Epigenetic Silencing of Mir-130A Ameliorates Hemangioma by Targeting Tissue Factor Pathway Inhibitor 2 Through FAK/PI3K/Rac1/Mdm2 Signaling

INTERNATIONAL JOURNAL OF ONCOLOGY 50: 1821-1831, 2017

Epigenetic silencing of miR-130a ameliorates hemangioma by targeting tissue factor pathway inhibitor 2 through FAK/PI3K/Rac1/mdm2 signaling

Feng Gao, Fa-gang Wang, Ren-rong Liu, Feng Xue, Jian Zhang, Guang-qi Xu, Jian-hai Bi, Zhen Meng and Ran Huo

Department of Aesthetic, Plastic and Burn Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shangdong 250021, P.R. China

Received December 31, 2016; Accepted March 13, 2017

DOI: 10.3892/ijo.2017.3943

Abstract. Hemangiomas are the most common vascular growth, restrained angiogenesis by decreasing the expression tumors that occur frequently in prematures and females. of angiogenesis markers and the percentage of CD31+ and microRNA (miR)-130a is associated with the growth and CD34+. Taken together, our research indicated that miR-130a invasion in many tumors, and its role in hemangiomas has functions as an oncogene by targeting TFPI2, miR-130a inhi- not been addressed so far. The present study revealed that bition reduced the growth and angiogenesis of hemangioma miR‑130a was overexpressed in infantile hemangioma tissues by inactivating the FAK/PI3K/Rac1/mdm2 pathway. Thus, compared with matched tumor-adjacent tissues. The inhibitor miR-130a may serve as a potential therapeutic strategy for the of miR-130a restrained cell growth and induced cell apop- treatment of hemangioma. tosis in vitro. miR‑130a inhibitor also induced a cell cycle arrest at G2/M phase. Further studies revealed that tissue Introduction factor pathway inhibitor 2 (TFPI2) was a novel miR-130a target, due to miR-130a bound directly to its 3'-untranslated Infantile hemangioma (IH) is a common benign tumor in region and miR-130a inhibitor enhanced the expression of childhood, and more frequent in prematures and low birth TFPI2. Contrary to the effects of miR-130a inhibitor, TFPI2 weight infants (1). IH has three distinctive clinical and histo- siRNA strongly promoted cell growth and colony formation, logical phases: an initial proliferative phase with rapid growth whereas TFPI2 overexpression contributed to the suppressing and undeveloped blood channels, a plateau and the final effect of miR-130a inhibitor in cell viability. Furthermore, involuted phase with scattered capillaries and large draining miR-130a inhibitor reduced the activation of focal adhesion vessels (2). IH is characterized by the spontaneous regression kinase (FAK)/phosphoinositide 3-kinase (PI3K)/Rac1/anti- of blood vessels. IHs are disfiguring in some cases, which is mouse double minute (mdm2) pathway proteins, inhibited also the leading cause of morbidity in affected children (2,3). the expression and nuclear translocation of mdm2. Moreover, Recently, advances have been made in the treatment of IH FAK overexpression prevented miR-130a inhibitor-induced with clear curative effect, such as propranolol (4), nadolol (5) cell cycle arrest and decrease of cell viability. In vivo experi- and pulsed dye laser (6). However, the main clinical problem ments, miR-130a inhibition effectively suppressed the tumor is still the lack of reliable parameters to treat IH lesions in patients. Therefore, the identification of genetic and epigenetic alterations in IH lesions will help to establish molecular mechanisms of this desease. Correspondence to: Dr Ran Huo, Department of Aesthetic, In recent years, miRNAs are considered as ideal Plastic and Burn Surgery, Shandong Provincial Hospital Affiliated biomarkers for cancer identification, for the properties that to Shandong University, No. 324 Jingwu Road, Jinan, Shandong miRNAs are highly conserved in the genome and have a 250021, P.R. China relatively high degree of tissue specificity (7). Abnormal E-mail: [email protected] expression of miRNAs is deeply associated with numerous pathological processes, such as angiogenesis (8), carcinogen- Abbreviations: IH, infantile hemangioma; miR, microRNA; esis (9) and inflammation (10). The microRNA (miR)-130 TFPI2, tissue factor pathway inhibitor 2; PCNA, proliferating cell family is composed of miR-130b, miR-301a and miR-301b, nuclear antigen; FAK, focal adhesion kinase; PI3K, phosphoinositide 3-kinase; mdm2, mouse double minute; VEGF, vascular endothelial which share a common seed sequence (9). miR-130a is growth factor; MMP, matrix metalloproteinase located at chromosome 11q12, close to the 11q13 area (11). miR-130a has been found to be involved in multiple types of Key words: hemangioma, microRNA-130a, tissue factor pathway human diseases, including gastric (12), cervical cancer (13), inhibitor 2, focal adhesion kinase, angiogenesis chronic myeloid leukemia (14) and metabolism-related inflammation (15). Besides, reports indicate that miR-130a 1822 Gao et al: silencing of miR-130a ameliorates hemangioma promote the proliferation, migration and tube formation of Northern blot analysis. The expression levels of miR-130a in vascular endothelial cells (16). However, the comprehensive hemangioma samples, adjacent normal tissues, HUVEC-12, effect of the miR-130a on tumor progression of hemangioma XPTS-1 and EOMA cells were further determined by northern has not been analyzed. blot assay according to previously described procedures (25). Tissue factor pathway inhibitor 2 (TFPI2) is secreted as a glycosylated protein with a short acidic amino-terminal region, The transfection of miR-130a mimic/inhibitor. The miR-130a three tandem Kunitztype domains and a carboxy-terminal mimic, negative control of miRNA mimic, miR-130a inhibitor tail (17). TFPI2 belongs to a Kunitz family of serine protease and negative control of miRNA inhibitor were purchased from inhibitors that contain one or more Kunitz-type domains. The Shanghai GenePharma, Co., Ltd. (Shanghai, China), and trans- family has more than 20 members including pancreatic trypsin fected into XPTS-1 and EOMA cells at a final concentration inhibitor (aprotinin) and the homolog of TFPI2 (18). A report of 100 nM with Lipofectamine 2000 (Invitrogen) according indicated that human TFPI2 was first identified in the placenta to the manufacturer's instructions. After 6 h, the cells were to inhibit the amidolytic activity of plasmin (19). Recent returned to normal medium and cultured for an additional reports suggested that the downregulation were associated 48 (for RNA isolation) or 72 h (for protein extraction). with the development of multiple deseases, including atherosclerotic plaque (20), pancreatic carcinoma (21), hepatocellular Colony formation assay. XPTS-1 and EOMA cells were carcinoma (22) and cervical cancer (23). Considering TFPI2 is transfected with miR-130a inhibitor or control. After 48 h of secreted into the extracellular matrix (ECM) to help regulate infection, the cells were plated in 6-well plates at 200/well and ECM remodeling, the role of TFPI2 involved in hemangioma grown for 2 weeks. Then, the cells were washed twice with is worthy of investigation. phosphate-buffered saline (PBS), fixed with methanol/acetic The present study explored the expression of miR-130a acid (3:1, vol/vol) and stained with 0.5% crystal violet. The in hemangioma tissues and cell lines. We found that the number of colonies was counted under the microscope. inhibition of miR-130a effectively suppressed the growth of hemangioma by targeting TFPI2 and inactivating focal Cell apoptosis assay and cell cycle analysis. XPTS-1 (5x103) adhesion kinase (FAK)/phosphoinositide 3-kinase (PI3K)/ and EOMA cells were plated into each well of 96-well plates, Rac1/anti-mouse double minute (mdm2) pathway. This study respectively. Six hours later, the cells were treated with also revealed an inhibitory effect of angiogenesis via down- miR-control or miR-130a inhibitor. The procedures of cell regulating miR‑130a. cycle analysis were previously described (21). Cell numbers were determined by Scepter™ 2.0 Handheld Automated Cell Materials and methods Counter.

