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Introduction

Hemangioma is a benign tumor that is characterized by endothelial hyperplasia in the blood vessels, which primarily occurs in infants and female patients (1,2). Patients with hemangioma may suffer from disease-related syndromes such as PHACE and LUMBAR syndromes, or exhibit skin lesions during the disease growth phase (2,3). To reduce the risk of complications experienced by patients with hemangioma, early identification by physical examination, ultrasound or MRI allows for immediate intervention with appropriate measures (3,4). Propranolol treatment, laser therapy and surgical resection are among the potential treatment options available for patients with hemangioma. However, certain patients with hemangioma do not respond well to these treatment strategies (2,5,6). Therefore, to optimize the management protocols of patients with hemangioma, it is necessary to explore the pathogenic mechanism of this disease to identify novel treatment targets.

MicroRNAs (miRNAs/miRs) are a class of endogenous, non-coding RNAs ~22 nucleotides in length, which mainly mediate the post-transcriptional regulation of mRNA (7). Among miRNAs, miR-203 has been shown to serve a key role in cell proliferation, migration and/or invasion in several malignancies (8-10). By decreasing the expression of regulator of G-protein signaling 17 (RGS17), miR-203 has been shown to suppress cell proliferation and invasion in non-small cell lung cancer (8). In addition, miR-203 has been reported to inhibit the proliferation and invasion of osteosarcoma cells, but promote their apoptosis, by targeting runt-related transcription factor 2 (RUNX2) expression (9). miR-203 has also been reported to inhibit epithelial-mesenchymal transition and invasion of renal cell carcinoma cells by inactivating the PI3K/AKT signaling pathway (10). However, whether miR-203a-3p exhibits similar effects on hemangioma remains unclear and the molecular mechanism requires further investigation.

Vascular epithelial growth factor A (VEGFA) is a critical regulator of a number of cellular processes, including proliferation, apoptosis and invasion (11-13). Notably, it has been demonstrated that VEGFA can serve a role in the pathogenesis of hemangioma in several ways, including through the promotion of angiogenesis (14). In addition, GG genotype of +405 G/C VEGFA polymorphism is associated with a lower risk for infantile hemangioma (15). The PI3K/AKT pathway has been shown to regulate various biological processes, including cell survival, mobility and malignant transformation (16,17). In particular, this signaling pathway has been reported to be associated with the pathogenesis and progression of hemangioma (18).

Our previous study revealed that miR-203a-3p was involved in the regulation of hemangioma progression mediated by long non-coding RNA MEG8(19). Therefore, the present study aimed to explore the effects of miR-203a-3p on endothelial cell proliferation, apoptosis and invasion in hemangioma, with specific focus on its downstream targets and associated signaling pathways.

Materials and methods

Cell lines

Human primary hemangioma endothelial cells (HemECs) and the 293T cell line were purchased from the BeNa Culture Collection. HemECs were maintained in complete endothelial cell medium (Gibco; Thermo Fisher Scientific, Inc.), whereas the 293T cells were maintained in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). All cells were cultured in a humidified incubator with 5% CO2 at 37˚C. Ethics approval for the use of HemECs was waived by the Ethics Committee of the Affiliated Hospital of Hebei University of Engineering (Handan, China).

Cell transfection

miR-203a-3p mimics, the miR-203a-3p inhibitor and their corresponding negative controls (NCs) were purchased from Changchun Changsheng Gene Biotechnology Co., Ltd. The VEGFA overexpression plasmid (pEX2-VEGFA) and the empty NC plasmid (pEX2-NC) were purchased from Shenzhen Morecell Biomedical Co., Ltd. HemECs (1x106 cells) were transfected with 50 nM miR-203a-3p mimics, 50 nM miR-203a-3p inhibitors, 50 nM NC mimics, 50 nM NC inhibitors, 0.8 µg pEX2-VEGF or 0.8 µg pEX2-NC by Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol at 37˚C for 6 h. The sequences for miR-203a-3p or NC mimics and inhibitors used were as follows: miR-203a-3p mimics, 5'-GUGAAAUGUUUAGGACCACUAG-3'; miR-203a-3p inhibitors, 5'-CUAGUGGUCCUAAACAUUUCAC-3'; NC mimics, 5'-UUGUACUACACAAAAGUACUG-3' and NC inhibitors, 5'-CAGUACUUUUGUGUAGUACAA-3'.

miR-203a-3p manipulation

The cultured HemECs were divided into the following five groups: i) Control group (without any treatment); ii) NC mimic group (transfected with NC mimics); iii) miR mimic group (transfected with miR-203a-3p mimics); iv) NC inhibitor group (transfected with the NC inhibitor); and v) miR inhibitor group (transfected with the miR-203a-3p inhibitor). Reverse transcription-quantitative PCR (RT-qPCR), cell proliferation, apoptosis, invasion and western blotting assays were performed following transfection.

