Tissue samples
A group of 5 pairs of PCa and matched non-tumor normal tissues were collected from Huashan Hospital, Fudan University. To deep confirm, another cohort of prostate tissues were obtained from prostate needle biopsies in Huashan Hospital, Fudan University. Our study was permitted by the ethics committee of Huashan Hospital, Fudan University (ethics approval no.2011-009) and written informed consent was obtained from all patients. All tissue was histologically identified by pathological section. If diagnosed as prostate adenocarcinoma, the Gleason score, PSA value, TNM stage and recurrence were according to the NCCN guideline(36). Otherwise, the tissues are recognized as normal contrast. A subset of patients had matched PCa tissues and normal tissues available in the qPCR. The initial screening step (Table 1) was conducted with microarray chip assay. Another cohort screening information which was considered as the validation of the expanded clinical samples (Table 2) was listed with the qPCR.
RNA extraction and purification
Total RNA was extracted and purified using mirVana™ miRNA Isolation Kit (Cat#AM1561, Ambion, Austin, TX, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).
RNA labeling
rRNA was amplified and labeled by Low Input Quick Amp WT Labeling Kit (Cat.# 5190-2943, Agilent technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat.# 74106, QIAGEN, GmBH, Germany).
Array hybridization
Each slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat.# 5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat.# G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat.# 121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat.# 5188-5327, Agilent technologies, Santa Clara, CA, US), followed the manufacturer’s instructions. Differentially expressed lncRNAs were analyzed with independent samples t-test. LncRNAs with ≥ 2.0 fold-changes (FC) and p < 0.05 were selected as lncRNAs with significant differential expression.
Data acquisition
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution = 3 μm, PMT 100%, 20bit. Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Limma packages in R.
Bioinformatics analysis
LncRNA targets identified with profiling data were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses based on their correlated mRNAs using GO (http://www.geneongoloty.org/) and KOBAS software (KEGG Orthology-Based Annotation System, https://www.kegg.jp/)(37, 38). The differentially expressed lncRNAs-targeted miRNAs were sought and predicted by miRanda software (http://miranda.org.uk/) coupled with statistical analysis. The lncRNAs expression profile microarray chip assay, besides data and bioinformatics analysis were carried out by Shanghai Biotechnology Corporation (Shanghai, China).
qPCR analysis
Total RNA from another normal tissues (9 samples) and PCa tissues (7 samples) sustained by pathology after perineal prostate biopsy guided by ultrasound was prepared by Trizol Isolation Reagent (Invitrogen). Dimethylcarbinol, ethanol and trichloromethane were of analytic grade. DNase I, SYBR Green Realtime PCR Master Mix Plus and the ReverTra Ace qPCR RT Kit are from Toyobo Co. Japan. The reverse transcription kit steps are strictly followed to transcribe to cDNA. cDNA was used as template, and hGAPDH as internal parameter. The primer concentration was set as 0.4 μmol/L. Three parallel samples were set for each sample, tested as 15 μl system used for amplification. For qPCR solution, THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) was utilized. qPCR was performed on the LightCycler 96 (Roche, Indianapolis, IN, USA) following the instruction. The reaction conditions of qPCR were: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 20 s and totally 40 cycles. Assay numbers got involved in the top 10 up-regulated and down-regulated expression of lncRNAs and GAPDH, respectively. The sequences of primer are listed in the Table 3. Differentiated gene expression was calculated by the comparative Ct method.
The co-expression network of lncRNA-miRNA-mRNA
Spearman correlation was calculated between the abundance of each lncRNA against each miRNA with the criteria of relative expression levels. Once the predicted pairs of lncRNA-miRNA relation were determined, and further filtered by comparison with the theoretical databases. The theoretical databases included ENCORI, lncBase, miRcode for lncRNA-miRNA relations and miRcode, ENCORI, TarBase, miRTarBase, miRDB, miRanda, miRecords for miRNA-mRNA relations. For Agilent chip GPL22120, adopt multiple IDs from different sources, should be correspond to one unique ID, and we map the all IDs in GPL22120 to RNA Central (https://www.rnacentral.org/, v14)(39). All the lncRNA ID listed in figure were started by “URS” which was the acronym of Unique RNA Sequence and combined with 10 numbers and/or English letters.
Survival Curve Analysis
We used the GEPIA (Gene Expression Profiling Interactive Analysis, http://gepia.cancer-pku.cn/) as tool to search for the survival curve of the top 10 upregulated and downregulated lncRNAs original gene(40).
Statistical analysis
All data are shown as mean ± standard deviation (SD). Statistical significance was determined using Student's t-test by SPSS 13.0 and Graphpad Prism 5. p < 0.05 was considered statistically significant.