Cell culture
Human non-small cell lung cancer cell lines (A549, H23, H226, H292, H358, H460, H522, H1299, Hcc95, Hcc827, and Hcc1438) were obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) or DMEM/HIGH GLUCOSE (A549; Hyclone) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (Hyclone). The Hcc95 cells were supplemented HEPS. The cells were maintained at 37 oC in a humidified atmosphere containing 5% CO2.
Transfection
A synthetic hsa-miR-7-5p mimic (Genolution, Seoul, Korea) was designed according to registered in miRBase database. miRNA inhibitor targeting hsa-miR-7-5p (5’-UGG AAG ACU AGU GAU UUU GUU GUU-3’) and an siRNA targeting LINC00240 (5’-CUA CAU UUG AGC AUA GUA U-3’) were synthesized by Bioneer Co. (Bioneer, Daejeon, Korea). Cell transfection was performed using Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The synthesized full-length LINC00240 sequence was subcloned into pcDNA3.1 vectors and, cell transfection was performed using a Lipofectamine 3000 transfection kit (Invitrogen) by following the manufacturer’s protocol.
Target labeling and hybridization to microarray
The synthesis of target cRNA probes and hybridization were performed using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technology, USA) according to the manufacturer’s instructions. Briefly, each 0.2 ug total RNA sample was mixed with T7 promoter primer mix and incubated at 65 oC for 10min. cDNA master mix (5X First strand buffer, 0.1M DTT, 10mM dNTP mix, RNase-Out, and MMLV-RT) was prepared and added to the reaction mixture. The samples were incubated at 40 oC for 2 hours and then the RT and dsDNA syntheses were terminated by incubating at 65 oC for 15 min.
The transcription master mix was prepared according to the manufacturer’s protocol (4X Transcription buffer, 0.1M DTT, NTP mix, 50% PEG, RNase-Out, inorganic pyrophosphatase, T7-RNA polymerase, and cyanine 3/5-CTP). Transcription of the dsDNA was performed by adding the transcription master mix to the dsDNA reaction samples and incubating at 40 oC for 2 hours. The amplified and labeled cRNA was purified on an RNase mini column (Qiagen) according to the manufacturer’s protocol. Labeled cRNA target was quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).
After determining the labeling efficiency, each 825ng of cyanine 3-labeled and cyanine 5-labeled cRNA target was mixed and the cRNA fragmentation was performed by adding a 10X blocking agent and 25X fragmentation buffer and incubating at 60 oC for 30min. The fragmented cRNA was resuspended in 2X hybridization buffer and directly pipetted onto an assembled Agilent Human Whole Genome 60K V3 microarray. The arrays were hybridized at 65 oC for 17 h using an Agilent Hybridization oven (Agilent Technology) as described previously [13]. The hybridized microarrays were washed according to the manufacturer’s washing protocol (Agilent Technology).
Data acquisition and analysis
The hybridization images were analyzed by an Agilent DNA Microarray Scanner (Agilent Technology) and data quantification was performed using Agilent Feature Extraction software 9.3.2.1 (Agilent Technology) as described previously [14]. The average fluorescence intensity for each spot was calculated and the local background was subtracted. All data normalization and the selection of fold-changed genes were performed using GeneSpringGX 7.3.1 (Agilent Technology). The genes were filtered to remove flag-out genes in each experiment. Intensity-dependent normalization (LOWESS) was performed, where the ratio was reduced to the residual of the Lowess fit of the intensity vs. ratio curve. The average of the normalized ratios was calculated by dividing the average of the normalized signal channel intensity by the average of the normalized control channel intensity. Genes changed >2.0-fold were selected and considered significant genes. The microarray result has been deposited into the Gene Expression Omnibus (GEO).
The functional annotation of genes was performed according to Gene OntologyTM Consortium (http://www.geneontology.org/index.shtml) by GeneSpringGX 7.3.1.
