1.

Activation of ESM1 by SOX4 transcription promotes IH progression in nude mice

The results of subcutaneous tumour formation in nude mice showed that, compared with that in the control group, the tumour volume in the low SOX4 group was significantly smaller, while the tumour volume in the ESM1 overexpression group was significantly larger (Fig. 11A). Compared with that in the low SOX4 group, the tumour volume in the low SOX4 + overexpressing ESM1 cotransfection group was partially restored (Fig. 11A). HE and CD31 antibody-labeled IHC staining of the tumour tissue from each group was performed to further analyze the differences in the MVD. HE staining revealed that the capillaries in the tumour tissues of the control group were evenly distributed and that the number of capillaries was moderate, while the capillaries in the tumour tissues of the SOX4 group were sparsely distributed, the number of capillaries decreased significantly, and interstitial fiber tissue hyperplasia was obvious (Fig. 11B). In the ESM1-overexpressing group, the capillaries were closely arranged, and the number of capillaries increased significantly (Fig. 11B). The density and number of capillaries in the tumours cotransfected with low SOX4 + overexpressing ESM1 were between those in the control group and those in the low SOX4 group (Fig. 11B). The MVD of the tumour tissues further showed that compared with that in the control group, the MVD of the tumour tissues was significantly decreased in the SOX4 knockout group and significantly increased in the ESM1 overexpression group (Fig. 11C). Compared with that in the low SOX4 group, the MVD of tumours in the low SOX4 + overexpressing ESM1 cotransfection group was reversed to a certain extent (Fig. 11C). According to the study results, SOX4 can promote the progression of tumours in nude mice by affecting cell proliferation and blood vessel formation, but some of its effects can also be inhibited by ESM1.

Currently, the pathogenesis of IH is not fully understood. It has been reported that IH may result from the clonal expansion of stem cells due to key gene mutations in somatic cells [38, 39]. Other studies have suggested that tissue hypoxia and the RAS may also be independent and significant risk factors for IH [40–42]. To identify key genes potentially related to IH pathogenesis, we conducted RNA-seq on IH 3D microtumours and 2D planar cells and analyzed the upregulated genes in addition to those from IH tissues in the early proliferating and involuting phases. The results indicated a potential close association between the transcription factor SOX4 and the onset and progression of IH.

SOX4 is a critical developmental transcription factor that regulates cell fate, differentiation, and progenitor cell development. SOX4 is an essential gene in mouse heart development, and complete SOX4 knockout results in embryonic lethality. Additionally, impaired B lymphocyte development has been observed in SOX4-knockout mouse embryos, suggesting a potential link to vascular system development [43]. SOX4 plays diverse roles in different tumours. While it acts as a protumour factor in breast cancer, lung cancer, glioma, and other cancers by promoting cell proliferation, survival, and migration and inhibiting apoptosis [44–47], it inhibits tumour growth in bladder cancer, hepatocellular carcinoma, glioblastoma multiforme, and other cancers [48–49]. Nevertheless, the literature on the role of SOX4 in IH remains scarce, and relevant mechanistic or functional studies to effectively describe the potential biological functions of SOX4 in IH progression are lacking. We observed significantly greater expression of SOX4 in microtumours than in 2D ordinary cells. Additionally, the expression of SOX4 was significantly greater in proliferating IH tissues than in involuting phase tissues, suggesting that SOX4 may play a crucial role in the pelletizing process of CD31 + HemECs and the progression of IH. Furthermore, we discovered that SOX4 substantially promotes CD31 + HemEC proliferation, migration, angiogenesis, and other behaviors. In a study by Li et al. [50], differentially expressed epigenetic genes in IH tissues and adjacent normal tissues were analyzed, revealing that SOX4 may act as a transcription factor that plays a vital role in the progression of IH. Subsequent research on human umbilical vein endothelial cells (HUVECs) suggested that SOX4 could promote IH progression by inhibiting apoptosis and promoting angiogenesis. It is speculated that SOX4 promotes tumour progression through mechanisms such as cell proliferation and survival promotion, apoptosis inhibition, and EMT promotion. These findings align with our results, suggesting that SOX4 may promote IH growth by enhancing cell proliferation, migration, angiogenesis, and other behaviors. Our study is the first to investigate the differential expression of SOX4 between IH microtumours and IH proliferative tissues, suggesting that SOX4 may be a crucial regulatory factor for the rapid growth of IH during the proliferative phase. It could serve as a diagnostic biomarker and therapeutic target for IH in the future. Furthermore, our study revealed that SOX4 significantly upregulated VEGF-A and MMP2 in CD31 + HemECs. Angiogenesis is crucial for the development of IH, and the rapid growth of IH is primarily driven by the proliferation of vascular endothelial cells to form abnormal hemangioma clusters. VEGF-A, the most effective angiogenic factor involved in the progression of IH, may stimulate the proliferation and migration of endothelial cells by activating MMP2, ultimately leading to abnormal angiogenesis[51]. Additionally, VEGF-A may directly promote endothelial cell formation by inducing calcium ion influx through phospholipase C activation. It may also stimulate endothelial cell mitosis and induce blood vessel formation.

