NSCLC and adjacent non-tumor lung tissues were obtained from patients during surgery in Kunming Medical University. The study was approved by the Ethics Committee for Human Medicine Research, Kunming Institute of Zoology, Chinese Academy of Sciences (Permit Number: SYDW-2012010). Written informed consent was obtained from the patients before obtaining samples for this study.
The animal experiments were carried out in strict accordance with the guidelines and approved by the Ethics Committee for Animal Experimentation, Kunming Institute of Zoology (Permit Number: SYDW-2012010).
NSCLC tissue samples and cell lines
Samples of primary NSCLC tissues were obtained from 30 patients during surgery. None of the patients had been treated before undergoing surgery. All of these samples were diagnosed according to the classification system of the World Health Organization and were staged using the criteria of the International Union Against Cancer [18, 19]. Samples of ten lung tissues, which were obtained from patients with lung bullous disease and lung inflammatory pseudotumors who were undergoing surgery, were used as the “noncancerous tissues.” The clinical characteristics of patients are listed in Additional file 1.
The lung cancer cell lines EPLC-32M1 (SCC), A549 (AC), 801D (LCC), NCIH-292 (mucoepidermoid carcinoma), NCI-H460 (LCC) and 16HBE (immortalized human bronchial epithelial cells) have been described previously [2, 3]. And details of all cell lines were provided in Additional file 2.
Generation of stable subcellular MCRS1 knockdown lines
The MCRS1 silencing vector was previously described . A target sequence (GCTGAAGAACAACGGTGAT) was designed and produced for MCRS1 silencing, and a luciferase-targeting shRNA oligonucleotide was used as the negative control. The complementary strands of the oligonucleotides were annealed and were ligated into the prelinearized RNAi-Ready pSIREN-RetroQ vector (Clontech, Palo Alto, CA, USA). The MCRS1-silencing and negative control vectors were transfected into RetroPack PT-67 cells using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the retrovirus-containing medium was filtered, and the retroviruses were used to infect the cells of NSCLC lines in the presence of 10 μg/ml Polybrene (Sigma, St. Louis, MO, USA). Forty-eight hours after infection, the cells were selected using puromycin (Sigma).
The miR-155 mimics, 2′-O-methylated miRNA antisense oligonucleotides (ASO) and their cognate controls that were used for transient transfection, were purchased from the RiBoBio Company (Guangzhou, China). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In each case, 100 nmol/L of the miRNA mimic and 150 nmol/L of the ASO were used.
To generate the Rb1 (retinoblastoma-related gene 1) luciferase reporter plasmid (pGL-Rb1), the 3′ untranslated region (3′-UTR) encompassing the miR-155 binding site was cloned between the KpnI and XhoI restriction sites of the pGL-3 basic vector (Promega, Madison, WI, USA) using a Polymerase Chain Reaction (PCR)-generated fragment. The mutation in the miR-155 binding site of the human Rb1 3′-UTR was generated using overlapping PCR. For the ectopic expression of Rb1, the coding region of human Rb1 was cloned into the pCDH-CMV-MCS-EF1-GFP-T2A-Puro vector (System Biosciences, California, USA). Subsequently, virus packaging and infection were performed according to manufacturer’s instructions. The contents of all of these constructs were confirmed by sequencing. The primers used are shown in Additional file 3.
MiRNA target prediction and luciferase reporter assays
The miRWalk database was used to predict the target genes of miRNAs . The genes that had both downregulated expression after MCRS1 silencing and had been predicted to be targets of miR-155 were chosen for analysis using the reporter assay.
Each reporter construct and the Renilla luciferase expression plasmid (pRL-TK) was co-transfected into cells with the miR-155 mimic or its negative control using Lipofectamine 2000. The pRL-TK plasmid was used as an internal control. Forty-eight hours after transfection, the levels of luciferase activity were determined using the Dual-Luciferase Reporter System (Promega) according to the manufacturer’s instructions.