Luciferase reporter assay

The binding sites between miR-203a-3p and VEGF were predicted using RNAhybrid v22 (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid). The 3'untranslated regions of VEGFA containing miR-203a-3p-binding wild type (WT) and mutant type (MT) sites were inserted into the pGLuc vector, which was synthesized by Beyotime Institute of Biotechnology. The 0.8 µg VEGFA-WT or 0.8 µg VEGFA-MT plasmids were then co-transfected with the 50 nM miR-203a-3p mimics or 50 nM NC mimics into 1x106 293T cells using Lipofectamine 2000. Following cell culture for 48 h, the cells were harvested and assessed using a luciferase assay kit (Beyotime Institute of Biotechnology) according to the kit's protocol. The luciferase enzyme activity was normalized to Renilla luciferase enzyme activity.

VEGFA regulation experiment

HemECs were cultured and divided into the following five groups: i) Control group (without any treatment), ii) NC mimic + pEX2-NC group (co-transfected with NC mimics and the pEX2-NC plasmid), iii) miR mimic + pEX2-NC group (co-transfected with miR-203a-3p mimics and the pEX2-NC plasmid), iv) NC mimic + pEX2-VEGFA group (co-transfected with NC mimics and the pEX2-VEGFA plasmid) and v) miR mimic + pEX2-VEGFA group (co-transfected with miR-203a-3p mimics and the pEX2-VEGFA plasmid). Further experiments, namely RT-qPCR, western blotting, cell proliferation, apoptosis and invasion assays, were performed following transfection.

RT-qPCR

Total RNA was extracted from the HemECs using a RNeasy Mini kit (Qiagen China Co. Ltd.) 48 h after transfection. The RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the kit's protocol. qPCR was performed using a SYBR®-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR was carried out with following procedure: 95˚C hold for 10 min; 40 cycles of denaturation at 95˚C for 15 sec and annealing/extension at 61˚C for 30 sec. The expression levels of miR-203a-3p and VEGFA were evaluated using the 2-ΔΔCq method (20) with U6 or β-actin used as endogenous controls, respectively. The primer sequences are listed in Table SI.

Cell proliferation assay

Cell Counting Kit-8 (CCK-8; MilliporeSigma) was utilized to assess the proliferation of HemECs. Briefly, HemECs were plated in 96-well plates (2x103 cells/well). At 0, 24, 48 and 72 h following transfection, 10 µl CCK-8 reagent was added before the mixture was incubated for an additional 2 h at 37˚C. The optical density values at 450 nm were subsequently evaluated using an automated enzyme immunoassay analyzer (BioTek China).

Cell apoptosis assay

A TUNEL apoptosis kit (Beyotime Institute of Biotechnology) was used for detecting the apoptosis of HemECs following transfection. Briefly, transfected HemECs were plated into 24-well plates (1x104 cells/well) and cultured for 48 h. The HemECs were then fixed with 4% paraformaldehyde (Beyotime Institute of Biotechnology) for 30 min at 37˚C and incubated with 0.1% Triton X-100 (Beyotime Institute of Biotechnology) for 5 min at 4˚C. Subsequently, 50 µl TUNEL solution was added and the cells were incubated for 1 h at 37˚C, followed by DAPI (Sangon Biotech Co., Ltd.) staining for 15 min at room temperature. After adding Antifade Mounting Medium (Beyotime Institute of Biotechnology), the cells were sealed and observed under an inverted fluorescence microscope (Motic China Group Co., Ltd.) with 3 random fields being chosen.

Cell invasion assay

The invasive ability of HemECs following transfection was assessed using Transwell chambers. Briefly, 48 h following transfection, HemECs, which were suspended in 1% FBS containing complete endothelial cell medium, were seeded into the upper Matrigel-coated chambers (Corning, Inc.) at a density of 5x104 cells/well, whilst complete endothelial cell medium containing 10% FBS was added into the lower chamber. The upper chamber was coated by 70 µl Matrigel, which was diluted with complete endothelial cell medium at a ratio of 1:8, at 37˚C for 1 h. The cells were cultured for 24 h before their invasive ability was assessed by staining with 0.5% crystal violet (MilliporeSigma) for 15 min at room temperature after being fixed with 4% paraformaldehyde (MilliporeSigma). The images were taken under an inverted light microscope (Motic China Group Co., Ltd.).

