MicroRNA-195-5p inhibits the progression of hemangioma via targeting SKI.

Hemangioma (HA), which is characterized by aberrant endothelial cell proliferation in blood vessels, is a common tumor during infancy. MicroRNAs (miRNAs/miRs) collectively participate in the development of HA; however, the potential roles of miR-195-5p in HA are not completely understood. The aim of the present study was to investigate the roles of miR-195-5p in HA. In the present study, miR-195-5p was found to be downregulated in HA cells, such as the XPTS-1 human infantile hemangioma-derived endothelial cell line and the EOMA hemangioendothelioma cell line. Overexpression of miR-195-5p was shown to suppress HA cell viability, colony formation and proliferation, and induced HA cell apoptosis. Furthermore, miR-195-5p downregulated Bcl-2 expression and upregulated Bax and Bcl-2 expression levels. V-ski sarcoma viral oncogene homolog (SKI) was identified as a target of miR-195-5p. Co-transfection of miR-195-5p mimics and SKI 3'-untranslated region wild-type decreased HA cell luciferase activity. SKI overexpression alleviated the miR-195-5p-induced decrease in HA cell proliferation and increased HA cell apoptosis. In addition, the regulatory role of miR-195-5p on the expression of Bcl-2, Bax and poly(ADP-ribose) polymerase was reversed by SKI. Collectively, the results of the present study demonstrated that miR-195-5p suppressed HA progression and its effects were mediated via SKI. Therefore, the miR-195-5p/SKI axis may represent a novel therapeutic target for HA. Copyright: © Sun et al.

Introduction

Hemangioma (HA), which is characterized by aberrant endothelial cell proliferation in blood vessels, is a common tumor during infancy (1), with an incidence of 5-10% (2). Although HAs generally stop progressing within 5-10 years, their development during the first year is rapid, resulting in 10-15% of HA cases being life-threatening (3). At present, the cause of HA is not completely understood (4). Therefore, identifying the potential mechanisms underlying the development and progression of HA may aid with the design of a therapeutic strategy for HA.

MicroRNAs (miRNAs/miRs) are a family of short non-coding RNAs that are 18-25 nucleotides in length (5). miRNAs serve a crucial role at the post-transcriptional level and regulate gene expression via binding to the 3'-untranslated region (3'-UTR) of target genes (6). Aberrant expression of miRNAs may contribute to abnormal cell activities, including proliferation, apoptosis, migration, invasion and autophagy, which further induce the initiation and development of various tumors, including HA (7,8). For example, miR-501 has been found to be upregulated in HA tissues and cells, whereas miR-501 knockdown inhibits the proliferation and migration of HA cells (9). In addition, miR-187-3p overexpression was found to promote the chemosensitivity of HA-derived stem cells to propranolol (10). miR-195-5p, as a member of the miR-15/107 family, participates in the progression of various types of cancer (11-13). A previous study demonstrated that the expression of miR-195-5p in patients with HA (n=56) was decreased to 0.48 compared with healthy controls (n=31), and this downregulation was associated with the migration and invasion of HA cells (14). However, the possible roles of miR-195-5p in HA have yet to be fully elucidated.

V-ski sarcoma viral oncogene homolog (SKI) was first identified as the transforming protein of the avian Sloan-Kettering retrovirus and its overexpression promotes oncogenic transformation (15,16), indicating that SKI possesses oncogenic potential. For example, SKI overexpression was shown to maintain the pluripotency of pancreatic cancer cells (17). miR-127-3p-activated TGF-β inhibits the progression of glioblastoma via downregulating SKI (15). SKI participates in the progression of cancer via regulating various cellular behaviors, including proliferation, apoptosis, migration and autophagy (18). SKI knockout suppresses osteosarcoma cell proliferation and migration (19). Moreover, in HA, overexpression of SKI, a transcriptional co-repressor, contributes to the uncontrolled proliferation and transformation of endothelial cells via inactivating TGF-β signaling (20). However, the roles of SKI in HA are not completely understood.

