Cell culturing and human tissue microarray
A549, PC-9 and NCI-H1299 cells were obtained from American Type Culture Collection (ATCC) and maintained by following the culture instructions of ATCC. Human lung adenocarcinoma tissue microarray (HLugA020PG01) was obtained from Shanghai Outdo Biotech Company. There were 84 lung adenocarcinoma tissues and 70 adjacent normal tissues. The clinical data of patients were showed in Additional file 1: Table S1. And the association of DKC1 expression and patient’s survival was analyzed.
DKC1 expression in LUAD samples from GEO and TCGA databases
The mRNA level of DKC1 in 83 LUAD tissues and paired adjacent normal tissues from the Gene Expression Omnibus (GEO) database (GSE75037) was examined. GEO databases (GSE12930 and GSE102016) were used to examine the mRNA level of Dkc1 in inflammatory non-transformed lung. (http://www.ncbi.nlm.nih.gov/geo/). GSE12930 shows gene expression in smoking induced inflammation non-transformed mouse lung [13]. GSE102016 shows gene expression in non-transformed lung from phytol ameliorated benzo(a)pyrene (B[a]P) induced lung cancer mouse model with lipopolysaccharides (LPS) induced inflammation [14]. In addition, we searched The Cancer Genome Atlas Program (TCGA) database (https://cancergenome.nih.gov/) to examine DKC1 expression in LUAD tissues. There were 483 tumor tissues and 59 adjacent normal tissues. The extracted data were normalized and processed by log2 transformation.
Generation of stable knockdown or overexpression cells
shRNA for DKC1 (shDKC1-1, -2) was cloned into lentiviral pLKO.1 construct (Sigma-Aldrich, St. Louis, USA). The sequence of the shDKC1-1 is CCGGGCTCAGTGAAATGCTGTAGAACTC GAGTTCTACAGCATTTCACTGAGCTTTTTG and the sequence of the shDKC1-2 is CCGGTAT GTTGACTACAGTGAGTCTCTCGAGAGACTCACTGTAGTCAACATATTTTTG. The coding sequence of DKC1 was cloned into lentiviral pCDH-EF1-T2A-zeocin vector (System Bioscience, CA, USA). The virus particles were produced according to the lentivirus packaging protocol of Addgene. After shRNA lentivirus infection of lung adenocarcinoma cells lines (A549 and PC-9), puromycin (Thermo Fisher Scientific, Waltham, USA) selection was applied for at least 7 days to obtain stable knockdown cells. The overexpression lentivirus infected DKC1 stable knockdown cells or NCI-H1299 cells were selected with zeocin (Thermo Fisher Scientific, Waltham, USA) for at least 7 days to obtain ectopically expressed cells.
Cell proliferation, cell cycle and cell apoptosis assay
For cell growth curve, 1500 cells were plated in one well of 96-well plate with at least 3 repeats for each cell and Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan) was used to do continuous detection for 4–5 days. For colony formation assay, 1000 cells were plated in one well of 6-well plate with at least 3 repeats for each cell and 7–9 days later were stained with crystal violet. Image J was used for colony number counting. For pyrazofurin (SML1502, Sigma-Aldrich, St. Louis, USA) affecting cell growth, 1500 cells were plated in one well of 96-well plate, with at least 3 repeats for each condition and the next day pyrazofurin or vehicle was added. CCK-8 was used to do the continuous detection for 4–5 days. For cell cycle, one million of cells were harvested and fixed using pre-cold 70% ethanol at 4 ℃, overnight. The next day, centrifuge and discard the supernatant, and wash the cells with PBS twice. Then add the propidium iodide (PI) staining solution (C1052, Beyotime Biotechnology, shanghai, China) and incubate cells at room temperature for 15 min. Then go for the flow cytometric analysis. For cell apoptosis, the DKC1 knockdown A549 and PC-9 cells and control cells were harvested for apoptosis markers (P21 and γH2A.X) analysis using western blot.
Western blot
The cells were harvested and lysed with RIPA lysis buffer (9806, Cell Signaling Technology, Boston, USA) containing protease inhibitors cocktail (B14002, Bimake, Houston, USA) and phosphatase inhibitor (B15001, Bimake, Houston, USA). After centrifugation, the protein concentration was measured using BCA protein assay kit (23,225, Thermo Fisher, Waltham, USA) and 30 μg lysates protein were subjected to 12% SDS-PAGE. The following antibodies were used for western blot: DKC1 (sc-373956, Santa Cruz Biotechnology, CA, USA), P21 (sc-6246, Santa Cruz Biotechnology, CA, USA), γH2A.X (2595, Cell Signaling Technology, Boston, USA), GAPDH (RM2002, Beijing Ray Antibody Biotech, China).