Recent studies have confirmed the significant involvement of the PI3K/AKT signaling pathway in the occurrence and development of IH[52–54]. In our study, we discovered that SOX4 can promote the expression of key proteins within the PI3K/AKT signaling pathway, thereby facilitating the progression of IH. Notably, Mehta et al.[55] reported that amplification of the SOX4 gene promotes the PI3K/AKT signaling pathway in breast cancer, highlighting its potential as a therapeutic target and biomarker within this signaling pathway. Similarly, other studies have revealed that SOX4 regulates the PI3K/AKT signaling pathway to promote the progression of acute lymphoblastic leukemia, pancreatic cancer, and colon cancer via transcriptional activation of LEMD1[56–58]. These findings align with our findings, which demonstrate for the first time that SOX4 can contribute to the progression of IH by activating the PI3K/AKT signaling pathway. However, the precise mechanism through which SOX4 regulates this pathway remains unclear. We hypothesize that the transcription factor SOX4 may activate the PI3K/AKT signaling pathway in cells by binding to key downstream target genes, thereby promoting the development of IH (Fig. 12).

ESM-1 is secreted by vascular endothelial cells and is primarily involved in angiogenesis, cell adhesion, and the inflammatory response. Numerous studies have reported a close association between ESM1 and angiogenesis, suggesting its involvement in the progression of various malignant tumours [59]. In hepatocellular carcinoma, ESM1 significantly enhances tumour angiogenesis, particularly in the early stages [60]. Studies on breast cancer have demonstrated that ESM1 can activate the AKT/NF-κB signaling pathway to promote cell proliferation, migration, and invasion [61]. Similarly, in esophageal cancer, ESM1 acts as an independent prognostic factor and promotes cell proliferation and migration through the JAK signaling pathway; silencing ESM1 significantly inhibits tumour cell proliferation and migration [62]. Additionally, ESM1 is highly expressed in head and neck squamous cell carcinoma and may contribute to tumour progression through the RAS-MAPK-ERK signaling pathway [63]. Thus, it is speculated that ESM1 may promote the progression of malignant tumours by enhancing angiogenesis. In our study, by conducting RNA-seq on SOX4-knockdown CD31 + HemECs, we discovered that ESM1 may be downstream of SOX4 transcriptional regulation. This finding was further confirmed by database prediction and ChIP-PCR, which confirmed the direct regulatory relationship between SOX4 and the ESM1 promoter region. Subsequently, we examined the impact of ESM1 regulation alone on the biological behavior of CD31 + HemECs. Our studies revealed that ESM1 enhances cell proliferation, migration, and angiogenesis, thereby promoting the progression of IH. These findings align with previous reports, indicating that ESM1 promotes the occurrence and development of IH through cell proliferation, migration, and angiogenesis. Moreover, ESM1 could be explored as a potential therapeutic target for IH. Furthermore, we observed that ESM1 promotes the activation of the PI3K/AKT signaling pathway, thereby contributing to the progression of IH. Similar observations have been made in cervical cancer, where ESM1 activates the PI3K/AKT signaling pathway and EMT, leading to increased proliferation, migration, and invasion of cancer cells [64]. Additionally, a recent study by Yang et al. [37] revealed that ESM1 induces angiogenesis in colorectal cancer by activating the PI3K/AKT/mTOR pathway, thus accelerating tumour progression. These studies support our findings and suggest that ESM1 also promotes the development of IH by activating the PI3K/AKT signaling pathway.

