From healthy controls and the uninvolved and involved skin of PS patients, 4–6 mm punch skin biopsies were collected. PS patients enrolled in this study received no systemic, photo or topical therapy in the 4 weeks prior to sample collection. Biopsies were stored in RNAlater (Qiagen) at −80°C prior to RNA extraction.
Small RNA library preparation and sequencing
RNA was extracted with the miRNeasy Mini Kit (Qiagen), with on-column DNase I digestion. RNA was prepared for sequencing on the Illumina GAIIx platform with the Small RNA Sample Prep Kit (Illumina) according to the manufacturer's instructions (protocol v1.5). This protocol required the use of a proprietary 3′ adapter that has a high affinity for Dicer cleavage products. Briefly, 3′ and 5′ adapters were ligated to 1 μg of total RNA. cDNA was synthesized with SuperScript II Reverse Transcriptase (Invitrogen) and subjected to 12 cycles of PCR amplification with high-fidelity Phusion Polymerase (Finnzymes Oy). Each library was loaded on a single Illumina lane at 20 pm and subjected to 36 cycles of sequencing.
Read processing and mapping
Each deep sequencing library was processed independently. Reads with a 3′ adapter substring <6 nt or trimmed sequence length <17 nt were removed from the data set. Trimmed reads were mapped to multiple human sequencing databases with Bowtie: miRNA precursors (miRBase v.16, http://www.mirbase.org/ftp.shtml
, last access date: 8-3-11), ncRNAs (fRNAdb, http://www.ncrna.org/frnadb/download
, last access date: 8-3-11) and the hg19 build of the human genome (UCSC Genome Browser, http://genome.ucsc.edu/cgi-bin/hgTables?command=start
, last access date: 8-3-11; 60
). Reads that mapped to miRNA precursors were attributed to mature miRNAs if they aligned to the annotated mature sequences with 3 nt up- and downstream extensions.
Novel miRNA prediction
Qualified reads that aligned to the hg19 build of the human genome were subjected to our novel miRNA prediction pipeline. Any reads that mapped to previously described miRNA loci were removed, and loci that shared adjacent reads within a gap of ≤30 nt were merged. For each locus, a series of overlapping DNA sequence segments was extracted for secondary structure analysis with RNAfold (http://www.tbi.univie.ac.at/~ivo/RNA/
, last access date: 8-3-11; 67
). The starting sequence segment extended 220 nt upstream of the locus, and subsequent segments were extracted by a sliding window of 250 nt, with an increment of 100 nt, until the window reached 220 nt downstream of the sequence reads. Segments lacking stems of at least 18 nt and segments lacking reads that mapped to any of their stems were excluded. Candidate miRNAs were prioritized based on (i) the occurrence of sequencing reads on the stem of a predicted hairpin structure (minimum free energy less than −18 kcal/mol); (ii) the presence of miRNA* reads on the opposite stem of the hairpin; (iii) the presence of 3′ overhangs on the highest likelihood miRNA/miRNA* duplex; and (iv) the evidence of spliceosome-mediated precursor processing based on alignment to intron–exon boundaries.
Novel miRNA conservation
Homology searches were performed in eight species: M. mulatta
, M. musculus
, C. l. familiaris
, L. africana
, Monodelphis domestica
, Gallus gallus
, Xenopus tropicalis
and Danio rerio
. The novel miRNA precursor sequences, defined as the maximal extension of sequences bearing the stem-loop structure, were mapped to each species' genome using blastn (http://blast.ncbi.nlm.nih.gov/Blast.cgi/
, last access date: 8-3-11) with a minimal word-size of seven. Blastn hits that were reported in any given species shared >90% sequence similarity and had a ratio of alignment length to the precursor length of >0.9. Secondary structure of aligned sequences was determined with RNAfold. Aligned sequences were classified as conserved novel miRNAs if the following criteria were met: (i) the highest likelihood secondary structure was a hairpin with a minimum free energy less than −18 kcal/mol, (ii) the mature miRNA sequence was derived from the hairpin stem, and (iii) the miRNA seed region (nt 2–7) was perfectly conserved.
Digital gene expression
Reads that aligned perfectly to mature known and novel miRNAs with 3 nt extension were subjected to DGE analysis. Reads that mapped to multiple mature miRNAs were attributed to all potential derivative miRNAs. Read counts in each skin category (NN, PN and NN) were normalized to adjust for slight variation in total read count between categories. Let N be the number of qualified reads that aligned to hg19, C the number of categories and M the number of qualified miRNA reads. Thus, the normalized number of reads for each miRNA in a given category is (Ntotal * Mcategory)/(C* Ncategory). A detection filter was applied such that miRNAs that were represented by fewer than 268 raw reads in the cumulative data set were removed. Fold changes were calculated from normalized read counts, and Pearson's χ2 test with Bonferroni correction was applied to determine significance.
qRT-PCR of mature miRNAs was performed with TaqMan miRNA assays according to the manufacturer's instructions (Life Technologies). Briefly, 5 ng of total RNA was reverse-transcribed in a 7.5 μl reaction with the TaqMan MicroRNA Reverse Transcription Kit (Life Technologies), and 0.67 μl of cDNA was added to triplicate 10 μl PCR reactions. PCR was performed on a 7900HT thermocycler (Life Technologies). miRNA expression was normalized to the endogenous snoRNA, Z30. Relative expression levels were calculated according to the 2−ΔΔCt
method as follows: 100 * 2((Ct Z30) − (Ct miRNA))
). Significance was determined with one-way ANOVA and post hoc
two tailed t
miRNA overexpression constructs
miRNA overexpression constructs were generated from the pEP-miR cloning and expression vector (Cell Bio Labs). Briefly, miRNA precursors ±100 nt were amplified from genomic DNA. PCR products were cloned into the BamHI and NheI sites of the vector. Transformants were selected on 1 μg/ml ampicillin and selected transformants were validated by Sanger sequencing.
