Tumor and nontumor tissue samples were obtained from 89 patients with NSCLC. All participants provided written informed consent. The study was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH-IRB-980524). The baseline characteristics of the patients with NSCLC (age, sex, smoking status, Eastern Cooperative Oncology Group performance status (ECOG PS), histology, differentiation grade, and TNM stage) were collected from chart records.
RNA extraction and qRT-PCR
Total RNA extraction, complementary DNA (cDNA) generation, and polymerase chain reaction (PCR) were performed according to manufacturer protocals. The detailed procedures and primer sequences are listed in Additional file 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Cell culture and shRNA transfection
H520, H1299, and REH cells were cultured in RPMI medium (GIBCO BRL, Gaithersburg, MD, USA) supplemented with 10 % fetal bovine serum (GIBCO BRL) at 37 °C in a 5 % CO2 atmosphere. TUG1 shRNA, scramble RNA, and mock were obtained from the National Research Program for Biopharmaceuticals and were transfected into H520 and H1299 cells (400 ng TUG1 shRNA added to 600 μL of cells, 2 × 105 cells/mL) by using Lipofectamine 2000 (Life Technologies). Transfection efficiency was determined through quantitative reverse transcription polymerase chain reaction (qRT-PCR).
Cell proliferation assay
Cell proliferation assay was used to examine whether TUG1 knockdown affects the viability of NSCLC cells. In brief, transfected H520 and H1299 cells were plated in 96-well plates (2 × 105 cells/mL, 100 μL/well). After 48 h, cell proliferation and viability were examined using the MTT assay. All experiments were performed in triplicate.
Circular chromosome conformation capture
The 4C experiment included the following basic procedures: formaldehyde cross-linking, and digestion and ligation of the known bait chromatin (i.e., TUG1 in this study) and the unknown sequences. Circular chimeric chromatin was then decross-linked, and the unknown sequences were amplified with inverse PCR by using the bait-specific primers. We followed the 4C method described by Stadhouders et al. , which involved secondary digestion and ligation between decross-linked and inverse PCR amplification. Secondary digestion is advantageous because it decreases the size of the DNA circles, enabling efficient PCR amplification of fragments. Six-base-recognizing (six-cutter) enzymes, which perform well on cross-linked chromatin, are generally recommended for primary digestion; any four-cutter enzyme that is insensitive to mammalian DNA methylation and has high religation efficiency can be used for secondary digestion. In addition, the final combination of primary and secondary restriction enzymes generates a suitable bait fragment for designing bait-specific primers for inverse PCR, depending on their compatibility . Based on our bait gene sequence (TUG1), a six-cutter enzyme (HindIII) was used as the primary digestive enzyme, and a four-cutter enzyme (CviQI) that can generate a bait sequence suitable for further primer design in HindIII-digested TUG1 fragments was used as the secondary digestive enzyme. In brief, REH cells were cross-linked with 1 % formaldehyde for 10 min at room temperature to preserve the three-dimensional nuclear architecture. HindIII was used to digest cross-linked chromatin (primary digestion). The digested chromatin was then ligated using the T4 DNA ligase. The digested and ligated chromatin was then decross-linked and submitted to the second restriction digestion by using CviQI to reduce the size of the fragments. Inverse PCR reactions were performed using TUG1-specific primers harboring Illumina adapter sequences to amplify the genomic DNA fragments ligated to TUG1 (first PCR: forward 5’-gtctccgatagtgcacacagc-3’, reverse 5’-gaccatctccttcaggacca-3’; nested PCR: forward 5’-cattcagccaatcacaaagct-3’, reverse 5’-cagatttatgacatagttccttccaa-3’).
The PCR products were purified using the Qiagen Mini-Elute kit. After purification, the amplicon was prepared for sequencing by using a Truseq DNA library preparation kit (Illumina). According to the TruSeq DNA Sample Preparation protocol, 100-ng purified amplicon pools were repaired to generate blunt-ended, 5’-phosphorylated DNA, and an A-tailing reaction compatible with the adapter ligation strategy was performed. The ligation product was purified by sample purification beads. To enrich the library, an enhanced PCR mix was used to perform PCR amplification. The size distribution of the library was verified using the High-Sensitivity DNA Kit (Agilent), and the concentration of the library was quantified using the GeneRead Library Quant Kit (Qiagen). The library was diluted and sequenced with 500 paired-end cycles on the Illumina MiSeq by following the standard protocol.
Bioinformatic analysis of TUG1 4C-sequencing data
Removing known fragments from sequencing reads
Removal of known fragments from sequencing reads involved three steps. First, forward and reverse sequencing reads were merged into one sequence when the length of the overlapping region between the forward and reverse sequencing reads was more than 20 nt. Second, BLAST, a widely used bioinformatic software of sequence searching, was used to identify the location of the known fragments and primers . The known fragments were located in the regions between the primers and the cutting site of the enzymes. The alignment similarity for BLAST was set at 95 %. Finally, results from Blast showed the location of known fragments and primers. These primers and known fragments of sequencing reads were removed from the sequencing reads. The remaining region of the sequencing reads was labeled “unknown fragments.”
Identifying potential TUG1 interaction regions
The Bowtie2 software is an efficient tool for aligning sequencing reads against reference sequences . Bowtie2 was used to align unknown fragments against human genome sequences (Grch38.p2 was used in the present study). Subsequently, fourSig, a software suite for analyzing and visualizing 4C-seq data, was used to identify the potential TUG1 interactive regions . The command “bamToReTab.pl –H 210 500 300 bowtie2_output hind3_site.txt nla3_site.txt NONE > foursig_output” was used to obtain the primary results from fourSig. The primary results were then used to scan for potential interaction regions. The window size was set at 50 (i.e., 50 fragments in the window). After scanning for the interaction regions, fourSig provided three categories of regions, which were defined as potential TUG1 interaction regions. The regions and their associated genes were listed and then annotated according to the genomic location of known human genes. These TUG1 interactive regions, having over 100 reads of coverage, were used as TUG1 interaction region candidates.
RNA immunoprecipitation (RIP)
RNA immunoprecipitation (RIP) assays were performed using ChIP-IT (Active Motif, Carlsbad, CA, USA), according to the Active Motif protocol. Anti-EED (Aviva Systems Biology, San Diego, CA, USA), anti-EZH2 (Cell Signaling Technology, Danvers, MA, USA) antibodies, and IgG were used. The detailed procedures are listed in Additional file 2.
DNA ChIP assays were performed using ChIP-IT (Active Motif, Carlsbad, CA, USA), according to manufacturer instructions. Anti-EED (Aviva Systems Biology, San Diego, CA, USA), anti-EZH2 (Cell Signaling Technology, Danvers, MA, USA) antibodies, and IgG were used. The detailed procedures are listed in Additional file 2. Six sets of primers were designed to amplify the CELF1 promotor region. The primer sequences are listed in Additional file 3: Table S1.
The differences in RNA expression between tumor and nontumor tissues from NSCLC patients were analyzed using a paired t test. The association between relative TUG1 RNA expression levels and clinical parameters (age, sex, smoking status, ECOG PS, histology, differentiation grade, and TNM stage) was analyzed using a t test and ANOVA. TUG1 shRNA and scramble shRNA transfected cells in the MTT assay were compared and analyzed through an independent samples t test. Statistical analyses were performed using SAS version 9.3; p < 0.05 was considered statistically significant.