Cell cultures
Four human lung adenocarcinoma cell lines were used in this study. PC-9 with an exon 19 in-frame deletion was provided from Immuno-Biological Laboratories (Gunma, Japan). NCI-HCC827 (HCC827) with a deletion in exon 19, NCI-H1975 (L858R/T790M), and H1650 with a deletion in exon 19 and PTEN null were purchased from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in RPMI1640 (Wako Pure Chemical Industries, Osaka, Japan) including 10% fetal bovine serum (BioWest, Nuaillé, France), and 1% penicillin and streptomycin (Wako Pure Chemical Industries) at 37°C in a 5% CO2 incubator as previously described12 . All cells were constantly examined for the absence of mycoplasma, and each experiment was performed independently three times for each condition.
Drugs and cell viability assay
Afatinib and osimertinib were obtained from Selleck Chemicals (Houston, TX, USA). We used the MTS assay to evaluate the sensitivity of the human lung adenocarcinoma cell lines to afatinib and osimertinib as previously described12 . For the MTS assay, 5,000 cells per well were seeded in 96-well tissue culture plates and incubated for 24 h. Subsequently, the cells were treated with various concentrations of EGFR-TKIs or vehicle (dimethyl sulfoxide) at 37°C for 72 h. Viability experiments were performed using the Cell Counting Kit 8 (Promega Corporation, Madison, WI, USA) and a microplate reader (Infinite M200 PRO; Tecan Group Ltd., Männedorf, Switzerland) according to the instructions provided by the manufacturer. The IC50 value was defined as the concentration of drug required for 50% inhibition of growth. The corrected absorbance of each sample was calculated by comparing with that of the untreated control. Each experiment was carried out thrice.
Western blotting analysis
Protein extraction, two-dimensional polyacrylamide gel electrophoresis, and transfer to nitrocellulose membrane were carried out as previously described11,12,15 . The membrane was incubated with the following antibodies: cleaved CASP3, cleaved PARP, PARP, p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, p-STAT3, STAT3, eIF4A3, p-4E-BP1, mTOR, p-P70-S6, MUC1, and MUC1-C purchased from Cell Signaling Technology (Danvers, MA, USA). The antibody to glyceraldehyde-3-phosphate dehydrogenase was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
RNA extraction and microarray analysis
Total RNA from cells was extracted by TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) as previously described10,30 or ISOGEN (NIPPON GENE, Tokyo, Japan).Gene expression microarray analysis was performed using a SurePrint G3 Human Gene Expression 8x60K v3 (Takara Bio, Shiga, Japan).
Real-time qRT-PCR
The cDNA of CRNDE, DGCR5, and eIF4A3 was utilized for qRT-PCR analysis using the THUNDERBIRD SYBR qPCR/RT Set III (TOYOBO, Osaka, Japan) according to the instructions provided by the manufacturer. The expression of these genes was examined through the TaqMan Gene Expression Assay (Thermo Fisher Scientific)31,32. Gene expression levels were quantified using the 2−ΔΔCt method.
siRNA transfection
The siRNA targeting CRNDE and negative control were purchased from Thermo Fisher Scientific. All siRNAs were treated with Lipofectamine RNAiMAX (Thermo Fisher Scientific) transfection reagent 24 hours after seeding, according to the instructions provided by the manufacturer. We transfected the siRNA complexes into cells at a final concentration of 50 nM. After 6 hours from the addition of the siRNA complexes, cells were seeded in RPMI1640 containing 10% fetal bovine serum, and 1% penicillin and streptomycin and incubated at 37°C for 48 hours.
Statistical analysis
Differences in categorical outcomes were assessed by the chi-squared test. The statistical significance of differences decided with the standard Student’s t-test. P-value of < 0.05 was considered statistically significant. Analyses were carried out by the statistical software JMP 9 (SAS Institute, Cary, NC, USA).
Bioinformatics
The transcriptome (consisting of mRNAs and lncRNAs) was used as a reference for mapping. LncRNA sequencing data were extracted from LNCipedia33 (https://lncipedia.org/downloads/lncipedia_5_0.fasta), including Refseq, Ensembl, Gencode, Broad Institute (Human Body Map lincRNAs), NONCODE and FANTOM CAT to establish a lncRNA database. The mRNA sequencing data were extracted from Gencode34 (ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_28/gencode.v28.transcripts.fa.gz) to establish a mRNA database. The sequencing data were extracted from the probe information site of Agilent Inc. The candidate RNA, with more than two-fold change in levels between parental cells and resistant sublines, was detected. The protein linked to the candidate RNA was detected using RNA-Protein binding data (http://starbase.sysu.edu.cn/starbase2/index.php)35.