Microtumour and subcutaneous tumour formation experiments in nude mice revealed that low SOX4 expression alone inhibited tumour growth, that overexpression of ESM1 promoted tumour growth, and that simultaneous overexpression of ESM1 and low SOX4 partially restored tumour growth. Therefore, ESM1 can be considered a new therapeutic target for IH. ESM-1 has been reported to be associated with drug resistance [65]. In patients with prolactinoma, the ESM1 microvascular density was significantly greater in bromocriptine-resistant patients than in bromocriptine-sensitive patients. Knockdown of ESM1 with interfering RNA significantly enhanced the sensitivity of rat prolactinoma cell lines to dopamine agonists. Additionally, ESM1 knockdown significantly increased the sensitivity of HUVECs to bevacizumab. treatment [66]. Thus, ESM1 has potential for further study as a target for propranolol treatment in patients with drug-resistant IH. Recent studies have also linked ESM1 to angiogenesis. Research on the mechanism of angiogenesis has revealed a positive feedback loop between VEGF-A and ESM1, where VEGF-A stimulates ESM1 expression through phosphorylation and activation of vascular epidermal growth factor receptor 2 (VEGFR-2)[67]. Conversely, ESM1 directly binds to fibronectin, replacing fibronectin-bound VEGF-A, thereby enhancing the bioavailability of VEGF-A and its mediated signaling [68]. Therefore, blocking the interaction between ESM1 and VEGF-A is expected to become a new strategy for inhibiting angiogenesis, and targeted inhibition of ESM1 may control the progression of IH in the future.

However, there are limitations to this study. First, the clinical data and tissue samples were obtained from a single center, and the sample size was relatively small. In the next phase, collaboration with multiple centers will be pursued to establish a clinical sample database and expand the number of samples to enhance the accuracy of the results. Second, this study did not investigate the effect of drugs (inhibitors) related to the SOX4-ESM1 signaling axis on IH progression, which will be the focus of our future research.

SOX4 plays a crucial role in the initiation and progression of IH, and SOX4/ESM1 may serve as a novel biomarker and potential therapeutic target for IH, providing a new approach for the future management and treatment of IH.

Infantile hemangioma (IH); three-dimensional (3D) hemangioma endothelial cells (HemECs); vascular endothelial growth factor A (VEGF-A); human umbilical vein endothelial cells (HUVECs); decellularized extracellular matrix (dECM); epithelial-mesenchymal transition (EMT); glucose transport albumin 1 (GLUT-1); hematoxylin-eosin (HE); immunohistochemistry (IHC); phosphate buffer (PBS); bicinchoninic acid (BCA); polyvinylidene fluoride (PVDF); tris buffered saline tween (TBST); chromatin immunoprecipitation sequencing (ChIP-seq); vascular endothelial growth factor A (VEGF-A); matrix metalloproteinase 2 (MMP2); microvessel density (MVD); renin-angiotensin system (RAS); vascular epidermal growth factor receptor 2 (VEGFR-2); gene ontology (GO); Kyoto Encyclopedia of Genes and Genomes (KEGG)

Acknowledgments

We thank Li Li and Chunjuan Bao (Institute of Clinical Pathology, West China Hospital) for performing the histological staining.