Western blot analysis

Total proteins from HemECs at 48 h following transfection were lysed using RIPA buffer (MilliporeSigma) and quantified using bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Of the extracted proteins, 20 µg per lane were separated by 4-20% SDS-PAGE and transferred onto nitrocellulose membranes (Pall Life Sciences). After blocking with 5% BSA (MilliporeSigma) at 37˚C for 1 h, the membranes were incubated at 4˚C overnight with primary antibodies (all obtained from Invitrogen; Thermo Fisher Scientific, Inc.) against Bcl-2 (1:1,000 dilution; cat. no. PA5-11379), cleaved (C)-caspase 3 (1:1,000; cat. no. PA5-96077), VEGFA (1:1,000; cat. no. PA5-16754), phosphorylated (p)-PI3K (1:1,000; cat. no. PA5-118549), PI3K (1:1,000; cat. no. PA5-99518), p-AKT (1:1,000; cat. no. MA5-38243), AKT (1:1,000; cat. no. 44-609G) and β-actin (1:2,000; cat. no. PA5-85291). Subsequently, the membranes were incubated with a HRP-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. 65-6120; Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 1 h. The bands were visualized using the ECL-PLUS reagent (Thermo Fisher Scientific, Inc.). Densitometric quantification was completed by ImageJ 1.8 (National Institutes of Health).

Statistical analysis

GraphPad Prism 7.0 (GraphPad Software, Inc.) was used to analyze the data, which were triplicates and exhibited as mean ± standard deviation. One-way ANOVA followed by Tukey's post-hoc test was used for comparisons among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of miR-203a-3p on the proliferation, apoptosis and invasion of HemECs

The expression levels of miR-203a-3p were significantly increased following transfection of the cells with miR-203a-3p mimics compared with those in the NC mimics group (P<0.001; Fig. 1A). By contrast, miR-203a-3p expression was significantly decreased following the transfection of HemECs with the miR-203a-3p inhibitor compared with that in the NC inhibitor group (P<0.05; Fig. 1A). These observations suggested that transfection was successful. In addition, compared with those in their corresponding NC groups, miR-203a-3p mimics and miR-203a-3p inhibitor transfection induced significant decreases and increases in cell proliferation at 48 and 72 h, respectively (all P<0.05; Fig. 1B). Subsequently, it was revealed that apoptosis was significantly increased by transfection with the miR-203a-3p mimics (P<0.001) but significantly alleviated by the miR-203a-3p inhibitor (P<0.05; Fig. 1C and D), compared with that in their corresponding NC groups. Furthermore, the expression levels of apoptotic markers Bcl-2 and C-caspase 3 were measured; miR-203a-3p mimics transfection significantly suppressed Bcl-2 expression whilst increasing that of C-caspase 3, whereas transfection with the miR-203a-3p inhibitor resulted in the opposite effects compared with miR-203a mimics (all P<0.05; Fig. 1E-G). The number of invasive cells was significantly decreased in cells transfected with miR-203a-3p mimics (P<0.01) but significantly elevated in cells transfected with the miR-203a-3p inhibitor (P<0.05; Fig. 2A and B), compared with that in their corresponding NC groups.

Figure 1 miR-203a-3p suppresses the proliferation and promotes apoptosis of HemECs. (A) miR-203a-3p expression, (B) cell proliferation and (C) cell apoptosis rate were measured in HemECs following transfection. (D) Representative images of TUNEL staining in C (magnification, x200). (E) Representative images of Bcl-2 and C-caspase 3 expression by western blot analysis. Relative expression levels of (F) Bcl-2 and (G) C-caspase 3 were semi-quantified. *P<0.05, **P<0.01 and ***P<0.001. miR-203a-3p, microRNA-203a-3p; HemECs, human hemangioma endothelial cells; NS, non-significant; C-, cleaved; NC, negative control; OD, optical density.