Hemangioma samples. Twenty pairs of human hemangioma Western blotting. Cell samples were lysed in lysis buffer and adjacent normal tissues were obtained from Shandong (Beyotime Institute of Biotechnology, Haimen, China). The Provincial Hospital Affiliated to Shandong University samples mixed with loading buffer were incubated in boiling (Shandong, China). The tissues were frozen in liquid nitrogen water for 10 min. protein (20-30 µg) was separated through and stored at -80˚C until use. Written informed consent for SDS-PAGE and then transferred onto polyvinylidene fluo- tissue donation (for research purposes) was obtained from the ride (PVDF) membranes (Millipore, Billerica, MA, USA). patients, and the protocol was approved by the Institutional The following primary antibodies were used: anti-TFPI2, Review Board of the Provincial Affiliated Hospital of the anti-Ki‑67, anti-proliferating cell nuclear antigen (PCNA), Shandong University. anti-FAK, anti-PI3K, anti-Rac1, anti-mdm2, anti-p-FAK, anti-p-PI3K, anti-p-Rac1, anti-p-mdm2, anti-vascular Cell lines. The human vascular endothelial HUVEC-12 endothelial growth factor (VEGF), anti-matrix metallopro- cells, human IH cell lines XPTS-1 and mouse hemangioen- teinase (MMP)-2, anti-MMP-9 and anti-GAPDH (Abcam, dothelioma cells (EOMA) were obtained from the American Hangzhou, China), which was used as the internal reference. Type Culture Collection (ATCC; Manassas, VA, USA). After incubation with the appropriate horseradish peroxidase These cell lines were cultured in Dulbecco's modified Eagle's (HRP)-conjugated secondary antibody, proteins were detected medium (DMEM) plus 10% fetal bovine serum (fbs; Life using a ChemiDoc XRS imaging system and Quantity One Technologies, Inc., Grand Island, NY, USA) and grown at 37˚C analysis software (Bio-Rad Laboratories, San Francisco, CA, in a 5% CO2 atmosphere. USA).