The aim of the present study was to investigate the possible roles of miR-195-5p in HA, in order to determine whether miR-195-5p can inhibit HA cell proliferation and apoptosis, and whether miR-195-5p may be of value as a novel biomarker for the treatment of infantile HA.

Materials and methods

Cell culture

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.

Cell transfection

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.

Reverse transcription-quantitative PCR (RT-qPCR)

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'.

Western blotting

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).

Cell Counting Kit-8 (CCK-8) assay

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.

Colony formation assay

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).

EdU assay

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.

Flow cytometry

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).

TUNEL assay

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).

Dual luciferase reporter assay

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.

Statistical analysis

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.

Results

miR-195-5p expression is decreased in HA cells

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).

Figure 1

miR-195-5p is downregulated in HA cells. Reverse transcription-quantitative PCR analysis revealed decreased miR-195-5p expression levels in (A) EOMA cells and (B) XPTS-1 cells compared with HUVECs. **P<0.01 vs. control. miR, microRNA; HA, hemangioma.

miR-195-5p overexpression inhibits HA cell proliferation

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).

Figure 2

miR-195-5p suppresses the proliferation of HA cells. miR-195-5p expression levels in (A) EOMA cells and (B) XPTS-1 cells were measured via reverse transcription-quantitative PCR analysis and found to be significantly upregulated by miR-195-5p mimics. (C) EOMA and (D) XPTS-1 cell viability was determined via the Cell Counting Kit-8 assay and was found to be significantly reduces by miR-195-5p overexpression. Colony numbers of (E) EOMA and (F) XPTS-1 cells were detected using the colony formation assay and found to be significantly decreased by miR-195-5p mimics. The proliferation of (G) EOMA and (H) XPTS-1 cells was assessed by EdU assays and found to be suppressed by miR195-5p overexpression. Scale bar, 20 µm. **P<0.01 vs. NC. miR, microRNA; HA, hemangioma; NC, negative control.

miR-195-5p promotes HA cell apoptosis

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).

Figure 3

miR-195-5p increases HA cell apoptosis. Apoptosis of (A) EOMA and (B) XPTS-1 cells was detected via flow cytometry. Apoptosis of (C) EOMA and (D) XPTS-1 cells was detected by TUNEL assays. Cell apoptosis was significantly increased by miR-195-5p mimics. Scale bar, 20 µm. **P<0.01 vs. NC. miR, microRNA; HA, hemangioma; NC, negative control.

miR-195-5p directly targets SKI

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).

Figure 4

miR-195-5p directly targets SKI in HA cells. (A) Binding sites between miR-195-5p and SKI. Cell luciferase activity in (B) EOMA and (C) XPTS-1 cells was measured by dual luciferase reporter assay. SKI expression was increased in (D) EOMA and (E) XPTS-1 cells. (F) SKI protein expression levels were measured via western blotting and were found to be decreased following overexpression of miR-195-5p. (G and H) SKI mRNA expression levels were determined via reverse transcription-quantitative PCR analysis and found to be increased following transfection with SKI overexpression plasmids. (I and J) SKI mRNA expression levels were determined via reverse transcription-quantitative PCR analysis and were found to be significantly downregulated by miR-195-5p overexpression, and this effect was alleviated by SKI overexpression plasmids. **P<0.01 vs. control or NC; ##P<0.01 vs. miR-195-5p mimics. HA, hemangioma; miR, microRNA; SKI, v-ski sarcoma viral oncogene homolog; WT, wild-type.

miR-195-5p inhibits the proliferation of XPTS-1 and EOMA cells by binding to SKI

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).

Figure 5

miR-195-5p inhibits the proliferation of HA cells via targeting SKI. (A) EOMA and (B) XPTS-1 cell viability was determined by the Cell Counting Kit-8 assay. (C) EOMA and (D) XPTS-1 cell proliferation was determined by performing colony formation assays. The decrease in colony numbers induced by miR-195-5p was reversed by SKI. (E) EOMA and (F) XPTS-1 cell proliferation was detected by performing EdU assays. SKI overexpression alleviated the reduction in EdU-positive cell numbers induced by miR-195-5p. Scale bar, 20 µm. **P<0.01 vs. control or NC; ##P<0.01 vs. miR-195-5p mimics. HA, hemangioma; miR, microRNA; SKI, v-ski sarcoma viral oncogene homolog.

miR-195-5p promotes the apoptosis of XPTS-1 and EOMA cells by binding to SKI

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).