Figure 2 miR-203a-3p decreases the number of invasive HemECs. (A) Representative images of invasive cells by crystal violet staining (magnification, x200). (B) Invasive cell count in HemECs following transfection. *P<0.05 and **P<0.01. miR-203a-3p, microRNA-203a-3p; HemECs, human hemangioma endothelial cells; NC, negative control.

miR-203a-3p regulates VEGFA expression and the PI3K/AKT signaling pathway in HemECs

A binding site on the 3'untranslated region of VEGFA mRNA for miR-203a-3p was predicted (Fig. 3A). Therefore, VEGFA-MT and VEGFA-WT sequences were designed based on this binding site. The relative luciferase activity was significantly decreased (P<0.001) in cells co-transfected with VEGFA-WT and miR-203a-3p mimics compared with that in cells transfected with the NC mimics, suggesting direct binding between VEGFA mRNA and miR-203a-3p; however, the relative luciferase activity did not differ between cells co-transfected with VEGFA-MT and miR-203a-3p mimics compared with that in cells transfected with the NC mimics (Fig. 3B). In addition, miR-203a-3p mimics transfection significantly decreased the expression levels of VEGFA, whereas transfection with the miR-203a-3p inhibitor exerted opposite effects (all P<0.05; Fig. 3C-E), compared with those in their corresponding NC groups. These results suggested that miR-203a-3p may negatively regulate VEGFA expression. In addition, compared with those in their corresponding NC groups, miR-203a-3p overexpression significantly decreased the phosphorylation levels of PI3K and AKT, whereas miR-203a-3p knockdown resulted in the opposite effects (all P<0.05; Fig. 3F and G).

Figure 3 miR-203a-3p downregulates VEGFA expression and PI3K/AKT signaling in HemECs. (A) Design of the WT and MT sequences of VEGFA for luciferase reporter gene assays. (B) Comparison of relative luciferase activity among the WT and MT groups after miR-203 mimics co-transfection. (C) Comparison of VEGFA mRNA expression levels among the groups after the manipulation of miR-203 expression. (D) Detection of the VEGFA protein expression by western blotting after the manipulation of miR-203 expression, (E) which was semi-quantified. (F) Detection of PI3K and AKT phosphorylation by western blot analysis. (G) Semi-quantification of the p-PI3K/PI3K and p-AKT/AKT ratios among the groups in HemECs following the manipulation of miR-203 expression. *P<0.05, **P<0.01 and ***P<0.001. miR-203a-3p, microRNA-203a-3p; VEGF, vascular endothelial growth factor; WT, wild type; MT, mutant type; p-, phosphorylated; HemECs, human hemangioma endothelial cells; NS, non-significant; NC, negative control.

miR-203a-3p regulates proliferation, apoptosis and invasion by inhibiting VEGFA in HemECs

Transfection of HemECs with the VEGFA overexpression plasmids significantly increased the expression levels of VEGFA (all P<0.001; Fig. S1A-C) and attenuated miR-203a-3p mimics-induced reduction of VEGFA (all P<0.001; Fig. 4A-C), suggesting successful transfection. Subsequently, overexpression of VEGFA significantly increased cell proliferation (all P<0.05; Fig. S1D) and also significantly reversed the miR-203a-3p mimics-induced decreases in cell proliferation after 48 and 72 h in HemECs (all P<0.01; Fig. 4D). By contrast, VEGFA overexpression significantly inhibited cell apoptosis (P<0.01; Fig. S1E and F), in addition to significantly reversing the miR-203a-3p mimics-induced increases in HemEC apoptosis (P<0.001; Fig. 4E and F). The number of invasive cells was found to be significantly increased by VEGFA overexpression (P<0.01; Fig. S1G and H), which also significantly reversed the miR-203a-3p mimics-induced reductions in invasive cell numbers (P<0.01; Fig. 5A and B).

Figure 4 VEGFA is implicated in miR-203a-3p-mediated HemEC proliferation and apoptosis. (A) Comparison of VEGFA mRNA expression levels among groups after miR-203-3p and VEGFA co-transfection. (B) Measurement of VEGFA protein expression by western blotting. (C) Semi-quantification of VEGFA protein expression among groups after miR-203-3p and VEGFA co-transfection. Comparison of (D) cell proliferation and (E) cell apoptosis rates after miR-203-3p and VEGFA co-transfection. (F) Representative TUNEL staining images of HemECs in E (magnification, x200). *P<0.05, **P<0.01 and ***P<0.001. miR-203a-3p, microRNA-203a-3p; VEGF, vascular endothelial growth factor; HemECs, human hemangioma endothelial cells; NS, non-significant; NC, negative control; OD, optical density.