Quantitative real-time polymerase chain reaction (qRT-PCR). Luciferase reporter assay. The cDNA of TFPI2 was ampli- The total RNA was extracted from TRIzol reagent (Invitrogen, fied by PCR, and general method of recombinant DNA was Carlsbad, CA, USA) following the manufacturer's instructions. used to clone the wild-type (WT) 3'-UTR and mutant (MUT) The RT-PCR primers for miR-130a and U6 were purchased from 3'-UTR of TFPI2. The WT and MUT sequences of TFPI2 GeneCopoeia , Inc. (San Diego, CA, USA). The PCR primers for were then cloned into a pMIR-Report luciferase reporter TFPI2 were: forward, 5'-GTCGATTCTGCTGCTTTTCC-3' vector (Ambion, Austin, TX, USA) to generate constructs and reverse, 5'-ATGGAATTTTCTTTGGTGCG-3' (440 bp) of Luc-TFPI2 and Luc-TFPI2-mut, followed by DNA (24). GAPDH and U6 SnRNA were used as the internal sequencing verification. The pMIR-Report control vector control of the mRNA or miRNA, respectively. Fold-change of (Luc-control) was also cloned. All three vectors were trans- miR-130a was calculated by the 2-ΔΔCt method. fected into XPTS-1 cells/EOMA cells in 6-well plates with INTERNATIONAL JOURNAL OF ONCOLOGY 50: 1821-1831, 2017 1823 a Lipofectamine 2000 reagent kit (Sigma-Aldrich, St. Louis, MO, USA). After 1 day, the luciferase activity was measured using a luciferase reporter assay system (Promega, Madison, WI, USA). All relative luciferase activities were normalized to the condition of Luc-control with β-galactosidase transfection. siRNA transfection. The small interfering RNA (siRNA), to genetically downregulate TFPI2 (TFPI2-siRNA), as well as its non-specific scramble siRNA (NC-siRNA), were purchased from Guangzhou RiboBio, Co., Ltd. (Guangzhou, China). Transfection of siRNAs into XPTS-1 cells was conducted using Lipofectamine 2000 reagent kit. In XPTS-1 cells, 100 nM siRNAs (TFPI2-siRNA and NC-siRNA) were used. The efficiency of siRNA interference was confirmed by western blot analysis 48 h after transfection.

Overexpression of TFPI2 and FAK. TFPI2 and FAK overexpression was achieved by PCR amplification using their cDNA as templates, separately, and the TFPI2 and FAK expressing vectors were constructed by inserting their cDNA into pcDNA 3.1 vector (Sigma-Aldrich). The recombinant plasmids and other agents were co-transfected into 3x106 XPTS-1 cells using a Nucleofector instrument. Forty-eight hours later, subsequent experiments were performed on the cells. The experiment was replicated thrice for data calculations.

Immunofluorescence staining.Fluorescent cells were cultured on an 8-well chamber culture slides (Becton-Dickinson, Bedford, MA, USA). After 8 h, cells were fixed in 3% para- formaldehyde in PBS at room temperature for 8 min, then permeabilized with 0.2% Triton X-100 for 15 min at room temperature. After washing in PBS, the cells were incubated with primary mouse anti-FAK monoclonal antibody (1 mg/ml; Figure 1. miR-130a is overexpressed in hemangioma tissues and cell lines. Transduction Laboratories, Lexington, KY, USA) at 4˚C over- (A) The relative expression of miR-130a in 20 human hemangioma tissues and adjacent non-tumor tissues was measured by qPCR. (B) The expres- night. After washing, cells were incubated with biotinylated sion of miR-130a in human vascular endothelial cells (HUVEC-12) and goat anti-mouse IgG (Pierce, Rockford, IL, USA) at room hemangioma cell lines (XPTS-1 and EOMA) was measured by qRT-PCR. temperature for 1 h. The immunoreactivity was revealed using The expression profile of miR-130a in hemangioma tissues (C) and cell lines Alexa568-conjugated streptavidin (Molecular Probes, Eugene, (D) was measured using northern blot assay. U6 was used as endogenous references. Data are represented as the mean ± SD of three experiments. OR, USA) and cells were counterstained with 10 mg/ml DAPI. **P<0.01 vs. non-tumor tissues, ***P<0.001 vs. HUVEC-12. The cells were examined under a Nikon fluorescence microscope (Image Systems, Columbia, MD, USA).

