Cell lines and cell culture
Human lung squamous carcinoma cell line SK-MES–1, H226 was provided by Cancer Institute of Tongji University Medical School, China. The cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin.
Tumor samples
All the tumor samples in the study were collected from patients recruited from Shanghai Pulmonary Hospital, between July 2012 and June 2015. The patients were newly diagnosed and histological confirmed lung squamous cell carcinoma and adenocarcinoma. Patients with a previous medical history of cancer, radiotherapy or chemotherapy were excluded. This study was approved by the Ethics Committee of the Tongji University.
High Content Screening (HCS)
The Lentivirus expressing shRNA against negative control (NC), ILK, PTEN, MAP3K14, MYD88, SRF, IRAK1, BIRC5, PIK3C2A, PTP4A3, BTK, NLK, RAF1, STAT3, SRC, AURKA, IRAK4, ERBB2IP, CDH were purchased from Genechem technology, China. All the Lentivirus carried GFP genes. SK-MES–1 cells were transfected with Lentivirus shRNA (Non-targeting shRNA, PSC1369, PSC1446, PSC14867mix, PSC14872, PSC3584, PSC14896mix, PSC14359, PSC14907, PSC1675, PSC14821mix, PSC1786, PSC1809, PSC8012, PSC4913, PSC2260, PSC14817mix, PSC4899, PSC3306) respectively at the same time, and then after2 or 3 days of transfection, when the fluorescence rate reached 80%, cells were collected to subsequent experiments. Cell growth were accessed using the Cellomics ArrayScan HCS system (Thermo Fisher Scientific, Pittsburgh, PA, United States).These high-throughput RNAi experiments were done by Genechem (Shanghai, China).
Cell proliferation analysis
The cells (3×103/well) were seeded into 96-well plates in triplicate and were exposed to various concentrations of erlotinib. After 72 hours, 20μl of 3-(4,5-dimethylthiazol–2-yl)–2,5-diphenyltetrazolium bromide (MTT) solution (5mg/ml) was added to each well and incubated. After 4 hours, crystalline formation was dissolved with Dimethyl sulfoxide (DMSO) and the absorbance at 530nm was read using the microplate-reader for ELISA MK–2 Labsystems Dragon. Percent survival was calculated as: (mean absorbance of the replicate wells containing drugs - mean absorbance of the replicate background wells) / (mean absorbance of the replicate drug-free wells - mean absorbance of the replicate background wells). The test was performed independently 3 times. All the results derived from triplicate experiments yielded almost similar results.
Apoptosis analysis
Flow cytometry and transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) Kit (Promega, USA) were used for apoptosis assay. In flow cytometry assay, SK-MES–1 cells were plated in 6-well plates. Twenty-four hours later, 2µmol/L erlotinib was added into experimental wells and incubated for another 72 hours. The cells were harvested, washed with PBS and resuspended in 500 µl binding buffer. The cells were stained with 5 μl of Annexin V-PE and incubated for 5 min at room temperature in the dark. Quantification of apoptosis was determined by flow cytometry.
In TUNEL assay, cells were seeded in 24-well plates and exposed to 2µmol/L erlotinib for another 72 h. Apoptosis was assessed by the TUNEL assay kit (GENMED, China) following the manufacturer’s protocol. Apoptotic index (AI) (%) was calculated by the formula: positive staining cells / tumor cells number × 100%.
Western blotting
Cells were washed twice with ice-cold PBS and lysed in 0.1ml of lysis buffer on ice for 30 min. Insoluble debris was removed by centrifuging at 13,000 rpm for 15 min at 4°C. Electrophoresis and blotting procedures were done according to methods described previously[15–17]. Intrinsic antibody against human ILK (R﹠D, USA) was used according to the manufacturer’s instructions. Blotting quantification was done with an Odyssey® Infrared Imaging system (LI-COR, USA).
Quantitative polymerase chain reaction (qPCR)
1μg cDNA was used for qPCR analysis using Lightcylcer (Roche, Switzerland) following the manufacturer’s instructions. GAPDH was used as the internal control. Amplifications were carried out in the 20µl reaction mixtures in the following conditions 95℃ for 2min and followed by 40 cycles of 95℃ for 20 s, 55℃ for 20 s and 72℃ for 35s; and then 72℃ for 3min. The copy numbers of ILK gene were determined by: target gene copy number 2-△△CT = (CT target gene -CT reference gene) experimental group - (CT target gene -CT reference gene) control group.
Cell cycle analysis
The cells were incubated in 6-well plates for 24 h, and the cell culture medium was replaced by fresh medium containing 10% FBS with or without erlotinib, and incubated for another 72 h. The cells were trypsinized, fixed in ice-cold 70% ethanol overnight, and stained with propidium iodide containing 1 mg/mL RNase (Sigma, USA), according to the instructions of the Cell Cycle Phase Determination Kit (Cayman Chemical Company, USA). Samples were analyzed on a flow cytometry (Becton Dickinson, USA). Cell cycle parameters from 10,000 events were analyzed using multi-cycle software.
Clone formation analysis
Cells (200/well) were seeded in 24-well plates and treated with erlotinib after 12 hours. After two weeks, the cells were stained with 1% methylrosanilinium chloride, and the number of visible colonies was counted. The relative clone formation ability was calculated as follows: (mean experimental clone numbers /mean control clone numbers) ×100%.
Signaling pathway microarray analysis
There were two lung SqCC cell groups: NC (SK-MES–1 cells treated with erlotinib), and KD (SK-MES–1 cells treated with erlotinib after ILK knockout). Total RNA were extracted from cells, and RNA probes were prepared and hybridized to the GeneChip primeview human 100 format (901838, Affymetrix GeneChip System; Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions by Genechem Inc.. For each sample, three biological replicates were performed. All arrays were washed, stained, and read by a GeneChip Scanner 3000 (Affymetrix). The fluorescence signal was excited at 570 nm, and data were collected on a confocal scanner at 3 lm resolution. Data were analyzed by GeneChip Operating Software 1.4.
Data analysis
The differentially expressed genes (DEGs) were selected based on Fold-change > 2 and P value < 0.05 to further study. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, significantly changed pathways were identified and connected in a pathway network (Path-net) to show the relationship between these pathways. GO analysis was used to organize DEGs into hierarchical categories. The list of DEGs, containing gene identifiers and corresponding expression values, was also uploaded into the IPA software (2012 Ingenuity Systems, Inc. http://www.ingenuity.com). The IPA software is based on computational algorithms that analyze the functional connectivity of the genes from information obtained within the IPA database. The “core analysis” function included in the software was used to interpret the differentially expressed data, which included biological processes, canonical pathways, upstream transcriptional regulators, and gene networks. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathway Knowledge Base (IPKB).
Statistical analyses
Values were expressed as mean ± SD. Statistical analyses were done by independent-samples t test. Differences were considered to be statistically significant if P < 0.05.