The human bronchial epithelial cell line HBE and lung cancer cell lines A549, SPC-A-1, LTEP-a-2, NCI-H520, NCI-H460, NCI-H1975, NCI-H358 and 95D were purchased from the cell bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and the American Type Culture Collection (ATCC, USA). HBE cells were cultured in DMEM medium (high-glucose) containing 10% fetal bovine serum (Gibco, USA) at 5% CO2 and 37℃, and other cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum.
Cell malignant transformation model
The malignant transformation model of HBE cells was established referring to our previous study . Briefly, the HBE cells were divided and assigned to either the 3-MCA-treated (purity ≥ 97.5%, as determined by high-performance liquid chromatography; Sigma, USA) group or the dimethylsulfoxide (DMSO) -treated group. According to the literature [17, 18], 1 and 10 µM of 3-MCA were determined to be used for the experiment. However, the results of 1 µM 3-MCA group were negative. Finally, 10 µM of 3-MCA was selected for continuous treatment of HBE cells for 45 generations. Cell samples from the 5th, 15th, 25th, 35th, and 45th passages were collected and named P5, P15, P25, P35, and P45, respectively. The malignancy of 3-MCA treated HBE cells were determined by the CCK-8 assay, soft agar assay and in vivo tumorigenicity assay to ensure successful establishment of the malignant transformation model.
Animal model and tissue samples
Chemical-induced rat lung cancer model was established as described in our previous study . All procedures were approved by the Animal Care Committee of Third Military Medical University and carried out instrict compliance with the relevant guidelines. Briefly, Wistar rats aged 6 weeks and weighing 180–220 g used in our experiments were randomly divided into two groups: experimental group and control group. Rats were treated with a single dose of 10 mg 3-MCA in iodized oil by left intra-bronchial instillation as the experimental group. Control rats were kept with iodized oil in the same conditions. Rats were sacrificed within 75 days after instillation. The left lungs were cut into two pieces, one of which was kept frozen for DNA, mRNA and protein extraction, and the other was fixed in 4% formaldehyde, embedded in paraffin, and routinely histopathologically evaluated as normal, precancerous, tumor according to diagnostic criteria described previously .
Human tissue samples
The lung tumor tissues and adjacent normal tissues of 75 patients with lung cancer randomly selected from the participants enrolled in our previous study . All participants signed informed consent and all procedures and implements were approved by the Ethics Committee of Third Military Medical University. Briefly, the tumor tissues and adjacent normal tissues were collected from the surgically removed tissues of patients with lung cancer who were operated in the First Affiliated Hospital of the Army Medical University (the Third Military Medical University). All the collected tumor tissues were verified by pathology. The lung tissues 5 cm away from the edge of tumor resection were taken as the adjacent normal control group. All the collected tissue samples were collected after the patients were informed in advance. The tissue samples were immediately frozen in liquid nitrogen and stored at − 80 ℃.
RNA extraction, real-time quantitative reverse-transcription polymerase chain reaction (qRT–PCR) and RT-PCR
Cells or tissue samples were collected and the total RNA was extracted by using TRIzol reagent. According to the manufacturer's instructions, reverse transcription of RNA (2–4 µg) was performed by the GoScript™ reverse transcription system (Promega, USA). PCR amplification of cDNA templates was performed using a Fluorometric Assay Kit (Promega, USA). Real-time quantitative RT-PCR and RT-PCR analyses were performed as previously described . The primer sequences of qRT-PCR and RT-PCR were shown in Supplementary Table S1 and Table S2. The 2−ΔΔCt method was used to analyze the results of qRT-PCR. All analyses were performed at least three times in triplicate.
Protein extraction and Western blot
A proper amount of cells or tissue samples were added to the RIPA protein lysate for lysis, and the protein was extracted after centrifugation at low temperature. Protein concentration was measured according to the instructions of BCA protein concentration test kit. After the preparation of SDS-PAGE gel, about 80 µg for each sample were taken to be electrophoretic and transferred to the PVDF membrane. The PVDF membrane was blocked with 5% skimmed milk powder and then incubated with primary antibodies at 4 ℃ overnight. The antibodies used in this study were anti-TET1 (1:1000; Sant Cruz, USA), anti-XRCC1 (1:800; Boster, China), anti-OGG1 (1:1000; Boster, China), anti-APEX1 (1:1000; Boster, China), anti-beta-actin (1:1000; Beyotime, China). The membrane was washed with TBST buffer for 3 times, then incubated secondary antibody at room temperature, and finally detected by ECL chemiluminescent substrate (Thermo, USA).
Immunohistochemistry was performed on serial sections and used to detect the expression of TET1 protein expression using protocols previously described . Thick sections with 4-µm thickness were incubated with anti-TET1 antibody. The immunohistochemical analyses were evaluated by two individuals who were blinded to the sample information. Nucleoplasm immunostaining was considered evidence of expression. Expression of the proteins was scored according to the staining intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and the proportion of positive cells (0, negative; 1, positive in < 25%; 2, positive in 25–49%; 3, positive in 50–74%; 4, positive in ≥ 75% of cells). If the multiplication of the two scores were more than 6, the expression of TET1 was considered to be high expression.
The P45 3-MCA treated HBE cells were inoculated into six-well plates at the concentration of 4 × 105 cells per well. Demethylation agent 5-aza-2’-deoxycytidine (5-aza-dC) (Sigma) with the concentration of 10 µM was used to continuously treat the cells in the six-well plates for 3 days. The cells were then collected and used to extract the DNA, RNA and protein. MeDIP-qPCR was used to detect the methylation level of TET1. QRT-PCR and Western blot were used to detect the mRNA and protein expression of TET1. The analyses were repeated at least three times.