Quantitative real-time PCR (qRT-PCR)
The total RNA was isolated from the cells using TRI Reagent (Ambion, Thermo Fisher Scientific) according to the manufacturer’s protocol. To determine the mRNA and lncRNA expression levels, cDNA was synthesized using M-MLV reverse transcriptase (Promega, Madison, WI, USA) after which qRT-PCR was performed in triplicate for the LINC00240, EGFR and GAPDH genes using iQ SYBR Green Supermix (BioRad Laboratories, Hercules, CA, USA), and a CFX Connect Real-Time PCR Detection System (BioRad Laboratories, Hercules) was used. The qRT-PCR conditions were, 95 oC for 3 min; 39 cycles of 95 oC for 10 s, 60 oC for 15 s, and 75 oC for 15 s. The primers used for the mRNA qRT-PCR were: LINC00240: forward: 5’-AGG TCA CCC ACC GGT CTG AA-3’, and reverse: 5’-TAG GCT GGG CTC AGC TGG AT-3’; EGFR: forward: 5’-CCA GAC TCT TTC GAT ACC CA-3’, and reverse: 5’-CTT CCT GGC TAG TCG GTG TA-3’; GAPDH: forward: 5’- ACA GTC AGC CGC ATC TTC TT-3’, and reverse: 5’- ACG ACC AAA TCC GTT GAC TC-3’. qRT-PCR for the miRNA for the miR-7-5p (Assay ID: 005723_mat) and RNU6B (Assay ID: 001093) expression levels was performed in triplicate using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA, USA) by following the manufacturer’s instructions. GAPDH and RNU6B were used as internal controls for normalization, respectively.
Invasion and migration assays
Invasion and migration assays were performed using 48-well micro-chemotaxis Boyden chambers that contained 12-μm-pore membranes (Neuroprobe, Gaithersburg, MD, USA) pre-coated with 10 μg/ml Matrigel (BD Bioscience, San Jose, CA, USA) for the invasion assay and 13 μg/ml collagen type I (Sigma Aldrich) for the migration assay as described previously [15]. The cells, H1299 (0.8 x 106 cells/ml) and Hcc1438 (1.3 x 106 cells/ml), were seeded in triplicate in the chambers, and incubated for 24-26 h. The resulting membranes were fixed and stained using Diff-Quik reagent (Sysmex Corporation, Kobe, Japan). The invaded and migrated cells were photographed under a light microscope, and the relative invasion and migration rates were calculated based on comparison to the negative controls.
Luciferase assay
To investigate whether LINC00240 directly interacted miR-7-5p, luciferase assay was performed. The fragment of LINC00240 containing the miR-7-5p binding sites were synthesis by PCR, and these are cloned the renilla luciferase reporter site of psiCHECK2 vectors. And we confirmed successful cloning using DNA sequencing. To luciferase assay, Hcc1438 cells were transfected with psiCHECK-2 plasmid containing position s 174-179 of the LINC00240 with or without mutations of binding site and with miR-7-5p mimic or negative control. At 48 hours after transfection, the luciferase assays were performed in manufacturer’s protocol using Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase activity was measured using a Synergy HTX microplate reader (BioTek, Winooski, VT, USA), and the results were normalized using the activity of firefly luciferase. All experiments were performed in duplicate.
The Cancer Genome Atlas (TCGA) program
The TCGA dataset of lung adenocarcinoma (AC) and lung squamous cell carcinoma (SQ) was downloaded from the TGCA Data Portal (https://tcga-data.nci.nih.gov/tcga/). The dataset contained a total of 1,016 RNA-seq data at level 3 (515 of AC and 501 of SQ RNA-seq data) as well as clinical data. Among them, 58 AC and 51 SQ cases had RNA-seq dataset of non-tumor pairs with complete clinical information. These cohorts were used for RNA expression analysis of LINC00240 and EGFR. Gene expression was measured by the Illumina HiSeq platform and presented as FPKM values. The dataset can be used for publication without restriction or constraint according to the publishing guidelines (https://cancergenome.nih.gov/publications/ publicationguidelines).
Statistical analysis
Each experiment was performed three times, and the data were expressed as mean ± standard deviation (SD). The results were analyzed using Student's t-test. P-values of ≤ 0.05 was considered statistically significant.