Hemangioma xenografts. Male athymic nude mice were analysis. Briefly, 5 µm-thick paraffin sections were deparaf- housed and manipulated according to the protocols approved finized in xylene and rehydrated in a 100, 95 and 75% ethanol by the Experimental Animal Center of the Shandong gradually. In order to quench the activity of endogenous

Provincial Hospital Affiliated to Shandong University. For peroxidase, the tissue sections were incubated in 30% H2O2 each mouse, 5x106 miR-130a-inhibiting XPTS-1 cells were for 30 min. After antigen retrieval in heated 10 mM citrate injected subcutaneously into the right scapula in 100 serum- buffer for 10 min, the tissue sections were immunostained free medium. After the development of a palpable tumor, the with mouse anti-human CD31 and CD34 primary antibody tumor volume was monitored every 5 days and assessed by overnight at 4˚C. Corresponding mouse horseradish peroxi- measuring the 2 perpendicular dimensions using a caliper and dase (HRP)-conjugated secondary antibody was added for 1 h the formula (a x b2)/2, where a is the larger and b is the smaller at room temperature. Images were obtained using an Olympus dimension of the tumor. At 25 days after inoculation, the mice IX71 inverted microscope. were sacrificed and tumor weights were assessed. A portion of each tumor was selected for western blotting for TFPI2 and Statistical analysis. All results are presented as mean ± SD key components of the FAK pathway and qRT-PCR analysis from a minimum of 3 replicates. Differences between groups for miR-130a. were evaluated by SPSS version 15.0 statistical software with Student's t-test when comparing only 2 groups or assessed by Immunohistochemistry. Tumor tissues were fixed in formalin, one-way ANOVA when >2 groups were compared. For the and then were embedded with paraffin for hemangioma C comparison of paired tissues, a paired Student's t-test was used 1824 Gao et al: silencing of miR-130a ameliorates hemangioma

Figure 2. The inhibition of miR-130a restrains hemangioma cell viability. (A) XPTS-1 and EOMA cells were transfected with miR-130a inhibitor, followed by qRT-PCR detection of miR-130a expression at 48 h after the treatment. (B) Northern blotting was used to confirm the effectiveness of miR-130a mimic transfection on XPTS-1 and EOMA cells. (C) The expression of Ki-76 and PCNA was detected by western blot assays in XPTS-1 and EOMA cells when miR-130a was inhibited for 48 h. The quantified expression of miR-130a, Ki-76 and PCNA in XPTS- (D) and EOMA cells (E) was statistically analyzed. (F) The number of colonies was counted under the microscope in XPTS-1 and EOMA cells transfected with miR-control or miR-130a inhibitor. Cell apoptosis was measured by flow cytometry (G) and was statistically analyzed in XPTS-1 and EOMA cells (H). (I) The distributions of G0/G1, S and G2/M phases were evaluated in XPTS-1 and EOMA cells transfected with miR-130a inhibitor for 48 h. (J) The statistical analysis of cell cycle distribution. The data are presented as means ± SD of three experiments. The statistically significant differences compared with the control are indicated: *P<0.05, **P<0.01; ***P<0.001 vs. inhibitor control group.

to determine statistical significance. The relationship between frozen hemangioma specimens and matched tumor-adjacent TFPI2 and miR-130a expression was explored by Spearman's normal tissues. The results of qPCR showed that the average correlation. Differences were considered statistically signifi- expression levels of miR-130a were significantly higher in cant at P<0.05. hemangioma tissues than that in adjacent normal tissues (P<0.01; Fig. 1A). Consistently, miR-130a expression was also Results increased in hemangioma cell lines (XPTS-1 and EOMA), compared with normal vascular endothelial cells (HUVEC-12) miR-130a is overexpressed in hemangioma tissues and cell (P<0.001; Fig. 1B). Northern blot analysis further confirmed lines. The expression of miR-130a was measured in 21 fresh- the increased expression of miR-130a in hemangioma speci- INTERNATIONAL JOURNAL OF ONCOLOGY 50: 1821-1831, 2017 1825

Figure 3. TFPI2 is a direct target of miR-130a. (A) Target relationship between miR-130a and TFPI2; TFPI2 expression was detected by immunoblot assays in XPTS-1 and EOMA cells transfected with miR-130a inhibitor (B) or mimic (C) for 48 h. Relative protein expression of TFPI2 in XPTS-1 and EOMA cells transfected with miR-130a inhibitor (D) or mimic (E) was quantified using Image-Pro Plus 6.0 software and normalized to GAPDH. (F) The mutated genes in TFPI2; XPTS-1 cells were transfected with luciferase reporter vectors containing wild-type or mutant TFPI2 3'UTR. The expression levels of luciferase were determined 48 h after transfection of miR-130a inhibitor or inhibitor control in XPTS-1 (G) and EOMA (H) cells. Data are presented as means ± SD of three experiments. The statistically significant differences compared with the control are indicated: **P<0.01, ***P<0.001.