Figure 6

miR-195-5p promotes apoptosis of HA cells via targeting SKI. (A) EOMA and (B) XPTS-1 cell apoptosis was determined via flow cytometry. (C) EOMA and (D) XPTS-1 cell apoptosis was assessed by TUNEL assays. Bax, Bcl-2 and PARP protein expression levels were measured via western blotting in (E) EOMA and (F) XPTS-1 cells. The regulatory effects of miR-195-5p on Bcl-2, Bax and PARP expression were alleviated by SKI. Scale bar, 20 µm. **P<0.01 vs. NC; ##P<0.01 vs. miR-195-5p mimics. miR, microRNA; HA, hemangioma; SKI, v-ski sarcoma viral oncogene homolog; PARP, poly(ADP-ribose) polymerase; NC, negative control.

Discussion

Hemangioma is one of the most common infantile tumors, with 10-15% of HA cases being life-threatening (1,2). The aim of the present study was to investigate the potential roles of miR-195-5p in HA. Previous studies revealed that miR-195-5p functions as a tumor suppressor (22,23) and abnormal miR-195-5p expression predicts poor prognosis (24). Zhang et al (14) revealed that miR-195-5p knockdown promoted HA cell migration and invasion. In the present study, miR-195-5p was found to be downregulated in HA cells, suggesting that miR-195-5p may have an antitumor function in HA. However, the potential mechanisms underlying the role of miR-195-5p in HA are not completely understood.

miRNAs participate in the initiation and progression of HA (9,10,25). Aberrantly expressed miRNAs in cancer may serve as oncogenes or tumor suppressor genes (8,9). miRNAs participate in the progression of cancer via regulating cellular behaviors, including cell proliferation and apoptosis (7,8,23,25). miR-195-5p was found to act as an antitumor miRNA in various types of cancer (11-13). In the present study, miR-195-5p inhibited HA cell proliferation and promoted HA cell apoptosis. Moreover, miR-195-5p overexpression decreased the expression level of pro-proliferative genes, including Bcl-2, and increased the expression levels of pro-apoptotic genes, including Bax and PARP (26). Therefore, the results of the present study suggested that miR-195-5p may act as an antitumor miRNA in HA, which was consistent with the findings of Zhang et al (14). The results of the present study indicated that miR-195-5p inhibited HA progression via regulating HA proliferation and apoptosis; however, the underlying molecular mechanism is not completely understood.

Accumulating evidence has revealed that miRNAs participate in the progression of cancer via binding to the 3'-UTR of their target genes (16). In the present study, SKI was predicted and verified as a target of miR-195-5p. miR-195-5p overexpression decreased SKI expression levels. SKI has been reported to serve as an oncogene in various tumors, including HA (15,17,20). However, the role of SKI in tumors is contradictory (27), as it exerts both oncogenic and tumor-suppressive effects. SKI, which is located at chromosome 1p36, is a tumor suppressor locus that is typically degraded in melanoma and neuroblastoma (28). Moreover, SKI functions as a transcriptional corepressor, inactivating TGF-β signaling pathways to modulate tumor development (29). Thus, the potential role of SKI in tumors may vary across different cell types and signaling pathways (19,20,30). In the present study, SKI was found to be upregulated in HA cells. SKI overexpression abrogated miR-195-5p-mediated effects on HA cell proliferation and apoptosis, and on the expression of apoptosis-related genes, including Bcl-2, Bax and PARP (26), which may be due to the pro-proliferative and anti-apoptotic effects of SKI in tumors. Collectively, the results of the present study suggested that miR-195-5p may regulate HA progression via targeting SKI.

In conclusion, miR-195-5p expression was found to be decreased in HA cells, whereas miR-195-5p overexpression inhibited HA cell proliferation and promoted HA cell apoptosis via targeting SKI. Therefore, the miR-195-5p/SKI axis may serve as a novel biomarker for HA.