Patient material and Ethics Statement
This research was approved by the Ethics Review Committee in Kumamoto University (No. 177). Skin specimens were obtained from 7 senile hemangioma, 3 venous malformation, 4 angiosarcoma, 4 venous lake, and 3 infantile hemangioma (). Seven control skin samples are obtained from routinely discarded skin of healthy human subjects undergoing skin graft. Control and patient samples were collected and processed immediately after resection in parallel. Written informed consent was obtained according to the Declaration of Helsinki.
Clinical data: ages of patients at the time of resection and location of the lesions.
RNA isolation and quantitative real-time polymerase chain reaction (PCR)
Skin specimens were obtained from 7 senile hemangioma, 3 venous malformation, 4 angiosarcoma, 4 venous lake, 3 infantile hemangioma and 7 healthy controls. They were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sliced. Total RNA isolation from paraffin-embedded section of normal skin and tumor tissues were performed with RNeasy FFPE kit (Qiagen, Valencia, CA) following the manufacturer's instructions. cDNA was synthesized from total RNA with PrimeScript RT rea(Takara Bio Inc, Shiga, Japan). Quantitative real-time PCR with a Takara Thermal Cycler Dice (TP800)® used primers and templates mixed with the SYBR Premix Ex gent Kit TaqII (Takara Bio Inc). Primer sets for vascular endothelial growth factor receptor (VEGFR) 1, VEGFR2 and GAPDH were purchased from SA Biosciences (Frederick, MD). Primers for HIF-1α, MEK1 and cyclin E1 were from Takara. These primer sets were prevalidated to generate single amplicons. MEK1 was amplified for 50 cycles of denaturation for 5s at 95°C, annealing for 30s at 70°C, whereas annealing temperature was set at 60°C for the other primers. Data generated from each PCR reaction were analyzed using Thermal Cycler Dice Real Time System ver2.10B (Takara Bio Inc). Specificity of reactions was determined by melting curve analysis. Transcript levels were normalized to GAPDH.
miRNA extraction and PCR array analysis of miRNA expression
Small RNAs were extracted using a miRNeasy FFPE kit (Qiagen). Then, RNAs were reverse-transcribed into first strand cDNA using an RT2 miRNA First Strand Kit (SABiosciences, Frederick, MD). For RT2 Profiler PCR Array (SABioscience), the cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human miRNAs. PCR was performed on a Takara Thermal Cycler Dice (TP800®) following the manufacture's protocol. Threshold cycle (Ct) for each miRNA was extracted using Thermal Cycler Dice Real Time System ver2.10B. The raw Ct was normalized using the values of small RNA housekeeping genes.
For quantitative real-time PCR, primers for mir-424, mir-1 or U6 (SABioscience) and templates were mixed with the SYBR Premix Ex TaqII (Takara Bio Inc). DNA was amplified for 50 cycles of denaturation for 5s at 95°C, annealing for 30s at 60°C. Data generated from each PCR reaction were analyzed using Thermal Cycler Dice Real Time System ver2.10B (Takara Bio Inc). Transcript levels were normalized to U6.
Paraffin sections were deparaffinized in xylen and rehydrated in a graded ethanol series. Antigens were retrieved by incubation with citrate buffer pH 6 for 5 min with microwave oven. Endogenous peroxidase activity was inhibited, after which sections were incubated with 5% milk for 30min and then reacted with the antibodies for α-smooth muscle actin or type IV collagen (Abcam, Cambridge, MA, 1
300) overnight at 4°C. After excess antibody was washed out with PBS, samples were incubated with HRP-labeled goat anti-mouse antibody (Nichirei, Tokyo, Japan) for 60min. The reaction was visualized by the diaminobenzidine substrate system (Dojin, Kumamoto, Japan). Slides were counterstained with Mayer's haematoxylin, and examined under a light microscope (OLYMPUS BX50, Tokyo, Japan).