mens (Fig. 1C) and cell lines (Fig. 1D). Taken together, these TFPI2 is a direct target of miR-130a. To explore the molec- results reveal that miR-130a is upregulated in human heman- ular mechanisms of miR-130a in hemangioma cell viability, gioma tissues and cell lines. putative miR-130a targets were predicted by TargetScan (http://www.targetscan.org/vert_71/). The results revealed The inhibition of miR-130a restrains hemangioma cell that TFPI2 was a potential target of miR-130a. The 3'-UTR viability. To investigate whether miR-130a was involved of TFPI2 mRNA contains a complementary site for the seed in regulating hemangioma cell growth, a synthetic region of miR-130a (Fig. 3A). To experimentally confirm inhibitor specific for miR-130a was employed to reduce TFPI2 as an authentic target of miR-130a, a synthetic mimic the level of endogenous miR-130a. The efficiency of this specific for miR-130a was employed to increased the level miR-130a inhibitor was confirmed by qPCR assay and of endogenous miR-130a. TFPI2 levels were immunoblot northern blot analysis (Fig. 2A and B). Two cell prolifer- analyzed adding miR-130a inhibitor or mimic in XPTS-1 and ating markers Ki-67 and PCNA were consistently found EOMA cells. The data indicated that miR-130a inhibition to be underexpressed in XPTS-1 and EOMA cells under induced the restoration of TFPI2 (P<0.01; Fig. 3B and D), miR-130a inhibitor treatment (P<0.05, P<0.01; Fig. 2C-E). whereas miR-130a overexpression caused the decline of TFPI2 Consistently, inhibition of miR‑130a significantly decreased expression (Fig. 3C and E). To further verify the regulatory colony formation in XPTS-1 and EOMA cells compared effect of miR-130a on TFPI2, the plasmid pMIR-Report- with their corresponding control (P<0.001; Fig. 2F). FACS TFPI2wt or pMIR-Report-TFPI2mut was transfected into analysis indicated that miR-130a inhibition induced elevated hemangioma cells together with miR-130a mimic (Fig. 3F). level of cell apoptosis (P<0.001; Fig. 2G and H) and an accu- Luciferase expression levels of pMIR-Report-TFPI2wt were mulation in G2/M phase (P<0.01; Fig. 2I and J), implying suppressed by miR-130a overexpressing. However, muta- the cell cycle arrest of hemangioma cells. The above results tion of the predicted binding site of miR-130a on the TFPI2 indicate that miR-130a inhibition reduces the viability of 3'-UTR rescued the luciferase activity in XPTS-1 and EOMA hemangioma in vitro. cells (P<0.001; Fig. 3G and H). Taken together, these results 1826 Gao et al: silencing of miR-130a ameliorates hemangioma

Figure 4. TFPI2 is involved in miR-130a-induced hemangioma cell growth promotion. The expression of TFPI2 in human hemangioma samples and paired normal tissues was measured by (A) qRT-PCR and (B) western blot analysis. (C) Relative expression of TFPI2 protein was statistical analyzed. (D) A statistically significant inverse correlation between miR-130a and TFPI2 mRNA levels in hemangioma specimens. (E) The expression of TFPI2 in XPTS-1 cells treated with miR-130a inhibitor or transfected with TFPI2 siRNA was detected by western blot assays; cell growth (F) and colony forming rate (G) were measured. (H) The expression of TFPI2 in XPTS-1 cells treated with miR-130a inhibitor or transfected with pcDNA-TFPI2 was detected by western blot assays. (I) Cell growth and (J) colony forming rate were measured. Data are presented as means ± SD of three experiments. The statistically significant differences compared with the control are indicated: *P<0.05; **P<0.01; ***P<0.001.

support the bioinformatics predictions indicating the TFPI2 in Fig. 4E-G, siRNA against TFPI2 enhanced cell growth 3'-UTR to be a direct target of miR-130a. and colony formation, which is contrary to those induced by miR‑130a inhibitor (P<0.001). Moreover, overexpression of TFPI2 is involved in miR-130a-induced hemangioma cell TFPI2 could strongly contribute to the inhibitive effect of growth promotion. To investigate whether TFPI2 serves as miR-130a inhibitor (P<0.001; Fig. 4H-J). These data suggest a critical mediator of miR-130a, we measured the expression that TFPI2 is associated with miR-130a-induced increased of TFPI2 in 21 pairs of clinical hemangioma samples using hemangioma cell viability. western blotting and qRT-PCR (Fig. 4A and B). Compared with paired normal tissues, hemangioma showed significantly lower Inhibition of miR-130a suppresses the activation of FAK/ TFPI2 expression (Fig. 4C). We also analyzed the correlation PI3K/Rac1/mdm2 signaling. To further investigate the mecha- between TFPI2 level and miR-130a expression in the same nism of miR-130a in hemangioma cell viability, the expression patients. As shown in Fig. 4D, a significant inverse correlation level of FAK signaling (FAK, PI3K, Rac1 and mdm2) and was observed (P<0.001, R=0.926). We further performed loss- their phosphorylated forms were measured. No significant of-function and gain-of-function studies by transfecting TFPI2 difference was observed in the expression of unphosphory- siRNA or pcDNA-TFPI2 plasmids in XPTS-1 cells. As shown lated signaling kinases (FAK, PI3K, Rac1 and mdm2) in INTERNATIONAL JOURNAL OF ONCOLOGY 50: 1821-1831, 2017 1827

