Patients and tissue samples
Ten de-identified tumor samples (each includes NSCLC and normal control patients) were obtained from the Shuang Ho Hospital Resource Center (New Taipei city, Taiwan). The patients in this study were free from any inflammatory conditions. Written informed consents were obtained from the patients and this study was approved by the ethics committee of Shuang Ho Hospital.
NSCLC cell lines and cell culture
Human adenocarcinoma cell lines H838, H23, and H2009 were purchased from American Type Culture Collection (Manassas, VA), while CL1-1 and CL 1–5 were kind gifts from Dr. Pan-Chyr Yang of National Taiwan University. Cells were cultured in RPMI 1640 medium (GIBCO, Gaithersburg, MD) containing 10 % fetal bovine serum (FBS) (Jacques Boy, Reims, France), 100 U/mL of penicillin (GIBCO) and 100 U/mL of streptomycin (GIBCO) at 37 °C in a 5 % CO2 atmosphere at 99 % humidity.
Generation of YKL-40 stable expression CL1-1 cell slines
The full length of YKL-40 cDNA was isolated directly from a NSCLC CL1-5 cDNA library using the polymerase chain reaction (PCR) and introduced restriction sites for BamHI and HindIII. After sequence confirmation, the full-length YKL-40 was cloned into the reporter vector pEGFP-C1 to yield the reporter plasmid. The NSCLC CL1-1 cells were transfected with 2 μg of YKL-40 or vector control DNA using TurboFect™ reagent (Fermentas, Glen Burnie, MD) as the delivery vehicle. Selection with 800 μg/ml of G418 was performed at 24 h after transfection, and the drug-resistant cell populations were used for subsequent studies.
Generation of YKL-40 stable knockdown CL1-5 cell lines
TurboFect™ (Fermentas, Glen Burnie, MD) was used to deliver plasmid DNA containing small hairpin RNA (shRNA) against human YKL-40(OriGene, Rockville, MD) into NSCLC CL1-5 cells in a 24-well culture plate. Selection with puromycin was performed at 24 h after transfection, and the drug-resistant cell populations were used for subsequent studies.
Generation of YKL-40 stable knockdown and re-overexpression CL1-5 cell lines
After the stable culture YKL-40 knockdown CL1-5 cells, the cells were transfected with 2 μg reporter vector (DsRed plasmid), which contained the full-length YKL-40 gene, using TurboFect™ reagent (Fermentas, Glen Burnie, MD) as the delivery vehicle. Selection with 800 μg/ml of G418 was performed at 24 h after transfection, and the drug-resistant cell populations were used for subsequent studies.
Migration assay
Cells were harvested and suspended in RPMI 1640 medium containing 10 % FBS at a concentration of 1 × 106 cells/mL. A transwell apparatus with 8 μm pore size membrane (Millipore, Billerica, MA) was used to analyze the migration activity. In brief, suspension of cells in 100 μL of serum-free RPMI 1640 medium were seeded into the upper chamber of the apparatus, 250 μL of RPMI containing 10 % FBS were added to the insert well, and the apparatus was incubated at 37 °C for 6 h. After incubation, the inner wall of the chambers were wiped with wet swabs to remove migrated cells. The outer wall of the chambers were gently rinsed with PBS and stained with Giemsa (Sigma-Aldrich, St. Louis, MO) for 10 mins. Finally, the membrane was rinsed and allowed to air-dry. The membrane in triplicate was photographed and the number of cells counted.
Invasion assay
Invasion assay was performed by a modification of the method described previously. A 100 μL Matrigel (Becton Dickinson, Franklin Lakes, NJ) was diluted to 1 mg/mL in serum-free RPMI. This solution was added to each upper chamber of the transwell with 8 μm pore size membrane. After solidification of Matrigel at 37 °C, approximately 1 × 105 cells in serum-free RPMI were seeded onto the Matrigel over the upper chamber, followed by the addition of 250 μL of RPMI containing 10 % FBS at the bottom insert-well. After incubation of the cells at 37 °C for 18 h, the inner wall of the chambers were wiped with wet swabs to remove the cells, while the outer wall of the chambers were gently rinsed with PBS and stained with the Giemsa stain solution for 10 mins. Finally, the membrane was rinsed and allowed to air-dry. The membrane in triplicate was photographed and the number of cells counted.
Western blot analysis
The electrophoretic experiments were performed by SDS-PAGE (10 % polyacrylamide unless specified otherwise) to analyze the YKL-40 and EMT related genes. Electrophoresis was conducted by a vertical gel electrophoresis device that was powered by (Mini PIII, Bio-Rad, Hercules, CA) a PAC 300 power supply (Bio-Rad). All SDS-PAGE samples (20 μg) were equilibrated in 10 mM Tris–HCl and 5 % SDS (pH 7.6) before loading.
Following complete separation, the gel was soaked briefly in a transfer buffer, which contained 25 mM Tris, 192 mM glycine, 20 % methanol, and 0.0375 % SDS (pH 8.3), for 30 s. The gel was then immediately electrotransfered to a nitrocellulose membrane (Hybond-ECL extra; Amersham) at 90 mA for 60 mins in a semi-dry Transfercell (Bio-Rad). The membrane was immersed in 2 % skim milk for 1 h with gentle agitation. After three washes with PBS for 5 mins each, the membrane was subjected to react with monoclonal or polyclonal antibodies and developed with chemiluminescence agents.
Reverse transcription polymerase chain reaction (RT-PCR)
The YKL-40 mRNA levels in the NSCLC cells was determined by RT-PCR. Total RNA was extracted from the cells using TRIzol (Invitrogen Corporation, San Diego, CA). Following spectrometric determination of RNA yield, cDNA was synthesized with oligo (dT) primer using Moloney Murine Leukemia Virus (MMLV) Reverse Transcriptase. An aliquot of cDNA was subjected to 35 cycles of PCR using a standard procedure of initiation at 65 °C for 5 mins, incubating at 37 °C for 2 mins, and inactivating at 70 °C for 15 mins. The amplified products were resolved in a 1 % agarose gel and visualized by Sybr safe staining.
Bioinformatics
The associations between gene expression levels and NSCLC patients prognosis were obtained from the PrognoScan database [16]. PrognoScan is a large collection of publicly available cancer microarray datasets with clinical annotation and a tool for assessing the biological relationship between gene expression and prognosis. In the PrognoScan database, association of gene expression with survival of patients was evaluated by the minimum P value approach. Briefly, patients were first arranged by expression levels of a given gene. They were then divided into high- and low-expression groups at all possible cutoff points, and the risk differences between any 2 groups were estimated by the log-rank test. Finally, the cutoff point that resulted the most pronounced P value was selected.
Meanwhile, the mRNA expression of YKL-40 among cancer patients and healthy population were mined from The Cancer Genome Atlas (TCGA) database and analysed using BoxPlotR.
Statistical analysis
Data are presented as mean ± standard deviation (SD) The difference between the groups were calculated by Student t test (2 tailed). A p value of < 0.05 was taken as the indication of statistical significance.