Figure 5 VEGFA is involved in miR-203a-3p-modulated HemEC invasion. (A) Representative images of invasive cells by crystal violet staining (magnification, x200). (B) Invasive cell count among groups following miR-203-3p and VEGFA co-transfection. *P<0.05 and **P<0.01. VEGF, vascular endothelial growth factor; miR-203a-3p, microRNA-203a-3p; HemECs, human hemangioma endothelial cells; NC, negative control.

miR-203a-3p inhibits the PI3K/AKT signaling pathway by targeting VEGFA in HemECs

To further explore if miR-203a-3p could regulate the PI3K/AKT signaling pathway through VEGFA, additional experiments were performed. Overexpression of VEGFA significantly increased the phosphorylation levels of PI3K (P<0.01) and AKT (P<0.05; Fig. S1I-K). In addition, overexpression of VEGFA significantly reversed the miR-203a-3p mimics-induced decreases in PI3K and AKT phosphorylation in HemECs (all P<0.01; Fig. 6A and B).

Figure 6 VEGFA is implicated in the miR-203a-3p-mediated PI3K/AKT signaling inhibition in HemECs. (A) Detection of PI3K and AKT phosphorylation by western blot analysis after miR-203-3p and VEGFA co-transfection. (B) Semi-quantification of p-PI3K/PI3K and p-AKT/AKT ratios among the different groups after miR-203-3p and VEGFA co-transfection. *P<0.05 and **P<0.01. VEGF, vascular endothelial growth factor; miR-203a-3p, microRNA-203a-3p; p-, phosphorylated; HemECs, human hemangioma endothelial cells.

Discussion

A number of miRNAs have been reported to be involved in the progression of hemangioma. In particular, miR-200c-3p has been reported to promote the proliferation of vascular endothelial cells by targeting the Notch signaling pathway, which serves a key role in the pathogenesis of hemangioma (21). In another previous study, downregulation of miR-556-3p expression was reported to suppress hemangioma cell proliferation whilst accelerating apoptosis by targeting VEGFC expression (22). Furthermore, miR-139-5p may negatively modulate cell proliferation and migration in hemangioma stem cells by targeting insulin-like growth factor 1(23). Other studies have also reported that some miRNAs can regulate the progression of hemangioma, including miR-382-5p and miR-125b-5p (24,25). Although these previous studies have revealed miRNAs can regulate the proliferation and apoptosis of hemangioma cells, it remains necessary to explore alternative biological pathways that are involved in the biological functions of hemangioma cells. This information could then be applied for designing potential treatment strategies for hemangioma. Our preliminary study revealed that miR-203 expression can be regulated by the lncRNA MEG8 and may participate in the modulation of hemangioma progression via inhibiting the Notch signaling pathway (19). However, the molecular mechanism by which miR-203a-3p can participate in modulation of hemangioma progression remains unclear. As a miRNA that has been extensively studied, miR-203 functions as a tumor suppressor in a variety of malignancies (26). Overexpression of miR-203 has been found to inhibit cell invasion and proliferation in hepatocellular carcinoma cells (27), whereas the expression levels of miR-203 have been revealed to be downregulated during lung cancer tumorigenesis (8). In lung cancer cells, miR-203 can suppress the invasion and proliferation of the A549 and Calu-1 cell lines by targeting RGS17 expression (8). In estrogen-dependent breast cancer cells, miR-203 has been reported to inhibit their viability and migration by suppressing the protein expression of estrogen receptor α (28). In addition, miR-203 knockdown has been revealed to inhibit cisplatin-induced cell apoptosis in osteosarcoma cells in vitro by targeting RUNX2 expression (29). Therefore, it is reasonable to hypothesize that miR-203 may participate in the regulation of cell viability, apoptosis and migration in hemangioma. In the present study, overexpression of miR-203a-3p decreased the proliferation and invasion of HemECs, whilst increasing apoptosis of these cells. By contrast, knocking down miR-203a-3p expression resulted in the opposite effects. Possible reasons for these findings could be that miR-203a-3p regulated activation of the VEGFA-mediated PI3K/AKT pathway upstream of cell proliferation, apoptosis and invasion, or that miR-203a-3p could have regulated cell invasion and proliferation by targeting RGS17 expression (8).

