Patient cohort and sample collection
Thirty-three Polish patients with NMSC were enrolled in this study (22 with BCC and 11 with SCC). The patients were surgically treated in the Department of Soft Tissue/Bone Sarcoma and Melanoma in the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology in Warsaw, Poland. The study was approved by local Bioethical Committee (permit number 13/2008). After surgical resection of an entire diseased area with a margin, small tissue samples were resected from both: the core of lesion and control normal skin from the border of excision and stored at −80 °C until use. General information about the examined group of patients and histopathological classification of carcinomas is provided in Additional file 1: Table S1.
Expression of GRHL genes in cancer samples
Total RNA was extracted from all collected fresh tissues (carcinoma and normal tissue from the border of excision) using RNeasy® Fibrous Tissue Mini Kit (Qiagen, cat. no. 74704) according to manufacturer’s instructions. The purity of RNA was determined using NanoDrop 2000 UV–vis spectrophotometer (Thermo Fisher Scientific). Assessment of RNA quality was performed with 2100 Bioanalyzer instrument and RNA 6000 Nano Kit (Agilent Technologies). Good quality samples (n = 27 pairs, cancer and normal tissue from the same patient) with RNA Integrity Number (RIN) higher than 5 were included in further analyses. RNA concentration was determined using the Qubit® 2.0 Fluorimeter and RNA BR Assay (Thermo Fisher Scientific, cat. no. Q10210).
Reverse transcription and real-time PCR
cDNA was synthesized from 250 ng of total RNA with SuperScript® VILO™ Master Mix (Invitrogen, cat. no. 11755050). The levels of expression of GRHL genes were assayed using Applied Biosystems chemistry: TaqMan® Fast Universal PCR Master Mix No AmpErase UNG (cat. no. 4352042) and TaqMan Gene Expression Assays (Assay ID: Hs01119372_m1 for GRHL1, Hs00227745_m1 for GRHL2, Hs00297962_m1 for GRHL3, Hs03929098_m1 for HPRT1 control). Real-time quantitative PCR was performed in 7900HT Fast Real-Time PCR System (Applied Biosystems). Gene expression was normalized to HPRT1 housekeeping gene and statistical differences were determined for relative expressions (2-∆∆Ct) with two-tailed Mann–Whitney U test with significance level < 0.05.
Interaction of miR-21–3p with 3′ untranslated region (UTR) of GRHL1
HaCaT cell line was bought from Cell Lines Service (cat. no. 300493). HEK293T cells were a kind gift from Ewelina Szymanska; the origin of this cell line is explained in a recent review article . HaCaT and HEK293T cells were routinely cultured in DMEM GlutaMAX medium (Thermo Fisher Scientific, cat. no. 10566–016) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, cat. no. 10270–106) and 100 IU/mL penicillin-streptomycin (Thermo Fisher Scientific, cat. no. 15140122) in a humidified incubator in an atmosphere of 95% air and 5% CO2 at 37 °C.
HaCaT cells treated with miR-21–3p mimic or hairpin inhibitor
60 nM miR-21–3p mimic or negative control (Ambion, mirVana cat. no. MC12979 and cat. No. 4464058) or 180 nM miR-21–3p hairpin inhibitor or negative control (Dharmacon, miRIDIAN cat. no. IH-301023-02 and IN-001005-01) were transfected into HaCaT cells using Lipofectamine 2000 or Lipofectamine 3000 transfection reagent (Invitrogen, cat. no. 11668019 or L3000–008). After 24 h cells were harvested with RNeasy Mini Kit (Qiagen, cat. no. 74104) or lysed in lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 1× Complete™ Protease Inhibitor Cocktail from Roche) for Western blot analysis. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, cat. no. 4368814). Real Time PCR was carried out with TaqMan Probes (Assay ID: Hs01119372_m1 for GRHL1, Hs00227745_m1 for GRHL2, Hs00297962_m1 for GRHL3, Hs00427620_m1 for TBP and Hs99999901_s1 for 18S). Histone deacetylase 8 – HDAC8 (Assay ID: Hs00954359_m1), which expression is regulated by miR-21–3p , was used as a control target. Each transfection was performed in duplicates and repeated three times. Gene expression was normalized to TBP housekeeping gene and statistical differences for relative expression (2-∆∆Ct) were determined with two-tailed Student’s t-test. For Western blot analysis 20 μg of total protein was separated on 12% SDS-PAGE gels and subsequently transferred to PVDF membrane. Membranes were blocked with 5% non-fat milk and incubated with primary antibody in blocking buffer. The following antibodies were used for immunoblotting: anti-GRHL1 (Sigma, cat. no. HPA005798), anti-rabbit IgG, HRP-linked (Cell Signaling, cat. no. 7074) and anti-β-actin (Sigma, cat. no. A3854). Quantification of protein abundance was carried out by ImageJ software and the relevant bands were normalized against the corresponding β-actin levels. Statistical analysis was performed using Student’s t-test.
