HUVECs, the XPTS-1 human infantile hemangioma-derived endothelial cell line, and the EOMA hemangioendothelioma cell line, were purchased from the ATCC. Cells were incubated in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Invitrogen; Thermo Fisher Scientific Inc.) and 1% penicillin/streptomycin at 37˚C with 5% CO₂.

miR-195-5p mimic (5'-UAGCAGCACAGAAAUAUUGGC-3') and its negative control (NC) (5'-UCACAACCUCCUAGAAAGAGUAGA-3') were obtained from Shanghai GenePharma Co., Ltd. SKI overexpression plasmids (OE) and its empty vector were synthesized and provided by Shanghai GenePharma Co., Ltd. XPTS-1 and EOMA cells were transfected with 50 nM of miR-195-5p mimics, NC mimics, SKI OE or SKI empty vector using Lipofectamine^® 2000 at 37˚C (Thermo Fisher Scientific, Inc.). After 48 h, the transfected cells were used in the following experiments.

Total RNA was isolated from HUVECs, XPTS-1 and EOMA cells using TRIzol^® reagent and a PureLink miRNA Isolation kit (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse-transcribed into cDNA using aa PrimeScript RT Reagent Kit (Takara Bio, Inc.) (Roche Diagnostics) according to the manufacturer's protocol. Subsequently, qPCR was performed using SYBR Premix Ex Taq kit (Takara Bio, Inc.) and the following thermocycling conditions: Initial denaturation for 5 min at 95˚C, followed by 40 cycles at 95˚C for 30 sec and 65˚C for 45 sec. miRNA and mRNA expression levels were quantified using the 2^-ΔΔCq method (21) and were normalized to the internal reference genes U6 and GAPDH, respectively. The sequences of the primers used were as follows: miR-195-5p, forward 5'-GGCTAGCAGCACAGAAAT-3' and reverse 5'-GTGCAGGGTCCGAGGT-3'; U6, forward 5'-CTCGCTTCGGCAGCACA-3' and reverse 5'-AACGCTTCACGAATTTGCGT-3'; SKI, forward 5'-CTTCCAATAAGAGCCTG-3' and reverse 5'-ATGAGGTAAAGGACGG-3'; GAPDH, forward 5'-TCCATGACAACTTTGGTATCG-3' and reverse 5'-GTCGCTGTTGAAGTCAGAGGA-3'.

Total protein was isolated from HUVECs, XPTS-1 and EOMA cells. Protein concentrations were determined using a BCA kit (Beyotime Institute of Biotechnology). Proteins (20 µg per lane) were separated via 10% SDS-PAGE and transferred onto PVDF membranes. Following blocking with 5% skimmed milk at room temperature for 2 h, the membranes were incubated at 4˚C overnight with primary antibodies, including anti-BAX (ab32503, 1:5,000), anti-Bcl-2 (ab182858, 1:2,000), anti-poly(ADP-ribose) polymerase 1 (anti-PARP1; ab191217, 1:1,000), anti-cleaved PARP (ab32064, 1:5,000) and then goat anti-rabbit IgG H&L secondary antibodies (ab6721, 1:10,000) at room temperature for 2 h in the dark. All antibodies were purchased from Abcam. Protein bands were visualized using chemiluminescence (MilliporeSigma) on ImageJ software (version 1.6; National Institutes of Health).

At 48 h post-transfection, XPTS-1 and EOMA cells were seeded into a 96-well plate (1x10⁴ cells/well). Subsequently, CCK-8 reagent (Dojindo Molecular Technologies, Inc.) was added to each well and incubated for 0, 12, 24, 48 or 72 h. Absorbance at the wavelength of 450 nm was measured using a microplate reader.

XPTS-1 and EOMA cells were collected and seeded into a 6-well plate (1x10³ cells/well) at 37˚C. Cells were fixed with 4% paraformaldehyde at 37˚C for 30 min and stained with 0.5% crystal violet solution at 37˚C for 15 min. The number of colonies (>50 cells) was observed in five fields of view under a light microscope (Nikon Corporation; magnification, x100).

HA cell proliferation was determined using an EdU assay kit (Invitrogen; Thermo Fisher Scientific, Inc.). Following incubation with 10 µM EdU reagent for 2 h at 37˚C, XPTS-1 and EOMA cells were fixed with 4% paraformaldehyde at room temperature for 10 min, and then treated with 1X Apollo^® Reaction Cocktail (Guangzhou RiboBio Co., Ltd.) and stained with Hoechst 33342 (Sigma-Aldrich; Merck KGaA) at room temperature for 5 min. Subsequently, the cells were stained with DAPI at room temperature for 10 min, and the stained cells were visualized using a fluorescence microscope (Nikon Corporation; magnification, x400). The ratio of EdU-positive cells to total cells was calculated as the proliferation index.

HA cell apoptosis was assessed using the Annexin V and PI Apoptosis Detection kit (BD Biosciences). At 48 h post-transfection, XPTS-1 and EOMA cells were trypsinized and harvested. Cells were washed with PBS and resuspended (2-3x10⁶ cells/ml). Following centrifugation at 716 x g for 10 min at room temperature, cells were incubated with 50 µg/ml Annexin V-FITC and PI in the dark at room temperature for 20 min. Late apoptotic cells were analyzed within 1 h of staining using a FACSCalibur flow cytometer (BD Biosciences) on using FlowJo 10.07 software (FlowJo LLC).