Figure 5. Inhibition of miR-130a suppresses the activation of FAK/PI3K/Rac1/mdm2 signaling. (A) The expression of FAK, PI3K, Rac1, mdm2 and their phosphorylated forms was measured by western blot analysis in XPTS-1 cells transfected with miR-130a inhibitor or inhibitor control. (B) The ratios of p-FAK/FAK, p-PI3K/PI3K, p-Rac1/Rac1 and p-mdm2/mdm2 was quantified in XPTS-1 cells transfected with miR-130a inhibitor or inhibitor control. (C) The expression of FAK was measured by western blot analysis in XPTS-1 cells transfected with miR-130a inhibitor with/or pcDNA-FAK. (D) Immunofluorescence staining of mdm2 in XPTS-1 cells were stained for mdm2 and counterstained with DAPI as indicated (40 µm). (E) The proliferation of XPTS-1 cells was determined by MTT assays when adding miR-130a inhibitor with/or pcDNA-FAK. (F) Cell apoptosis and the distributions of G0/G1, S and G2/M phases (G) were evaluated in XPTS-1 cells transfected with miR-130a inhibitor with/or pcDNA-FAK for 48 h. Data are presented as means ± SD of three experiments. The statistically significant differences compared with the control are indicated: *P<0.05, **P<0.01; ***P<0.001.

XPTS-1 cells transfected with miR-130a inhibitor. However, FAK transfection displayed reduced nuclear staining of mdm2 the expression of phosphorylated kinases (p-FAK, p-PI3K, under miR-130a inhibitor treatment (Fig. 5D). Shown by MTT p-Rac1 and p-mdm2) was strongly suppressed in XPTS-1 cells assays, miR-130a inhibitor counteracted the promoted prolif- treated with miR‑130a inhibitor transfection (Fig. 5A). The eration of XPTS-1 cells induced by pcDNA-FAK transfection decreased ratios of p-FAK/FAK, p-PI3K/PI3K, p-Rac1/Rac1 (P<0.01; Fig. 5E). Similarly, flow cytometric analysis demon- and p-mdm2/mdm2 further confirmed the inhibitory effect of strated that the reduced cell apoptosis (P<0.001; Fig. 5F) and miR-130a inhibitor in the activation of FAK signaling (P<0.05; G2/M arrest (P<0.01; Fig. 5G) by pcDNA-FAK transfection Fig. 5B). To show convincingly that miR-130a could regulate was prevented adding miR-130a inhibitor in XPTS-1 cells. FAK signaling, gain-of-function analyses were performed by These data clearly suggest that the inhibition of miR-130a overexpressing FAK in XPTS-1 cells transfected with miR- inactivates the FAK/PI3K/Rac1/mdm2 pathway to suppress 130a inhibitor (Fig. 5C). Cells in miR-130a inhibitor group XPTS-1 cell viability. showed minimal mdm2 nuclear staining, whereas cells transfected with pcDNA-FAK exhibited maximum cytoplasmic miR-130a inhibitor reduces tumor growth in hemangioma and nuclear staining of mdm2. Besides, cells with pcDNA- xenograft models. To further investigate the effect of 1828 Gao et al: silencing of miR-130a ameliorates hemangioma

Figure 6. miR-130a inhibitor reduces tumor growth in hemangioma xenograft models. (A) miR-130a-inhibiton or control XPTS-1 cells (106) were injected subcutaneously into nude mice (n=5). At 25 days after implantation, the mice were sacrificed and the tumors were recovered. (B) Volume of xenograft tumors. (C) Levels of miR-130a in xenograft tumor tissues as determined by qRT-PCR. The expressions of TFPI2 (D), Ki-67 and PCNA (E) were detected by western blotting. (F) Western blot analysis of FAK pathway proteins (phosphorylated FAK, PI3K, Rac1 and mdm2) in miR-130a-inhibition and control xenograft tumor tissues. Tumors 1, 2 and 3 were obtained from 3 different mice. Data are presented as means ± SD of three experiments. The statistically significant differences compared with the control are indicated: ***P<0.001.

