Patients and tissue specimens
We collected paired tumor and adjacent non-tumor tissues from 138 patients with ESCC (between 2007 and 2009), from the Department of Oncological Surgery of the Central Hospital of Shantou City, P.R. China. Cases were selected in this study only if a follow-up was obtained and clinical data were available. The follow-up for patients after esophageal resection was continued until their deaths and only patients that died from ESCC were included in the tumor-related deaths. Patients, suffering from severe post-operative complications, other tumors or died of other causes were excluded. This study was approved by the Ethical Committee of the Central Hospital of Shantou City and the Medical College of Shantou University, and written informed consent was obtained from all surgical patients to use resected samples and clinical data for research. The tissue specimens were snap frozen in liquid nitrogen shortly after resection and stored at − 80 °C until RNA extraction.
Cell lines and culture conditions
Human esophageal cancer cell lines KYSE150, KYSE180, KYSE450, KYSE70, KYSE140 and TE3 were kindly provided by Dr. Ming-Zhou Guo (Chinese PLA General Hospital, Beijing, China) and grown in RPMI 1640 medium (Invitrogen, California, USA), with both media supplemented with 10% FBS (Invitrogen, California, USA). The human immortalized esophageal cell line NE2 was kindly provided by Professor Sai-Wah Tsao (University of Hong Kong, China) and grown in defined keratinocyte serum-free medium (Gibco, Grand Island, NY, USA) and Cascade Biologics® EpiLife® (Life Technologies, Grand Island, NY, USA) in a 1:1 mixture. All cell lines were cultured at 37 °C in 5% CO2 and 95% air.
Human samples or cell lines were lysed using TRIzol® (15596-018, Life Technologies, Carlsbad, CA, USA) and total RNA was released and further purified with a PureLinkTM RNA Mini Kit (12183018A, Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol. The purity and concentration of RNA were determined by OD260/280 using spectrophotometer (NanoDrop ND-2000).
Quantitative RT-PCR (qRT-PCR)
For qRT-PCR, the reverse transcription (RT) reactions were carried out with a PrimeScriptTM RT reagent kit with gDNA Eraser (RR047A, TaKaRa, Dalian, China) according to the manufacture’s protocol. Reverse transcriptase reactions contained 1 μg total RNA. The 20 μl RT reaction mixture was incubated in a 2720 Thermal Cycler (Applied Biosystems). Quantitative PCR reactions were then performed on an ABI 7500 with SYBR® Premix Ex TaqTM (RR420A, TaKaRa, Dalian, China) in a 20 μl reaction volume, which also contained 2 μl cDNA and 0.8 μl PCR primer mix (forward and reverse primers at a final concentration of 0.2 μM each). The reactions were incubated at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 34 s. The Ct value of each candidate lncRNA was then normalized to the expression value of β-actin. Relative expression levels of the lncRNAs were calculated using the 2-ΔCt method. Specimens that had no amplification within 40 cycles were deleted. Sequences of primers for qRT-PCR of the lncRNAs are listed in Additional file 1: Table S1.
Cells were transfected with siRNAs against RP11-366H4.1.1, LINC00460 and AC093850.2, with scrambled siRNA used as a negative control. The procedures for siRNA transfection were performed according to the X-tremeGENE siRNA transfection reagent instructions (Sigma-Aldrich, St. Louis, MO). The sequences for RP11-366H4.1.1 were sense: 5’-ACACACAUCCUAGUUCUUUdtdt-3′, and antisense: 5’-AAAGAAC UAGGAUGUGUGUdtdt-3′. The sequences for LINC00460 were sense: 5’-GUCACCCCGAUUUAUGUUAdtdt-3′, and antisense: 5’-UAACAUAAAUCGGGGUGACdtdt-3′. The sequences for AC093850.2 were 5’-GGACAAUGAAGACUGAACUdtdt-3′, and antisense: 5’-AGUUCAGUCUUCAUUGUCCdtdt-3′. The negative control siRNA was sense: 5’-UUCUCCGAACGUGUCACGdtdt-3′, and antisense: 5’-CGUGACACG UUCGGAGAAdtdt-3′.