Authors’ contributions

Conceptualization, methodology, data curation,writing-original draft preparation, MK; investigation, data curation, methodology, formal analysis, software, validation,YL; , data curation, methodology,TQ; funding acquisition, supervision, writing-review & editing, YJ. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 82273556), the Key Project in the Science & Technology Program of Sichuan Province (grant numbers 2022YFS0233 and 2022YFS0225), the Project of Natural Science Foundation of Shandong Province (grant number ZR2022MH229), the ‘0 to 1’ Project of Sichuan University (grant number 2022SCUH0033), the Med-X Center for Informatics Funding Project (YGJC004), the 1·3·5 Project for Disciplines of Excellence-Clinical Research Incubation Project of West China Hospital of Sichuan University (grant numbers 2023HXFH004, 2020HXFH048 and 2019HXFH056), and the 1·3·5 Project for Disciplines of Excellence-Clinical Research Interdisciplinary Innovation Project of West China Hospital of Sichuan University (ZYJC21060).

Availability of data and materials

All the data analyzed in the present study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the West China Hospital of Sichuan University.

Consent for publication

All authors have read the manuscript and approved the final version.

Competing interests

The authors declare that there are no conflicts of interest.

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FigureS1.tif
Supplementary material Additional file 1: Figure. S1 HE and GLUT-1 IHC staining of IH samples.
TableS1..docx
Additional file 2: Table. S1 Clinical features of sixteen patients with infantile hemangioma.
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Additional file 3: Table. S2 SOX4/ESM1 plasmid construction sequence.
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Additional file 4: Table. S3 Gene primer sequence.
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Additional file 5: Table. S4 Prediction of SOX4 and ESM1 promoter sequence binding sites by JASPAR database.
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Additional file 6: Table. S5 ESM1 promoter primer sequence.
FigureS2.tif
Additional file 7: Figure. S2 The siRNA and plasmid of SOX4 were successfully constructed in CD31+ HemECs.
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Additional file 8: Table. S6 Differential expression of the top 10 down-regulated genes (si-SOX4 vs. si-NC).
FigureS3.tif
Additional file 9: Figure. S3 The siRNA and plasmid of ESM1 were successfully constructed in CD31+ HemECs.

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Targeting ESM1 via SOX4 promotes the progression of infantile hemangioma through the PI3K/AKT signaling pathway

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Abstract

Figures

Background

Methods

Patients and samples

Antibodies and reagents

Culture and treatment of 2D cells and 3D microtumours

Construction of siRNAs and overexpression plasmids

IHC/immunofluorescence staining

Western blot

qRT-PCR

CCK-8 assay

5-Ethynyl-29-deoxyuridine (EdU) assay

Scratch assay

Transwell assay

Apoptosis assay

Angiogenesis assay

Microtumour RNA-seq

The database predicted the promoter binding sites of SOX4 and ESM1

ChIP-PCR

Experiment of subcutaneous tumour formation in nude mice

Statistical analysis

Results

Screening for the gene SOX4, which may be closely related to the development of IH

SOX4 was highly expressed in IH tissues during the proliferating phase

SOX4 may play a role by activating the PI3K/AKT signaling pathway

RNA-seq and enrichment analysis results of SOX4-knockdown CD31 + HemECs

Database prediction and ChIP-PCR confirmed that ESM1 was the downstream target gene of SOX4

ESM1 may play a role by activating the PI3K/AKT signaling pathway

SOX4-mediated transcriptional activation of ESM1 promotes CD31 + HemEC progression

SOX4-mediated transcriptional activation of ESM1 promotes 3D microtumour proliferation

Activation of ESM1 by SOX4 transcription promotes IH progression in nude mice

Discussion

Conclusions

Abbreviations

Declarations

References

Supplementary Files

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