In total, 100 µl of XPTS-1 and EOMA cells were plated into 24-well plates (~5x10⁷ cells/ml). After fixation 4% neutral formalin for 10 min at room temperature and permeabilization with 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4˚C, cells were stained with TUNEL reagent (50 µl) added to each well (the ratio of TdT enzyme to fluorescent labeling solution was 1:24) at 37˚C for 1 h using a TUNEL kit (Shanghai Runwell Technology Co., Ltd.) according to the manufacturer's protocols. Cells were mounted in antifade medium and stored at 2-8˚C and were then stained with DAPI at 4˚C for 10 min. Subsequently, stained cells in five fields were visualized using a fluorescence microscope (magnification, x400).

The target of miR-195-5p was predicted using TargetScan 7.2 (www.targetscan.org/vert_72). The psiCHECK-2 luciferase reporter vector SKI wild-type (wt) and mutant (mut) containing the binding sites of miR-195-5p were provided and synthesized by Shanghai GenePharma Co., Ltd. XPTS-1 and EOMA cells (1x10⁵ cells) were co-transfected with miR-195-5p mimic or NC mimic and psiSKI-wt or psiSKI-mut at a concentration of 50 nM using Lipofectamine^® 2000 for 48 h at 37˚C. Subsequently, the results were determined with a Dual Luciferase Reporter kit (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Each experiment was conducted thrice. Statistical analyses were performed using SPSS software (version 19.0; IBM Corp.). Data are presented as the mean ± SD. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Bonferroni's post hoc test. P<0.01 was considered to indicate a statistically significant difference.

RT-qPCR was performed to determine the expression levels of miR-195-5p. miR-195-5p was found to be significantly downregulated in XPTS-1 and EOMA cells, suggesting that miR-195-5p may have an antitumor function in HA (Fig. 1A and B).

To evaluate the roles of miR-195-5p in HA, the potential effects of miR-195-5p on HA cell viability, colony formation and proliferation were assessed. miR-195-5p mimics significantly upregulated miR-195-5p expression compared with the NC mimics group, suggesting successful transfection (Fig. 2A and B). miR-195-5p overexpression significantly suppressed HA cell viability compared with the NC mimics group (Fig. 2C and D). Consistent with the inhibitory effect of miR-195-5p on HA cell viability, miR-195-5p mimics significantly decreased the number of colonies compared with the NC mimics group (Fig. 2E and F). Moreover, miR195-5p overexpression markedly reduced the number of the EdU-positive cells, suggesting that miR-195-5p suppressed HA cell proliferation (Fig. 2G and H).

Flow cytometry and TUNEL assays were performed to detect HA cell apoptosis. The rate of HA cell apoptosis was significantly increased by miR-195-5p mimics (Fig. 3A and B). The TUNEL assay results were consistent with the flow cytometry results. miR-195-5p mimics markedly increased the number of TUNEL-positive cells (Fig. 3C and D).

To explore the mechanisms underlying miR-195-5p-mediated modulation of HA progression, the possible target of miR-195-5p was predicted. Among the identified targets, SKI was found to be closely involved in the progression of HA (20). However, the roles of SKI in cancer remain incompletely understood. Therefore, the present study investigated the roles of SKI in HA. TargetScan was used to predict the binding sites of miR-195-5p on its target SKI (Fig. 4A). The dual luciferase reporter assay was conducted to demonstrate whether miR-195-5p could directly target SKI. Luciferase activities were significantly decreased after co-treatment with miR-195-5p mimics and psiSKI-wt, whereas there was no significant difference in the psiSKI-mut group (Fig. 4B and C). The results indicated that SKI was directly targeted by miR-195-5p. Additionally, the expression levels of SKI were found to be increased in HA cells (Fig. 4D and E), whereas overexpression of miR-195-5p suppressed the protein levels of SKI (Fig. 4F), and SKI expression was increased following transfection with SKI overexpression plasmids (Fig. 4G and H). Moreover, SKI mRNA expression levels in HA cells were found to be significantly increased, whereas miR-195-5p overexpression significantly downregulated SKI expression levels, which was alleviated by SKI overexpression plasmids (Fig. 4I and J).

It was hypothesized that miR-195-5p may attenuate HA progression by binding to the 3'-UTR of SKI. Overexpression of SKI antagonized the effects of miR-195-5p on the viability of XPTS-1 and EOMA cells (Fig. 5A and B). The decrease in colony numbers induced by miR-195-5p was reversed by SKI (Fig. 5C and D). Furthermore, SKI overexpression alleviated the reduction in EdU-positive cell numbers induced by miR-195-5p (Fig. 5E and F).

The flow cytometry results demonstrated that the miR-195-5p-induced increases in HA cell apoptosis were reversed by SKI overexpression (Fig. 6A and B). The TUNEL assay results indicated that SKI abrogated the increase in the numbers of TUNEL-positive cells induced by miR-195-5p (Fig. 6C and D). Additionally, the regulatory effects of miR-195-5p on Bcl-2, Bax and PARP expression were alleviated by SKI (Fig. 6E and F).