In situ hybridization
In situ hybridization was performed with 5′-locked digoxigenin-labeled nucleic acid (LNA) probes complementary to human mature mir-424 and scrambled negative control (Exiqon, Vedbaek, Denmark) 
. Briefly, human tissues were deparaffinized and deproteinized with protease K for 5 min. Slides were then washed in 0.2% Glycine in PBS and fixed with 4% paraformaldehyde. Hybridization was performed at 48°C overnight followed by blocking with 2% fetal bovine serum and 2% bovine serum albumin in PBS and 0.1% Tween 20 (PBST) for 1 hour. The probe-target complex was detected immunologically by a digoxigenin antibody conjugated to alkaline phosphatase acting on the chromogen nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche Applied Science, Mannheim, Germany). Slides were counterstained with nuclear fast red, and examined under a light microscope (OLYMPUS BX50; Tokyo, Japan).
Paraffin sections were deparaffinized in xylen and rehydrated in a graded ethanol series. Antigens were retrieved by incubation with 0.1% trypsin at 37°C for 5 min. The slides were permeabilized in 0.5% Triton-PBS for 5min and blocked in 5% nonfat dry milk for 30min at room temperature. As the primary antibodies, rabbit anti-MEK1 (1
50, Santa Cruz Biotechnology) or rabbit anti-cyclin E1 (1
50, Santa Cruz Biotechnology) with mouse anti-CD34 (1
25, DakoCytomation, Carpinteria, CA) diluted in 5% milk in PBS, were applied to the sections. The sections were incubated overnight at 4°C, followed by PBS-0.05% Triton X-100 washes. Matching isotype IgG was used as a negative control. Then, Alexa Fluor 488 anti-rabbit (1
200, Invitrogen, Carlsbad, CA) and Rhodamine-conjugated anti-mouse secondary antibodies (1
100, Jackson ImmunoResearch, Suffolk, UK) were applied to the sections. After 1 hour at room temperature, sections were washed and mounted with VECTASHIELD mounting medium with DAPI (Vector, Burlingame, CA). Zeiss Axioskop 2 microscope (Carl Zeiss, Oberkochen, Germany) was used for fluorescence microscopy.
Human dermal microvascular ECs (HDMECs, CC-2543) were obtained from Lonza (Walkersville, MD). HDMECs were grown on 0.2% gelatin-coated dishes in EGM-2 (Clonetics, San Diego, CA) with 20% fetal bovine serum (Hyclone, Logan, UT) and Antibiotic-Antimycotic (Invitrogen, Carlsbad, CA) in 5% CO2
at 37°C 
. Cells were passaged 1
3 every 4 to 6 days and used between passages 3 to 9.
The transient transfection
siRNAs against MEK1 or cyclin E1 was purchased from Santa Cruz Biotechnology. miRNA inhibitors were from Qiagen. Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) was used as transfection reagent. For reverse transfection, siRNA (6pmol) or miRNA inhibitor (60pmol) mixed with transfection reagent were added when cells were plated, followed by incubation for 48–72 hours at 37°C in 5% CO2. Control experiments showed transcript levels for target genes or miRNAs to be reduced by >80% (data not shown).
Cell lysis and immunoblotting
HDMEC were washed with cold PBS twice and lysed in lysis buffer (Denaturin Cell Extraction Buffer, BIOSOURCE, Camarillo, CA). Aliquots of cell lysates (normalized for protein concentrations as measured by the Bio-Rad reagent) were separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked for 1 hour and incubated overnight at 4°C with antibody for MEK1 (Santa Cruz Biotechnology, Santa Cruz, CA) or cyclin E1 (Santa Cruz Biotechnology). The membranes were washed in Tris-buffered saline (TBS) and 0.1% Tween 20, incubated with secondary antibody, and washed again. The detection was performed using the ECL system (Amersham, Arlington Heights, IL) according to the manufacturer's recommendations. As a loading control, immunoblotting was also performed using antibodies against β-actin.
HDMECs were detached from the wells by trypsin treatment and counted using a Coulter® Particle Counter (Beckman Coulter, Fullerton, CA) 
Statistical analysis was carried out with the Mann-Whitney test for comparison of means. P values less than 0.05 were considered significant.