HEK293 cells were cultured in DMEM supplemented with 2 mm l-glutamine, 10 mg/ml penicillin–streptomycin and 10% fetal bovine serum at 37°C and 5% CO2. Transfections were performed in triplicate. Twenty-four hours prior to transfections, 1 × 105 cells were plated in each well of a 24-well plate. Transfections were performed with TransIT-LT1 transfection reagent according to the manufacturer's instructions (Mirus). Briefly, 750 ng of pEP-miR and 3.75 μl of LT-1 were incubated in 46.25 μl of RPMI for 30 min at 22°C before treatment. Cells were collected 48 h post-treatment.
miRNA northern blots
Thirty micrograms of total RNA was mixed with formamide loading dye and incubated at 65°C for 20 min. Samples were loaded on a pre-warmed 12% denaturing polyacrylamide gel (Sequagel), and run at 100 V until bromphenol blue reached the bottom of the gel. RNA was transferred onto a Genescreen Plus membrane (Perkin Elmer) with a Trans-Blot SD semi-dry transfer cell (Bio-Rad) at 250 mA for 15 min. The membrane was baked for 1 h at 80°C, pre-hybridized for 2h in PerfectHyb Plus (Sigma) at hybridization temperature and hybridized overnight with a 32P-labeled DNA probe. miR-203-AS probe sequence was 5′-CCAGTGGTTCTTAACAGTTCAA-3′. The membrane was washed three times in 0.1× SSC, 0.1% SDS at hybridization temperature and exposed for 3 days. For input control, the membrane was stripped with two 20 min applications of boiled 0.1% SDS with gentle agitation at 22°C, and re-hybridized with a 32P-labeled U6 snRNA LNA probe (Exiqon).
HEK293 cells were transfected with pEP-miR-null or pEP-miR-novel #117 constructs as described above, except that transfections were scaled up to generate one 10 cm plate per immunoprecipitation. Cells were washed three times in 1× PBS and UV-crosslinked once for 400 mJ/cm2 and again for 200 mJ/cm2, with gentle agitation in between. Cells were pelleted by centrifugation at 4000 r.p.m. for 5 min at 4°C. Cell pellets were washed once in 1× PBS and resuspended in 200 μl of 1× PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% nonidet P-40, supplemented with 1 U/μl RNasin (Promega) and 1× Complete Protease Inhibitor Cocktail (Roche). Lysates were incubated on ice for 10 min, and cleared by centrifugation at 10 000 r.p.m. for 10 min at 4°C. Each cleared lysate was added to 50 μl of protein G-coated Dynabeads (Invitrogen) which had been previously bound to 5 μg of anti-mouse Ago2/eIF2C2 monoclonal antibody (Abcam) or normal rabbit IgG (Cell Signaling Technology), according to the manufacturer's protocol, and incubated for 4 h at 4°C with rotation. Beads were washed three times with 1× PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% nonidet P-40 and three times with 5× PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% nonidet P-40. RNA extraction and qRT-PCR were performed as described above, except that the relative abundance of novel #117 in the Ago2 IP sample was calculated relative to the IgG IP sample, in lieu of an endogenous control.
Ten microliters of 2× SDS reducing buffer was added directly to washed beads following immunoprecipitation, and incubated at 95°C for 5 min. Samples were loaded on a pre-warmed 4–20% polyacrylamide gel, run at 200 V for 30 min and wet-transferred for 1 h at 100 V onto a 0.45 μm nitrocellulose membrane. The membrane was blocked in 1× TBST, 5% milk at 22°C for 1 h. A 1:500 dilution of Ago2/eIF2C monoclonal antibody (Abcam) in 1× TBST, 5% milk was applied to the membrane and incubated at 22°C for 3 h. The membrane was washed three times for 10 min in 1× TBST, 5% milk. A 1:5000 dilution of HRP-conjugated secondary antibody in 1× TBST, 5% milk was applied to the membrane and incubated at 22°C for 1 h. The membrane was washed three times for 10 min in 1× TBST, 5% milk. The blot was developed with 1 ml of Supersignal West Femto Chemiluminescent Substrate (Thermo Scientific).
miRNA in situ hybridization
miRNA in situ
hybridizations were performed as previously described (71
). Briefly, fresh skin biopsies were fixed in 10% formalin for 24–72 h and paraffin-embedded. Six micrometer sections were mounted on glass slides, deparaffinized and treated with 10 μg/ml proteinase K for 20 min at 37°C. Slides were hybridized with 20–60 nm
double-DIG-labeled LNA probes (Exiqon) overnight at 57°C (miR-135b, miR-205) or 50°C (miR-142). Slides were washed in 5× SSC, 1× SSC and 0.2× SSC for 10 min at hybridization temperature. Staining was performed with NBT/BCIP (Roche) for 90 min at 32°C followed by nuclear fast red counterstain (Vector Laboratories). An LNA probe with scrambled sequence was used as a negative control (Exiqon).
Reads that aligned to mature miRNAs with one mismatch were subjected to filters prior to editing analysis. Reads containing a low-quality mismatch [P(sequencing error) > 0.05] based on the single-base Illumina quality score were removed. 3′ terminal N → A or N → T mismatches were also removed. From the remaining pool of one mismatch reads, the relative frequencies of all possible substitutions at positions 1–20 of the miRNA relative to the 5′ end were calculated. Significance was determined with Pearson's χ2 test.