Cell lines and reagents
Human lung cancer cell lines PC9 and PC9/GR were cultured at 37°C in 5% CO2 in RPMI-1640 medium (WELGENE, Inc., Daegu, South Korea) containing 10% fetal bovine serum (FBS; WELGENE). The EGFR-TKI gefitinib was obtained from Tocris Bioscience (Iressa, 184475-35-2; Bristol, UK).
Plasmids pCDNA3.1-Mig-6-FLAG and FLAG-tagged full-length Mig-6 were used for transfection. Transfections with different DNA constructs were performed using Lipofectamine LTX with Plus reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Assays were conducted after incubation for 48 h.
Lentivirus production, infection, and establishment of stable cell lines knocking down cellular genes
Lentiviruses were prepared using human embryonic kidney 293T cells. PC9 and PC9GR cells were infected with lentivirus to knock down cellular human Mig-6 genes. The expression arrest pLKO lentiviral vector encoding a non-silencing control short hairpin RNA (shRNA) or Mig-6 shRNA was obtained from Sigma-Aldrich (St. Louis, MO, USA). Virus supernatant plus 2 ng/mL polybrene was applied to 70-80% confluent cells, and non-infected cells were eliminated though puromycin selection.
Western blot analysis
Cells were harvested and suspended in protein lysis buffer (Translab, Korea) and heated at 100°C for 10 min. Protein concentration was determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA; cat. no. 500-0006). Approximately 30 µg of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Darmstadt, Germany). The following antibodies were used: anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Mig-6 (GTX116560, GeneTex, Irvine, CA, USA), anti-p-EGFR (Tyr1068) (#2236, Cell Signaling Technology, Danvers, MA, USA), anti-p-EGFR (Tyr1045) (#2237, Cell Signaling Technology), anti-EGFR (sc-03, Santa Cruz Biotechnology), anti-p-AKT (#40605, Cell Signaling Technology), anti-AKT (sc-1619, Santa Cruz Biotechnology), anti-p-ERK (sc-7383, Santa Cruz Biotechnology), anti-ERK (#9102, Cell Signaling Technology), anti-E-cadherin (610182, BD Biosciences, San Jose, CA, USA), anti-ZO1 (ab59720, Abcam, Cambridge, UK), anti-vimentin (550513, BD Biosciences), and anti-PARP (#9542, Cell Signaling Technology). Blots were developed using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Waltham, MA, USA).
Phos-tag Immunoblot analysis
To check the phosphorylated status of Mig-6 in the lysates, we employed phos-tag SDS-PAGE. The lysate was loaded in 6% Acrylamide 100uM Mn2+-Phos-tagTM Acrylamide. To confirm that those slower migrating bands are really phosphorylated species of Mig-6, we treated a part of the lysate with protein phosphatase for 9 h.
Twenty-six pairs of lung tissue sections were cut from paraffin blocks. Tumor tissues were obtained from Chungnam national university hospital and Chonnam national university hospital. All methods were carried out in accordance with relevant guidelines and regulations.
Written informed consent was obtained from all patients and the study protocol was approved by the Clinical Research Ethics Committee of Chungnam national university hospital. Institutional review board (IRB) approved this research. IRB file number is 2015-07-001-002. All experiments were performed in accordance with relevant guidelines and regulations.
Immunohistochemical staining analysis
Tissue sections were mounted on the coated slides, deparaffinized with xylene, hydrated in serial solutions of alcohol, and heated in a pressure cooker containing 10 mmol/l sodium citrate (pH 6.0) for 3 minutes at full power for antigen retrieval. Endogenous peroxidase activity blocking was performed using 0.03% hydrogen peroxide containing sodium azide for 5 min. The sections were incubated at room temperature for 4 h with the rabbit polycloncal anti-ERRFI1 (Mig-6) antibody (1:50, ab198834, Abcam, Cambridge, United Kingdom). After washing, the samples were incubated in labelled polymer-HRP anti-mouse (Dako EnVision+system-HRP (DAB), Dako, Carpinteria, California, USA) for an additional 20 min at room temperature followed by additional washing. After rinsing, chromogen was developed for 2 min. The slides were then counterstained with Meyer's hematoxylin, dehydrated, and coverslipped. Immunohistochemical staining was scored to evaluate both intensity of immunohistochemical staining and proportion of stained tumor cells in each stained slide. The intensity was scored as 0 (negative), +1 (mild), +2 (moderate), +3 (marked) and proportions were scored ranged from 0 to 100%.
Cultured cells were fixed with 4% paraformaldehyde at room temperature, permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) and blocked with 3% FBS in PBS. Following overnight incubation at 4°C with the appropriate primary antibody and incubation in the dark with Alexa 594 Fluor dye-labeled secondary antibodies, immunofluorescence was detected using a fluorescence microscope (OLYMPUS, Tokyo, Japan).