Figure 7. miR-130a inhibitor suppresses angiogenesis. (A) Western blot analysis of FAK pathway proteins (phosphorylated FAK, PI3K, Rac1 and mdm2) in miR-130a-inhibition and control xenograft tumor tissues. Tumors 1, 2 and 3 were obtained from 3 different mice. (B) Level of surface antigens CD31 and CD34 was detected by immunohistochemical staining. (C) The percentage of surface antigens CD31+ and CD34+ was measured by flow cytometry and statistical analyzed (D and Ε). Data are presented as means ± SD. The statistically significant differences compared with the control are indicated: ***P<0.001. INTERNATIONAL JOURNAL OF ONCOLOGY 50: 1821-1831, 2017 1829 miR‑130a on tumor growth in vivo, miR-130a-downregulated of tumor cells. In the present study, the designed miR-130a and control XPTS-1 cells were injected subcutaneously into inhibitor was transfected in XPTS-1 and EOMA cells. The the scapula of each mouse (n=20). The tumor growth of the results exhibited that inhibition of miR-130a significantly miR‑130a-downregulated tumors was significantly suppressed decreased colony formation, induced increased level of cell (Fig. 6A). The average tumor volume of miR‑130a-down- apoptosis and caused an accumulation in G2/M phase. These regulated tumors was lower compared with control tumors findings suggest that miR-130a inhibition reduces the viability (Fig. 6B). The expressions of Ki-67 and PCNA were signifi- of hemangioma in vitro. cantly decreased in miR-130a inhibitor group compared with Numerous reports confirmed that miRNA exerts its control group (Fig. 6C). qRT-PCR and western blot analyses effect in pathological process via regulating target genes. For convincingly decreased miR-130a with elevated TFPI2 levels example, miR-130a exerted promoting effect on the metastasis in miR-130a-downregulated tumors (P<0.001; Fig. 6D and and epithelial-mesenchymal transition of osteosarcoma via E). Finally, we examined the effect of miR-130a inhibitor on targeting PTEN (32). miR-130a regulated cell migration and the expression of p-FAK and its downstream genes. As shown invasion via inhibition of Smad4 in gemcitabine-resistant in Fig. 6F, the levels of p-FAK, p-PI3K, p-Rac1and p-mdm2 hepatoma cells (33). According to the report of Brock et al (34) were all decreased in miR-130a-downregulated tumors. miR-130 enhanced hypoxia-induced smooth muscle prolif- These findings indicate that miR-130a inhibitor restrains eration and might be involved in the development of right tumor growth and the activation of FAK/PI3K/Rac1/mdm2 ventricular hypertrophy and vascular remodeling in pulmo- pathway in vivo. nary hypertension, by directly targeting tumor suppressor p21 (CDKN1A). On the basis of bioinformatics analysis, we miR-130a inhibitor suppresses angiogenesis. As the further predicted another miR-130a target, TFPI2, which has angiogenic process contribute to the pathogenesis of heman- been reported to regulate matrix metalloproteinase activa- gioma (26), we measured the effect of miR-130a inhibitor on tion and extracellular matrix degradation. The deficiency of angiogenesis in hemangioma xenograft models. The results TFPI-2 may accelerate initiation of atherosclerotic lesion in revealed that miR‑130a inhibitor effectively decreased the mice (20). Besides, methylation of TFPI2 has been indicated to level of angiogenesis-related markers (VEGF, MMP-2 and predict high risk of advanced tumor stage, early tumor recur- MMP-9) (Fig. 7A). Besides, immunohistochemistry for CD31+ rence, and poor prognosis of hepatocellular carcinoma (22). and CD34+ showed that the percentages of CD31+ and CD34+ From the aspect mentioned above, increasing the level of tumor cells were significantly decreased compared with TFPI2 via miR-130a inhibitor may offer a novel strategy to control tumors (Fig. 7B). Similarly, flow cytometric analysis prevent the oncogenic effect of miR-130a in hemangioma. convincingly showed that, compared with control group, In the present study, the experimental results confirmed that miR-130a inhibitor strongly reduced the percentages of CD31+ TFPI2 is a functional target of miR-130a in hemangioma cells and CD34+ cells in hemangioma xenograft models (P<0.001; by luciferase-reporter gene assays. The expression of TFPI2 Fig. 7C-E). These results indicate that miR-130a inhibitor was significantly decreased in miR-130a overexpressing cells. alleviates hemangioma via suppressing angiogenesis. Besides, a significant inverse correlation was observed between TFPI2 and miR-130a expression in patients. Moreover, the Discussion knockdown of TFPI2 promoted cell growth contrary to the effect induced by miR-130a inhibitor, whereas TFPI2 overex- Infantile hemangiomas (IHs) are vascular tumors, usually pression was able to strengthen the growth inhibitive effect absent at birth or present as a premonitory mark with rapid of miR-130a inhibitor. These results strongly suggest that post-natal growth followed by slow involution (27). Topical miR-130a-increased cell growth is partly mediated by the antibiotics, occlusive dressings, pulsed dye laser and triam- repression of TFPI2 