Cell migration and colony formation assays
After RP11-366H4.1.1, LINC460 or AC093850.2 was subjected to individual knockdown, KYSE150 or KYSE70 cell migration and colony formation assays were performed as previously described . Briefly, at 24 h post transfection, cells were starved for 12 h with serum-free medium (Invitrogen, California, USA) and then 5 × 104 cells were plated in serum-free medium in the upper well of a transwell chamber (24-well insert; pore size, 8 μm; BD Biosciences, Franklin Lakes, NJ, USA), and the lower chamber containing medium with 10% FBS. After 48 h, cells in the top chamber were removed with a cotton swab and only cells that migrated through the pores were fixed and stained in haematoxylin solution (Sigma-Aldrich, St. Louis, MO, USA) and counted. For colony formation, 500 cells per well in 24-well plate were incubated in medium supplemented with 10% FBS for ten days, and then colonies were stained with haematoxylin solution and observed.
URWScore and bioinformatics analysis
The URW-LPE method has been previously described in detail . Briefly, a seed composed of each dysregulated lncRNA/PCG (protein coding gene), combined with an edge composed of an extended co-expression relation, was used as a random walk. Differentially-expressed lncRNAs and PCGs in ESCC were used to construct an extended lncRNA-PCG co-expression network and the random walk was run for the network and the fold change (FC) values of each node on the network was regarded as the initial probability vector. The random walk was represented according to the formula: pt + 1 = (1-r) Wpt + rp0. W is represented by the adjacency matrix in the lncRNA-PCG co-expression network, pt is a vector representing the probability of the corresponding lncRNA /PCG nodes at step t and p0 is used as the initial probability vector. Thus, each lncRNA in the network would be given an URWScore value and lncRNAs with a higher URWScore value may possess more important biological functions in ESCC. In the lncRNA-PCG co-expression network, protein-coding genes highly associated with the higher URWScoring lncRNAs (Pearson correlation coefficient > 0.40, P < 0.05) were selected. The association of the lncRNAs with potential protein-coding genes was visualized by Cytoscape_v2.8.3 software . GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway function enrichment analyses for the co-expressed protein-coding genes were performed according to the DAVID database on line (https://david.ncifcrf.gov/) .
The 138 specimens were randomly separated into a training set (n = 77) and test set (n = 61). A multivariable Cox regression model in the training set, including age, gender, histologic grade, invasive depth, lymph node metastasis and therapies, was constructed . Comparisons between the two sets for clinicopathological characteristics was performed using the t-test, Fisher’s exact test and chi-squared test. Comparisons of the relative expression between tumor and paired adjacent normal tissues were performed using paired a t-test. Overall survival (OS) was measured from the date of surgery to death or the latest follow-up. Disease-free survival (DFS) was measured from the date of surgery to the first occurrence of any of the following events, including recurrence, distant metastasis or death from any cause without documentation of a cancer-related event . The optimal cut-off point of lncRNA expression (2-ΔΔCt, ΔΔCt = ΔCt tumor – ΔCt normal, ΔCt = Ct (selected lncRNA) – Ct (β-actin)) and risk score were assessed by the X-tile program . According to the cutoff value, the relative levels of lncRNAs from 138 paired ESCC samples and adjacent normal tissues was divided into high or low expression groups using X-tile and then probabilities of OS and DFS patients with ESCC were calculated by Kaplan-Meier analysis and compared using the log-rank test with SPSS19.0 (IBM, Armonk, New York, USA). A two-tailed P-value less than 0.05 was considered to have statistical significance. All analyses were performed using SPSS 19.0 (IBM, Armonk, New York, USA) for Windows.