Although the significance of this enrichment is yet unknown, it has been proposed that the functions of X-linked miRNAs may explain some of the gender difference observed in immunity and related diseases, as well as cancers [92]. In conclusion, our findings further underline the presence of cancer-specific miRNA profiles [93]. Through microRNAome- wide expression profiling of cancers, it should therefore be possible to develop specific, miRNA-based clinical markers for diagnosis and the selection of treatment options for various types of cancers. With regards to SCC, studies with larger sample numbers and follow-up data should establish the value of miR-361-5p as a diagnostic and prognostic marker. As comprehensive data on the expression of miR-361-5p are currently unavailable, further studies should also reveal its association with other tumor types and targets, either acting in the same, or in different pathways. Materials and Methods Ethics Statement The collection of specimens from clinically indicated excisions for this study was explicitly approved by the institutional review board (Kantonale Ethikkommission Z u¨rich). Informed consent (both written and verbal) was obtained from patients for the use of their skin samples in this research project. Plasmids A fragment comprising nucleotides 43,753,225 to 43,754,253 of human chromosome 6 (Build GRCh37/hg19, February 2009), containing nucleotides 926 to 1925 of the 39-UTR of human VEGFA (isoform a, NM_001025366.2), was amplified from HeLa S3 genomic DNA using KOD Hot Start DNA Polymerase (Novagen, Cat. No. 71086-3) and the following primer pair (restriction sites are underlined): 59-TCACTC- GAGGTCCCGGCGAAGAGAAGAG-39 (forward, containing XhoI site) and 59-CATGCGGCCGCTCAATGGAGAAGGA- GAAACCA (reverse, containing NotI site). The amplicon was cloned into pCR-Blunt II-TOPO (Invitrogen, Cat. No. K2800- 20). The mutation in the putative miR-361-5p recognition element was introduced using the QuikChange I site-directed mutagenesis kit (Stratagene) according to the manufacturer’s recommenda- tions. The following oligonucleotide and its reverse complement were used (the sequence complementary to the miRNA seed sequence is underlined): 59-GTGTGTATATATATATATA- TATGTTTATGTATATATGTGATGATAAAATAGA- CATTGCTATTCTGTTTTTTATATGTAAAAACAAA-39. Af- ter sequence verification, wild type and mutated VEGFA 39-UTR fragments were subcloned into psiCHECK-2 (Promega) via XhoI and NotI restriction sites. Cell Culture and Tissue Samples Primary human dermal blood vascular endothelial cells (BECs) and lymphatic endothelial cells (LECs) were isolated from neonatal foreskin as previously described [78,79]. HEK293 [74], A431 [76] (both from American Type Culture Collection, Cat. Nos. CRL- 1573 and CRL-1555, respectively), and HaCaT [77] (Cell Lines Service, Cat. No. 300493) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Invitrogen, Cat. No. 41966) supplemented with 10% fetal bovine serum (Invitrogen, Cat. No. 10270–106) and 1x antibiotic-antimycotic (Invitrogen, Cat. No. 15240–062). BECs and LECs were grown in endothelial basal medium (EBM; Lonza, Cat. No. CC-3121) supplemented with 20% fetal bovine serum, 1x antibiotic antimycotic, 2 mM L- glutamine (Invitrogen, Cat. No. 25030024), 10 mg/ml hydrocor- tisone (Sigma-Aldrich, Cat. No. H0396) and 25 mg/ml N-6,29-O- VEGFA and miR-361-5p Expression in Human SCC dibutyryladenosine 39,59-cyclic monophosphate (Sigma-Aldrich, Cat. No. D0627) for up to 11 passages in plastic dishes coated with type I collagen (50 mg/ml in PBS; Advanced Biomatrix, Cat. No. 5005-B). All cells were grown in 5% CO2 at 37uC. Squamous cell carcinoma (SCC) samples were obtained at the time of surgery. Normal skin was obtained from abdominoplastic reductive surgery. All specimens’ diagnoses were confirmed by a board-certified dermatohistopathologist. Four mm punch bi- opsies from SCC or normal skin were placed in preheated PBS at 60uC for 45 seconds, and then chilled on ice in 0.1% PBS for one minute, followed by mechanical separation of epidermis and dermis by scratching. The epidermis was homogenized in TRIzol reagent (Invitrogen, Cat. No. 15596–026) and stored at 280uC. RNA was extracted according to the manufacturer’s recommen- dations. Quantity and quality of extracted RNA was assessed by spectrophotometry with a NanoDrop 1000 (Thermo Fisher Scientific Inc.) and the 2100 Bioanalyzer (Agilent Technologies), respectively. All RNA samples had an RNA Integrity Number (RIN) of higher than 7.0. Luciferase Reporter Assays 20,000 HEK293 cells were reverse transfected with 20 ng of the indicated luciferase reporter constructs using polyethylenimine (Polysciences, Inc., Cat. No. 23966). For complex formation, DNA and polyethylenimine stock solution (1 mg/mL in water) were diluted to 20 mg/mL and 60 mg/mL, respectively, in Opti-MEM I (Invitrogen, Cat. No. 51985–026). Both solutions were incubated for 10 min at room temperature, and then mixed at equal volumes (mass ratio polyethylenimine to DNA = 3:1; final polyethylenimine concentration = 30 mg/mL). After incubation for 20 min at room temperature, solutions were diluted 1:10 in DMEM (Invitrogen, Cat. No. 41966) to achieve a final DNA concentration of 1 mg/ mL. 20 mL of the transfection mixes were added to each well of a 96-well plate, followed by the addition of 80 mL of cell suspension in DMEM (2.56105 cells/mL). 16 hours after trans- fection of the luciferase reporter plasmid, the cells were further transfected with 50 nM of Pre/Anti-miR-361-5p or Pre2/Anti- miR Negative Control #1 (Applied Biosystems, Table S4) by using siPORT NeoFX (Applied Biosystems) according to the manufac- turer’s recommendations. All transfections were performed in triplicate. After 48 hours, the medium was aspirated and cells were lysed with a mixture of 15 mL Luciferase Assay Reagent II (Promega) and 15 mL nuclease-free water (Invitrogen, Cat. No. 10977). Firefly luciferase activity was measured after 10 min. Then, 15 mL Stop & Glo Reagent (Promega) were added and Renilla luciferase activity was measured after 10 min. Luciferase activity measurements were performed in an LMAX II 384 luminometer (Molecular Devices), with 5 seconds in- tegration time. For each triplicate, the mean Renilla/firefly ratio was calculated. ELISA 20,000 A431 or HaCaT cells were reverse transfected with 10, 30, 50 or 100 nM Pre2/Anti-miR-361-5p or Pre2/Anti-miR Negative Control #1 (Applied Biosystems, Table S4) by using siPORT NeoFX (Applied Biosystems) according to the manufac- turer’s recommendations. Transfections were performed in triplicate in 96-well plates. Where indicated, TNF-a (Peprotech, Cat. No. 300–01A) was added at 10 ng/mL immediately after transfection to induce VEGFA expression. 24 hours after trans- fection, supernatants were collected and centrifuged to remove cell debris (1000 g for 3 min at room temperature). VEGFA protein levels were determined using the Human VEGF-A Platinum ELISA kit (eBioscience, Cat. No. BMS277) according to the PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49568