Transwell migration and invasion assays
Migration and invasion assays were performed using a transwell membrane (0.8 µm pore-size cell culture insert, FALCON, Abilene, TX, USA). A non-coated transwell membrane was used for the cell migration assay, and a transwell membrane coated with 2 mg/mL Matrigel was used for the cell invasion assay. A total of 2 × 104 cells were used for the migration assays. For the invasion assays, 2 × 105 cells in serum-free medium were seeded onto the upper chamber and 750 µL medium supplemented with 10% FBS was added to the lower chamber. After incubation for 16 h, cells that did not migrate through or invade the pores were removed with a cotton swab. Cells that exhibited migration and invasion (adhered to the lower surface) were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and counted using a light microscope in three randomly selected fields. The experiments were performed thrice in triplicate.
Scratch-wound healing and cell viability assays
Cells were seeded onto 6-well plates to 80–90% confluence and the cell monolayer was scratched in a straight line using a 200 μL pipette tip. Images were taken at 0 and 24 h after the scratch to calculate the cell migration rate.
Cell viability was counted using the CCK-8 assay kit (Dojindo Molecular Technologies, Inc., Rockville, MD, USA) following the manufacturer’s instructions. All experiments were performed three times in triplicate.
Reverse transcription polymerase chain reaction (RT-PCR)
Cells were collected for RNA extraction. Total RNA was isolated using TRIzol reagent (Invitrogen) following the manufacturer's instructions. cDNA was synthesized using oligo(dT) primers. The primers used for PCR amplification were as follows: hMig-6 (sense 5'-TGC ATT CTG CCC ATT ATT GA -3' and antisense 5'-AGG TAT GGT GGT CGT TCA GG -3'), hE-cadherin (sense 5'-GAA CTC AGC CAA GTG TAA AAG CC -3' and antisense 5'-GAG TCT GAA CTG ACT TCC GC -3'), hVimentin (sense 5'-AAA GTG CTG CCA AGA AC -3' and antisense 5'-AGC CTC AGA GAG GTC AGC AA -3'), and hGAPDH (sense 5'- ACC ACA GTC CAT GCC ATC AC -3' and antisense 5'- TCC ACC ACC CTG TTG CTG T -3'). PCR products were electrophoresed on a 1% agarose gel and visualized by ethidium bromide staining.
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay was performed in cells using an In Situ Cell Death Detection Kit, TMR red (Roche, Basel, Switzerland) according to the manufacturer’s protocol. Nuclei were visualized using 4’,6-diamidino-2-phenylindole (DAPI). TUNEL-positive (red) and DAPI-positive (blue) staining patterns were acquired with a fluorescence microscope (OLYMPUS) in three randomly selected fields. The experiments were performed thrice in triplicate.
Genomic and clinical data sets
All genomic data of lung adenocarcinoma were obtained from The Cancer Genome Atlas (TCGA) data portal (https://tcga-data.nci.nih.gov) and cancer browser (https://genome-cancer.ucsc.edu). Clinical data in patients with lung adenocarcinoma (n = 514), gene expression data from mRNA-seq, and protein expression data from Reverse Phase Protein Array (RPPA) data were analyzed. Clinical parameters included age, gender, smoking history, TNM stage, and EGFR mutation state.
Selection of specific gene signatures and functional enrichment analysis in relation to ERRFI1 expression
To investigate the role of Mig-6 in lung adenocarcinoma, patients were divided into two groups according to ERRFI1 expression. Based on the median value, 257 patients were included in the high ERRFI1 expression group and the low ERRFI1 expression group and Gene Ontology Consortium (GOC) was utilized to select specific gene signatures. Genes associated with the canonical pathway in the Gene Ontology (GO) database were analyzed. Patients with EGFR mutations were also analyzed separately. Gene Set Enrichment Analysis (GSEA) was utilized to enrich the mRNAs predicted to correlate with pathways in the hallmark and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.
Survival data of lung adenocarcinoma from TCGA project were obtained from TCGA data portal and cancer browser. Overall survival (OS) was measured from the date of diagnosis to the date of death or last follow-up. Survival was estimated using the Kaplan-Meier method, and survival rates were compared using the log-rank test.
The clinical pathological data were compared using the chi-square test and the paired t-test. Patients were divided into two groups according to ERRFI1 expression as described above. In order to select differentially expressed genes between the two groups, false discovery rate-adjusted P-values (<0.05) were used to correct for the Benjamini-Hochberg method. All in vitro experiments were repeated three times, and statistical significance was analyzed using a two-tailed student’s t-test or one-way analysis of variance followed by Tukey’s post hoc test. A P-value < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01). SPSS software (version 20; IBM Corp., Armonk, NY, USA) was used for all statistical analyses.