Luciferase 3’UTR reporter assay in HEK293T cells
HEK293T cells were plated in 24-well plates and transiently transfected with 50 ng of GRHL1_3’UTR, GRHL1_3’UTR_mut or negative control vector (GeneCopoeia, cat. no. HmiT055586-MT01 and cat. no. CmiT000001-MT01,) using Lipofectamine 2000 (Invitrogen, cat. no. 11668019) following the manufacturer’s protocol. At the same time, miR-21–3p mimics or negative control were co-transfected with reporter vector in a final concentration of 60 nM. The 3’UTR of human GRHL1 was mutated using a QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, cat. no. 200518). Cells were harvested 24 h after transfection using the reporter lysis buffer (Promega). Firefly and Renilla luciferase activities were analyzed at room temperature in a multimode reader Infinite M1000Pro (Tecan) using the Dual-Luciferase Reporter Assay System (Promega, cat. No. E1910). Relative luciferase activity was defined as the mean value of the firefly/Renilla normalized ratios obtained from 3 independent biological replicates. Statistical differences were indicated with 2-tailed Student’s t-test.
Expression of miR-21–3p and GRHL1 in cancer cell lines
Squamous cell carcinoma cell lines were purchased from the American Type Culture Collection: A-431 (cat. no. CRL-1555), CAL-27 (cat. no. CRL-2095), SCC-15 (cat. no. CRL-1623), SCC-25 (cat. no. CRL-1628). HaCaT cell line was bought from Cell Lines Service (cat. no. 300493). SCC-351 cell line was a kind gift from Agnieszka Kobielak; this cell line is also known as USC-HN1 and originates from the laboratory of Alan L. Epstein, Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA, and was first described by the members of his laboratory . All cell lines were cultured as described above for the HaCaT cell line. RNA extraction, cDNA synthesis and TaqMan assays were carried out as described above.
Mutations and polymorphisms in GRHL genes
DNA extraction, target enrichment and next generation sequencing
DNA from 10 to 15 mg of homogenized tissues (Bio-Gen PRO homogenizer) was extracted using QIAamp® DNA Mini Kit (Qiagen, cat. no. 51304) according to manufacturer’s instructions. The purity of DNA was determined with the NanoDrop 2000 UV–vis spectrophotometer (Thermo Fisher Scientific). Accurate DNA concentration was quantitated using the Qubit® 2.0 Fluorimeter and dsDNA BR Assay (Invitrogen; cat. no. Q32853). SureDesign HaloPlex Standard Wizard was employed to select the custom probe sequences based on target regions of GRHL genes, according to the hg19/GRCh37 assembly from UCSC database ( Feb, 2009 version); the list of analyzable regions is provided in Additional file 1: Table S2. Capture of the targeted regions was performed with reagent set from a custom design HaloPlex Target Enrichment System 1–500 kb (Agilent Technologies), according to the Protocol Version D (August 2012). Briefly, the protocol consisted of the four following steps: 1) digestion of genomic DNA (250 ng) by restriction enzymes in eight parallel reactions; 2) hybridization resulting in circularization of digested DNA fragments with complementary probes which incorporated indexes and Illumina sequencing motifs; 3) capture of targeted DNA using streptavidin beads and ligation of circularized fragments; 4) PCR amplification of captured target libraries. Paired-end sequencing of samples was performed on a MiSeq instrument (Illumina) in Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany.
NGS data processing
Sequence reads were resynchronized and trimmed to remove Illumina adapter sequences and only reads longer than 36 bp were kept. Sequences were further filtered with Trimmomatic  for low quality leading/trailing bases with phred quality lower than 20. Subsequently sequences were aligned to the human reference genome (version hg19) with Stampy . Additionally, the initial 5 bases were trimmed due to potential allele bias in case of single nucleotide polymorphisms (SNP) present in restriction enzyme cutting sites. SNPs were called with SAMtools mpileup algorithm with default parameters . Coverage cutoff value was 20.