expression. cinolone acetonide have been reported to be sometimes FAK acts as an adaptor for protein-protein interaction or useful to limit pain and accelerate healing (28,29). Despite functions at sites of cell attachment to the ECM, contributing the various clues that have emerged regarding pathogenesis, to focal-adhesion ‘scaffolding’, and also transmits adhesion- many questions still remain. The upregulation of miR-130a dependent signals into the cell interior (35). Rapid activation has been observed frequently in a variety of human malignan- of FAK/PI3K has been indicated to control actin reorganiza- cies, including gastric (7,12), bladder cancer (9), hepatocellular tion in colon cancer cells (36). Rac1, a small GTP-binding carcinoma (30) and chronic myeloid leukemia (14). Similarly, protein, plays a pivotal role in regulating actin dynamics. the elevated level of miR-130a was observed in human heman- Substantial research has indicated that the loss of Rac1 acti- gioma tissues and cell lines in this study, and we revealed a vation contributed to cell migration and apoptosis (37,38). miR-130a/TFPI2/FAK/PI3K/Rac1/mdm2 axis that is involved Kallergi et al (39) reported that activation of FAK/PI3K/Rac1 in promoting proliferation and angiogenesis in hemangioma. signaling regulated actin reorganization and inhibits cell The increased expression of miR-130a was reported to motility in malignant human breast and prostate epithelial be associated with tumor growth (7,9,12,31). Duan et al (7) cells. Inhibition of the FAK/PI3K pathway also caused less indicated that overexpressed miR-130 could promote prolif- mesenchymal-like characteristics and reduced the mobility, eration of SGC7901 cells, and miR-130 was considered as an migration and invasion of Hep2 cells (40). Considering the oncogene in gastric cancer. The miR-130/301 family has been effect of Rac1 in controlling actin cytoskeletal organization in reported to be a regulator of multiple pro-proliferative path- the cell, the decreased p-Rac1/Rac1 ratio in this study suggests ways in pulmonary hypertension (31). The research indicated that miR-130a inhibitor treatment inhibits hemangioma that the inhibition of miR-130a may prevent the proliferation viability may involve actin depolymerization. Reports also 1830 Gao et al: silencing of miR-130a ameliorates hemangioma revealed that inhibiting the phosphorylation of Akt and mdm2 6. Furuta S, Sato H, Tsuji S, Murakami F and Kitagawa H: Effective treatment for infantile hemangioma with long-pulsed dye laser could lead to the accumulation of p53 and induced apoptosis of with oral propranolol medication: A preliminary report. Pediatr cancer cells (41). In accordance with these reports, the present Surg Int 32: 857-862, 2016. study demonstrated that XPTS-1 cells with pcDNA-FAK 7. Duan J, Zhang H, Qu Y, Deng T, Huang D, Liu R, Zhang L, Bai M, Zhou L, Ying G, et al: Onco-miR-130 promotes cell transfection displayed reduced nuclear staining of mdm2 and proliferation and migration by targeting TGFβR2 in gastric cell proliferation, increased cell apoptosis and G2/M cell cycle cancer. Oncotarget 7: 44522-44533, 2016. arrest under miR-130a inhibitor treatment. These findings 8. Anand S, Majeti BK, Acevedo LM, Murphy EA, Mukthavaram R, Scheppke L, Huang M, Shields DJ, Lindquist JN, Lapinski PE, together with in vivo experiments in hemangioma xenograft et al: MicroRNA-132-mediated loss of p120RasGAP activates models convince that the inhibition of miR-130a inactivates the endothelium to facilitate pathological angiogenesis. Nat Med the FAK/PI3K/Rac1/mdm2 pathway to suppress hemangioma 16: 909-914, 2010. 9. Egawa H, Jingushi K, Hirono T, Ueda Y, Kitae K, Nakata W, viability. Fujita K, Uemura M, Nonomura N and Tsujikawa K: The miR-130 Angiogenesis is a complex process that depends on the family promotes cell migration and invasion in bladder cancer balance of pro- and anti-angiogenic factors that influence the through FAK and Akt phosphorylation by regulating PTEN. Sci Rep 6: 20574, 2016. quiescence or proliferative state of the endothelium in various 10. Yang Y, Yang L, Liang X and Zhu G: MicroRNA-155 promotes diseases (8). A report revealed that the formation of vascular atherosclerosis inflammation via targeting OS CS1. Cell Physiol Biochem 36: 1371-1381, 2015. tumors including hemangioma is partly related to elevated 11. Zheng SL, Stevens VL, Wiklund F, Isaacs SD, Sun J, Smith S, level of angiogenic growth factors, such as VEGF and MMPs, Pruett K, Wiley KE, Kim ST, Zhu Y, et al: Two independent which lead to the development of a disorganized blood vessel prostate cancer risk-associated loci at 11q13. Cancer Epidemiol Biomarkers Prev 18: 1815-1820, 2009. mass (42,43). According to the research of Zhao et al (44), 12. Jiang H, Yu WW, Wang LL and Peng Y: miR-130a acts as a TFPI-2 overexpression may strongly inhibit the proliferation potential diagnostic biomarker and promotes gastric cancer and migration of VSMCs and suppress MMP-2/9 activity, migration, invasion and proliferation by targeting RUNX3. 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