Quantitative real-time polymerase chain reaction (QRT-PCR) assays of miRNA and mRNA expression
The total RNA was extracted from cells and tissues using TRIzol reagent (Sigma). The qRT-PCR assays of mRNA and miRNA expression levels were conducted as previously described . The housekeeping genes GAPDH and U6 snRNA were used as internal controls for the mRNA and miRNA assays, respectively. The primers used are shown in Additional file 3.
Western blotting analysis
The preparation of protein-containing lysates and western blotting was conducted as described previously . Briefly, the proteins in the lysates were resolved using 10 % sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were incubated with the following antibodies: anti-Rb1 (sc-50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-MCRS1 (R36649; Sigma), and anti-GAPDH (G8795; Sigma). Finally, the membrane was incubated with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific Inc., Waltham, MA, USA), and the blots were exposed to X-ray film. The films were developed, scanned and analyzed using the Image J software (NIH, Bethesda, MD, USA). GAPDH was used as an internal control.
Cells were seeded at a density of 4 × 103 cells/well in 96-well plates. Seventy-two hours post-transfection, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to each well. After an incubation period, the medium was removed, and dimethyl sulfoxide (DMSO) was added. The absorbance at 570 nm (using 630 nm as a reference) was detected using a microplate reader (model 680, Bio-Rad Laboratories, Berkeley, CA, USA).
In vivo tumor growth assay
Sixteen female BALB/c nude mice (4 weeks old) were purchased from Vital River Laboratories (Beijing, China) and were housed under standard conditions. Overall, 1 × 106 control EPLC-32 M1 cells (without MCRS1 silencing) and MCRS1-knockdown EPLC-32 M1 cells were subcutaneously implanted into the left and right flanks of the same mouse, respectively. Tumor growth was assessed using calipers every five days from 4 days to 32 days post-implantation, and the tumor volumes were estimated using the following equation: 0.5 × length × width2. The mice were anesthetized with diethyl ether and sacrificed by cervical dislocation at 5 weeks post-implantation, and the tumor pairs were harvested and weighed.
cDNA microarray analysis
The total RNA was extracted from MCRS1 knockdowned cells and control cells using TRIzol reagent (Sigma). Agilent 60 K Human Gene Expression arrays were used for mRNA profiling. Quality control of the total RNA, the probe labeling, and the array hybridization, as well as the data extraction and normalization, were performed at the CapitalBio Corporation (Beijing, China; http://www.capitalbio.com). The differentially expressed genes were determined according to the ratio of their expression levels relative to those of the control cells (ratio > 2: upregulated genes; ratio < 0.5: downregulated genes). The differentially expressed genes are shown in Additional file 4.
Chromatin immunoprecipitation (ChIP) assays
The ChIP assays were performed using the EZ-Magna ChIP kit (Millipore, Merck KGaA, Darmstadt, Germany) as described previously . Briefly, the chromatin of the samples was sheared into fragments with an average length of approximately 250 bp by sonication on ice using a Bioruptor sonicator. Immunoprecipitation of the sonicated chromatin fragments was conducted using an anti-MCRS1 antibody (SC-376569; Santa Cruz Biotechnology) and normal mouse IgG antibodies (the negative control), and the input and immunoprecipitated DNA fragments were analyzed using PCR. The GAPDH promoter primers provided in the ChIP kit were used as the internal control. The primers used are shown in Additional file 3.
DNA copy number assays
Genomic DNA was extracted from tissues and cultured cells using standard methods. After digesting the samples using proteinase K, the genomic DNA was isolated using phenol/chloroform/isoamyl alcohol and ethanol precipitation. Then, based on the instructions of the qBiomarker Copy Number PCR assay kit, each sample DNA was diluted to the same concentration and was amplified using qRT-PCR. The results were analyzed using the qBiomarker Data Analysis program (http://pcrdataanalysis.sabiosciences.com/cnv/CNVanalysis.php). The DNA copy number was determined using the 2-△CT method. The primer set used to determine the MCRS1 copy number was as follows: 5′CACCAGAAGGCTCACTCTTCA3′ and 5′TGTCTGGTTGTCGAAGTCCGG3′. The qBiomarker Multicopy Reference Assay (MRef, QIAGEN, Suzhou, China) was used as the internal control.