SNPs of association
Distribution of SNPs in examined NMSC population was compared to the European population (data derived from 1000Genomes database ) and association p-value was determined with a χ2 – test or Fisher’s exact test. The p-value threshold for significance was adjusted with multiple-comparison correction (Bonferroni correction).
Predicted effects of SNPs in TF binding motifs
Mapping of SNPs to Encyclopedia of DNA Elements Consortium (ENCODE) regions  and transcription factor binding motifs was performed with the Nencki Genomics Database-Ensembl funcgen . The motif matches against DNA sequences were scored as the log-odds of the respective position weight matrix of the motif derived from the JASPAR database . For each SNP, the original reference sequence and the sequence modified by a single SNP were considered (interactions of multiple SNPs in the same motif were not considered). The differences between the log-odds scores were interpreted as the logarithm of the fold change of the binding energy. In cases where the odd scores of the mutated sequence were 0 (no motif match at all); the log odds differences were interpreted as infinite (log(x) approaches negative infinity as x approaches 0).
Genotyping of exonic SNPs rs141193530 and rs41268753 in a replication cohort
Formalin-fixed, paraffin-embedded (FFPE) materials from 177 Polish patients with non-melanoma skin cancers (144 with BCC and 32 with SCC) were randomly selected. The patients were surgically treated in the Department of Soft Tissue/Bone Sarcoma and Melanoma in the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland and the study was approved by the Bioethical Committee; permit number 13/2008.
DNA extraction from FFPE samples (5–10 slices of 10-μm-thick sections) was performed using NucleoSpin DNA FFPE XS (Macherey-Nagel), all steps were performed according to manufacturer’s instructions. DNA quantitation was carried out with Qubit™ dsDNA HS Assay Kit (Invitrogen, cat. no. Q32854). To obtain useful information, including high-quality sequence data, FFPE DNA samples were treated with NEBNext FFPE DNA Repair Mix (New England BioLabs, cat. no. M6630 L) according to manufacturer’s instructions.
To identify DNA samples with potential SNPs in their sequences, preliminary selection was performed with PCR-RFLP (restriction fragment length polymorphism). PCR amplification of genomic DNA was conducted using forward primer: 5’–CTTCAGGGGCAATGAGACGAC–3′ and reverse primer 5’–GCACATTGGGGATGAACAGC–3′, the annealing temperature for PCR reactions was 65 °C. For accurate replication of template, Q5® High-Fidelity DNA Polymerase was used (NEB, cat. no. M0492S) and PCR products (size 80 bp) were digested with BsaHI restriction enzyme (NEB, cat. no. R0556S), which digests only templates without examined SNPs (the presence of either rs141193530 but also rs41268753 will abolish the restriction site 5’-GRCGYC-3′). The digested PCR products were resolved in 2.5–3% High Resolution Agarose (EurX, Poland, cat. no. E0302–50) gels, stained with SimplySafe™ (EurX, Poland, cat. no. E4600–01) and visualized with G:Box (Syngene). The digested PCR product of each sample was compared to the same amount of non-digested product. Samples with positive outcome (product not digested or partially digested) were additionally genotyped by pyrosequencing.
To specify exact single nucleotide polymorphism in selected DNA samples, a pyrosequencing assay was designed to measure the relative quantification of nucleotide incorporation at the SNP sites: rs41268753 (C/T) and rs141193530 (C/G). PCR amplification primers for the genomic DNA templates were as follows: Fw_5’–biotinylated–CTTCAGGGGCAATGAGACGAC–3′ and Rv_5’–GCACATTGGGGATGAACAGC–3′, with primer annealing at 65 °C. The biotinylated PCR products (80 bp) were subject to pyrosequencing using internal pyrosequencing primer 5’-ATTGGGGATGAACAGCAC–3′. The sequence content analyzed was GGGTG[G/C]C[G/A]TCTCC. Pyrosequencing service was provided by A&A Biotechnology (Gdynia, Poland).
For analysis of single marker association, SNP frequencies in coding regions in control European non-Finnish population were obtained from the Exome Aggregation Consortium database (ExAC) . For calculating odds ratio, relative risk, confidence interval, significance level and other parameters, statistical methods appropriate for medical research were employed . General information about the considered group of patients is provided in Additional file 1: Table S3.