Cell transfection experiment
For overexpression, the cDNA of the human TET1 gene was cloned into the mammalian expression vector pCMV6-AC-GFP. For gene knockdown, TET1 shRNAs and a negative control shRNA were designed and synthesized, and then subcloned into shRNA-GFP vector. Cell transfection was performed as previously described . Briefly, cells were seeded in plats and cultured for 24 hours. Then, the mixed system of ViaFect™ transfection reagent (Promega, USA), the target plasmid, Opti-MEM medium and complete medium were prepared and replaced the medium in the culture dishes. After 48 h of transfection, the fluorescence of the cells was observed with an inverted fluorescence microscope. Cells were obtained for the extraction of RNA and protein. The stably cells were screened by G418. Stable cells with TET1 overexpression or knockdown were confirmed by RT-PCR and Western blot.
Cell proliferation assay
Cells were evenly seeded in 96-well plates and transfected after culture for 24 hours. The mixed system of CCK-8 solution and RPMI-1640 complete medium were prepared following the instructions of the CCK-8 kit (Dojindo, Japan). Then, the proliferation of cells was detected respectively, after transfected for 24 h, 48 h, 72 h and 96 h (named as D1, D2, D3 and D4). Finally, OD value was quantified by measuring the absorbance at 450 nm. All assays were performed at least three times in triplicate.
Clone formation experiment
Approximately 1000 cells transfected for 48 hours were seeded in six-well plates, and appropriate concentration of G418 was used to treat the cells for 14–21 days. When clones with a stable plasmid integrated into genomic DNA were formatted, 4% paraformaldehyde was used to fix the cells and crystal violet staining solution (Sigma) was used to stain the clones. Clones contained more than 50 cells were considered to be positive clones. All assays were performed at least three times in triplicate.
Soft agar assay
Soft agar assay was performed as previously described . Briefly, the lower layer agar and the upper layer agar were prepared in a 24-well plate. Cells were mixed with the upper layer agar, and incubate in a 37 °C cell incubator for 2–3 weeks. Clones that contained more than 50 cells were considered as positive clones and counted by phase-contrast microscopy. All assays were performed at least triplicate.
Scratch healing experiment
Cells were seeded in six-well plates, and transfected after cultured for 24 h. Toothpicks were used to scratch in the six-well plates after transfected for 24 h. After scratching for 0 h and 48 h, the same point of the scratched cells were took photos to observe the migration. Finally, the distance of cell migration was analyzed statistically. All assays were carried out at least three times in triplicate.
Transwell assay was performed as previously described . Briefly, approximately 2 × 104 cells that were transfected for 24 hours were seeded in the Transwell chamber (Corning, USA). Transwell chamber with or without Matrigel gel was used for the detection of cell invasion and cell migration, respectively. After culturing for another 24 h, the cells were fixed by 4% paraformaldehyde at room temperature and stained by crystal violet staining solution for 30 min. The inverted microscope was used to take photos of the stained cells. Finally, five different fields were randomly selected for analysis. All assays were performed at least three times in triplicate.
Nude mouse tumorigenesis experiment
All experimental procedures were approved by the Animal Ethics Committee of the Third Military Medical University. Nude mice with six-week-old were randomly divided into the experimental group and the control group with four animals in each group. Lung cancer cells with and without TET1 gene overexpression were harvested, and single-cell suspensions of 1 × 107 in 200 µL of phosphate-buffered saline were injected into the right abdominal wall of the nude mice and assigned to the experimental group and the control group, respectively. From the first day of injection, the major diameter (D) and the minor diameter (d) of tumor volume were measured every 5 days with a vernier caliper. The formula (D × d2) /2 was used to calculate the volume of the tumor. All mice were killed after one month. The tumors were surgically dissected, and were then embedded in paraffin for HE staining and immunohistochemistry analysis.
Methylated DNA immunopreciption (MeDIP) and hydroxymethylated DNA immunopreciption (hMeDIP)
According to the instructions of the EpiQuik™ methylated DNA immunoprecipitation kit (EpiGentek, USA) and EpiQuik™ hydroxymethylated DNA immunoprecipitation kit (EpiGentek, USA), MeDIP and hMeDIP were performed to detect the level of methylation and hydroxymethylation in the promoter of genes. Briefly, 1 µg of sonicated DNA was diluted and incubated with 150 µl of antibody buffer at room temperature for 90 minutes in strip wells. For MeDIP, normal mouse IgG as the negative control and anti-5-mC for samples were added in the antibody buffer. For hMeDIP, non-Immune IgG was added to the negative control well, anti-5-hmC antibody was added to the sample wells and the positive control wells. Next, the wells were washed once with TE buffer. DNA release buffer containing proteinase K was added to the samples followed by reverse buffer. At last, Filter column was used to elute purified DNA. DNA was analyzed by qRT-PCR, and primer sequences were provided in Supplementary Table S3. All analyses were performed at least triplicate and repeated three times.
Statistical analysis was performed using SPSS 19.0 software (SPSS, Inc., Chicago, IL). Measurement data were expressed as the mean value ± standard deviation (SD) and the significances among groups were analyzed using Student’s t test (normal distribution) or F test. P values less than 0.05 were considered statistically significant.