Quantitative PCR-based telomerase repeated amplification protocol
The Telomerase Repeated Amplification Protocol (TRAP) is one sensitive telomerase activity detection assay. The quantitative PCR-based TRAP assay (qTRAP) is a more convenient measurement methods [15]. DKC1 stable knockdown A549 and PC-9 cells or control cells were harvested and lysed on ice for 30 min using NETN buffer (40 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.5% NP40, 1 mM EDTA, 10% glycerol, 1 mM DTT, and protease inhibitors in nuclease-free water). 5000, 500 or 50 cell lysates were incubated in quantitative RT-PCR buffer (1708884AP, Bio-Rad, Hercules, USA) with TS primers and ACX primers at 30℃ for 30 min. After the primer extension step, then do PCR following the steps: 90 s at 94 °C; 40 cycles of 30 s at 94 °C and 60 °C each; 45 s at 72 °C. The products of qTRAP (5000 cells) were visualized on a 10% non-denaturing acrylamide gel.
Telomere length measurements
The mean telomere length was measured by a quantitative PCR-based technique [16] in DKC1 knockdown A549 and PC-9 cells. The mean telomere length was expressed as a ratio (T/S) of telomere repeat length (T) to the copy number of a single copy gene (S, β-Globin). The followings were the sequence of the PCR primers.
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T1: 5′-ACACTAAGGTTTGGGTTTGGGTTTG GGTTTGGGTTAGTGT-3'
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T2: 5′-TGTTAGGTATCC CTATCCCTATCCCTATCCCTATCCCTAACA-3'
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S1: 5′-CGGCGGCGGGCGGCGCGGGCTGGGCGGcttcatccacgttcaccttg-3'.
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S2: 5′-GCCCGGCCCGCCGCGCCCGTCCCGCCGgaggagaagtctgccgtt-3'.
Cytochemical detection of senescence-associated-β-galactosidase activity
DKC1 stable knockdown A549 and PC-9 cells or control cells (0.5 million) were plated in 6-well plate. Chromogenic β-gal substrate X-gal (C0602, Beyotime biotechnology, shanghai, China) were used to stain the fixed cells for 8 h. The cells were washed with phosphate-buffered saline (PBS) and imaged by bright field microscopy.
Subcutaneous xenograft
BABL/c female nude mice were obtained from Beijing Vital River Laboratory Animal Technology Company (Beijing) and randomly assigned into three groups (shCTL, shDKC1-1 and shDKC1-2). Each group contains 6 mice. 1.5 million DKC1 knockdown PC-9 cells or control cells were injected to 5–6 weeks-old mice subcutaneously. When the tumors were detectable, tumor length and width were measured every 3 days. Tumor volume was calculated by using the following formula: tumor volume = (length × width2)/2. After 30 days of injection, the tumor was removed and sent for immunohistochemistry analysis.
Immunohistochemistry (IHC) and survival analysis
The paraffin-embedded tissues were sectioned and after rehydration, antigen retrieval and blocking, the slides were incubated with the following antibodies: anti-DKC1 (sc-373956, Santa Cruz Biotechnology, CA, USA), anti-Ki67 (550,609, BD Biosciences, Franklin, USA) and anti-γH2A.X (2595, Cell Signaling Technology, Boston, USA), at 4 °C, overnight. Next day, after the incubation of HRP (horseradish peroxidase)-conjugated secondary antibody for 1 h at room temperature, the DAB (3, 3′-diaminobenzidine) staining was used for detecting HRP signaling (KIHC-5, Proteintech Group, Chicago, USA). Then counterstain slides with hematoxylin. The integral optical density (IOD) of each target was measured by Image-Pro Plus. The DKC1 expression in human lung adenocarcinoma tissues was interpreted independently by two pathologists. Two characteristics were used for scoring the expression of DKC1 in slices: overall stain intensity (with possible values ranging from 0 to 3) and a score representing the percentage of tumor cells that were stained (1, 0–25%; 2, 25–50%; 3, 50–75% and 4, > 75%). An IHC score was then calculated by multiplying the values of the two characteristics. Based on IHC score, patients were divided into two groups: high DKC1 expression (IHC score \(\geq\) 4) and low DKC1 expression (IHC score < 4) (Additional file 1: Table S1). Overall survival (OS) was estimated using the Kaplan–Meier method, and the log-rank test was used for statistical analysis.
Quantitative RT-PCR (qRT-PCR)
Total RNA was isolated using TRIzol reagent (15,596,026, Thermo Fisher Scientific, Waltham, USA) according to the introductions. The cDNA is synthesized using One-step gDNA Removal and cDNA Synthesis Kit (AE311, TransGen Biotech, Beijing, China). The qRT-PCR were performed using the SYBR Green SuperMix (1708884AP, Bio-Rad, Hercules, USA) and normalized by the expression level of GAPDH. The sequences of the primers for qRT-PCR were listed as follows.
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DKC1 F: 5′-ATGGCGGATGCGGAAGTAAT-3'
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DKC1 R: 5′-CCACTGAGACGTGTCCAACT-3'
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TERC F: 5′-ACCCTAACTGAGAAGGGCGTA-3'
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TERC R: 5′-AATGAACGGTGGAAGGCGG-3'
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GAPDH F: 5′-GAAGGTGAAGGTCGGAGTC-3'
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GAPDH R: 5′-GAAGATGGTGATGGGATTTC-3'
Statistics
GraphPad Prism was used to conduct the statistical analysis for most of the data unless indicated otherwise. Statistical significance was assessed by Student’s t-test or one-way ANOVA except when stated otherwise.