The VEGF family consists of cytokines known to be angiogenic factors that participate in blood vessel formation and lymphangiogenesis (30). VEGFA is the main member of this family of proteins, which has been revealed to regulate cell proliferation, apoptosis and invasion (11-13,31,32). In particular, VEGFC has also been reported to impact the progression of hemangioma (22). A previous study suggested that VEGFA serves a key role in the pathogenesis of hemangioma, specifically through the proliferation and invasion physiological pathways (24). In addition, the miR-206/VEGFA axis has been documented to serve an important role in the suppression of HemEC proliferation after the expression of metastasis-associated lung adenocarcinoma transcript 1 has been knocked down (33). It has also been shown that VEGFA is a master regulator of angiogenesis in hemangioma (14,34). In the present study, it was demonstrated that VEGFA could regulate hemangioma proliferation, apoptosis and invasion. According to previous studies, VEGFA is one of the targets of miR-203a-3p and has been shown to be a key regulatory mechanism during the pathogenesis of laryngeal carcinoma and progression of cervical cancer (35,36). In the present study, it was found that miR-203a-3p regulated the cellular functions of hemangioma through VEGFA. The reason could be attributed to VEGFA being a direct target of miR-203a-3p as previously reported (35).

AKT is a serine threonine kinase acting downstream of PI3K, which regulates a high number of critical cellular processes (37). Inhibition of the PI3K/AKT/mTOR signaling pathway has been shown to restore the autophagic process in epidermal keratinocyte cells from psoriatic mice (38). In addition, suppression of AKT/mTOR signaling has been observed to promote chemo-radiotherapy resistance in lung squamous cell carcinoma cells (39). In another study, AKT was found to serve a critical role during the life cycle of acute and persistent murine norovirus strains in macrophages (40). In addition to these cellular activities, it has been reported that the PI3K/AKT signaling pathway serves a key role in hemangioma. According to an earlier study, activation of the PI3K/AKT signaling pathway may affect tumorigenesis of lung hemangioma through the downstream mTOR (41). Furthermore, the PI3K/AKT pathway has been demonstrated to suppress hemangioma proliferation by downregulating the expression of proliferating cell nuclear antigen, which may further influence the progression of hemangioma (42). In the present study, it was found that the PI3K/AKT signaling pathway was regulated by miR-203a-3p in hemangioma, which was in line with the data previously reported in other tumors, such as renal cell carcinoma, papillary thyroid cancer and glioblastoma (10,43,44). In addition, it was confirmed that miR-203a-3p can directly suppress VEGFA expression and the PI3K/AKT pathway, which could suppress the proliferation of hemangioma, thereby uncovering the miR-203a-3pVEGFA/PI3K/AKT axis.

In conclusion, in the present study, miR-203a-3p overexpression was found to inhibit endothelial cell proliferation, suppress invasion and promote apoptosis by inactivating the VEGFA-mediated PI3K/AKT pathway in hemangioma. These findings may assist in identifying suitable targets for the management of hemangioma.

Supplementary Material

VEGFA can regulate cell proliferation, apoptosis, invasion and PI3K/AKT signaling in HemECs. (A) Comparison of VEGFA mRNA expression among the groups after VEGFA overexpression. (B) Detection of VEGFA protein expression by western blotting after VEGFA overexpression, (C) which was semi-quantified. (D) Cell proliferation and (E) apoptosis rates among the groups after VEGFA overexpression. (F) Representative images of TUNEL staining in E (magnification, x200). (G) Comparison of invaded cells among the groups after VEGFA overexpression. (H) Representative images of invasive cells after crystal violet staining (magnification, x200). (I) Detection of PI3K and AKT phosphorylation by western blotting after VEGFA overexpression. Comparison of (J) p-PI3K/PI3K and (K) p-AKT/AKT ratios, *P<0.05, **P<0.01 and ***P<0.001. NC, negative control; OD, optical density; p-, phosphorylated; VEGF, vascular endothelial growth factor; NS, not significant.

Primers used for reverse transcription quantitative PCR.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by funds received from the following grant: Scientific Research Fund Project of Hebei Provincial Health Commission, Molecular Mechanism of Propranolol Induced Apoptosis in Hemangioma Endothelial Cells (grant no. 20200582).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

ZH and LZ designed the present study. Material preparation and data collection were performed by YL and DD. ZH, LZ and JG analyzed the data. All authors contributed to the interpretation of data, methodology and reviewing and editing of the manuscript. ZH and LZ confirm the authenticity of all the raw data. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Ethics approval for the use of HemECs was waived for the present study by the Ethics Committee of the Affiliated Hospital of Hebei University of Engineering (Handan, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References