Ethics statement
This study was approved by the Ethics Committee of Harbin Medical University Cancer Hospital, following the Declaration of Helsinki. The informed consent was obtained from each eligible participant. The animals were treated in accordance with the standards of animal ethics.
Clinical samples
Twenty-seven CRC patients diagnosed in Harbin Medical University Cancer Hospital from August 2018 to August 2019 were recruited and the clinical characteristics of patients were shown in Supplementary Table 1. None of the patients received radiotherapy or preoperative chemotherapy. The tumor tissues and adjacent tissues were preserved in liquid nitrogen for subsequent analysis.
Cell culture and grouping
SW480 cells (CCL-228, American Type Culture Collection, Manassas, VA, USA), HCT-8 cells (CCL-244) and immortalized normal intestinal epithelial cells (NCM460 cells) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Solarbio Science & Technology Co., Ltd., Beijing, China) containing 10% fetal bovine serum (FBS) at 37 °C with 5% CO2. After the cells adhered to the wall, they were passaged and detached with 0.25% trypsin (Hyclone Company, Logan, UT, USA). The cells at logarithmic phase were used for the experiments.
125I seed irradiation: internal 125I seeds were obtained from HTA Co., Ltd. (Beijing, China). The 125I seeds at the doses of 0.4 mCi (14.53 MBq), 0.6 mCi (22.97 MBq) and 0.8 mCi (29.97 MBq) were used for experiments. The in vitro irradiation device was established by using 125I seed irradiation model. Human colon cancer cells (SW480 and HCT-8) were irradiated by different doses of 125I seeds for 72 h. The total radiation doses were 113 cGy, 162 cGy and 225 cGy, respectively. The control cell lines were not irradiated by 125I seeds in the same device. The in vitro irradiation model was established as described previously [22]. The specific operation was as follows: in the in vitro model, eight 125I seeds were evenly wound on the circumference of 30 mm in diameter, and one 125I seed was placed in the center to process the cells.
Cell grouping: mimic NC, miR-615 mimic, inhibitor NC and miR-615 inhibitor were purchased from GenePharma (Shanghai, China). The cell transfection was performed using the Lipofectamine 2000 (Invitrogen Inc., Carlsbad, CA, USA), with the final concentration of 100 nm. After transfection, the cells were collected to evaluate the transfection efficiency. Thereafter, the transfected cells were irradiated by corresponding dose of 125I seed for subsequent experiments.
Solvent treatment grouping: dimethyl sulphoxide (DMSO) group and 5-Aza group (DNA methyltransferase inhibitor; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). After 24 h of culture, the cells adhered to the wall and then were placed in the 5-Aza (final concentration was 2 μmol/L) or DMSO (final concentration was 2 μmol/L) culture fluids. The culture fluid was refreshed every 24 h [23].
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The cell viability was determined by measuring the ability of cells to convert thiazolyl blue tetrazolium bromide (CT02, Sigma-Aldrich) into blue violet crystalline methyl tetrazolium (formazan). Briefly, 20 μL MTT solution [5 mg/mL in phosphate-buffered saline (PBS)] was added into the 96-well plates and cultured with cells for 5 h. Then, the medium was replaced by DMSO (200 μL/well). The optical density (OD) value at 570 nm was measured by a microplate reader (VSERSA Max, Molecular Devices, CA, USA).
Colony formation assay
Briefly, 1.2% agar was heated and dissolved, and placed in 46 °C water bath for standby. The prepared CRC cells were counted and suspended in DMEM preheated at 40 °C. The 6-well plates were added with cell suspension (containing 1 × 103 cells) per well. Then, 125 μL preheated 1.2% agar was gently and rapidly mixed with the above cell suspension and sample in the 6-well plates, avoiding bubbles. After natural solidification, the mixture was put in the incubator at 37 °C with 5% CO2. After 8–10 days, the cell colonies were stained with 0.1% crystal violet (Sigma-Aldrich, Dorset, USA) and observed and counted under the inverted microscope.
Transwell assay
CRC cells were starved in serum-free medium for 24 h, then detached and washed with PBS twice. The cells were resuspended in serum-free Opti-MEMI (Invitrogen) containing 10 g/L bovine serum albumin (BSA; Sigma-Aldrich), and the cell density was adjusted to 3 × 104 cells/mL. This experiment used 8 μm 24-well Transwell chamber (Corning Glass Works, Corning, NY, USA). Before the experiment, each chamber was coated with 50 μL Matrigel (Sigma-Aldrich), with 3 chambers in each group and 100 μL cell suspension in each chamber. The basolateral chamber was added with 600 μL 10% RPMI1640 medium and incubated at 37 °C with 5% CO2. After 48 h, the cells were fixed with 4% paraformaldehyde for 30 min, treated with 0.2% Triton X-100 (Sigma-Aldrich) for 15 min and stained with 0.05% gentian violet for 5 min. The number of stained cells was counted under the inverted microscope (Leica DMi8-M, Germany). Five visual fields were randomly selected. The experiment was repeated for three times.
Flow cytometry
For apoptosis detection, 1 × 106 cells at logarithmic phase were collected and washed with cold PBS twice. The cells were suspended in 1 × Annexin buffer, added with 5 μL Annexin-VFITC (Becton Dickinson Bio-sciences) and placed in the dark at room temperature for 10 min. After mixing, the cells were placed in the dark at room temperature for 5 min and washed with cold PBS once. The cells were suspended in 300 μL 1 × Annexin. The apoptosis rate was detected by flow cytometry.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using TRIzol reagent (15,596,026, Invitrogen) and reverse transcribed into cDNA using Ncode ™ miRNA First-Strand cDNA Synthesis kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The synthesized cDNA was detected using Fast SYBR Green PCR kit (Applied Biosystems, Inc., Carlsbad, CA, USA). The primer sequences of miR-615 were synthesized by Sangon Biotech (Shanghai, China). Reverse primer was 5′-AGTTAAGAGTAGTGGGGAGATTAA-3′ and forward primer was 5′-AAATTTTTTTTCTTTATTTACCCC-3′.
Methylation-specific PCR (MSP)
The methylation level of miR-615 promoter region in HCT-8 cells or tumor tissues was detected using DNA Methylation-Gold™ kit (D5005, Zymo Research, Irvine, CA, USA). The primer sequences of methylation reaction for MSP amplification were miR-615-MD (5′-GGGCGGAGGCGTTTTTTTC-3′) and miR-615-MR (5′-CGACCGAAAAAAAAAAAAACGAAAACCG-3′). The primer sequences of unmethylation reaction were miR-615-UD (5′-AAAGTTTTTTGTTTGGGTGGAGGTGTTTTTTTTG-3′) and miR-615-UR (5′-ACCCACAACCAAAAAAAAAAAAAACAAAAACCA-3′). The primer sequences of miR-615 were synthesized by Sangon Biotech. The purified DNA was added into CT transformation reagent for denaturation and bisulfate transformation. The reaction column was used for desulfurization and purification. The purified DNA was used for subsequent PCR reaction. PCR products were subjected to agarose gel electrophoresis. Image analysis was performed by gel electrophoresis imaging and analysis system (Thermo Fisher Scientific).
Chromatin immunoprecipitation (ChIP) assay
HCT-8 cells were treated with 4% formaldehyde (Aladdin Biochemistry, Shanghai, China) (final formaldehyde concentration was 1%). The collected cells were broken by ultrasound and added with anti-Dnmt3b (ab2851, 1:50, Abcam Inc., Cambridge, MA, USA), anti-Dnmt1 (ab13537, 1:50, Abcam) and anti-Dnmt3a (ab2850, 1:50, Abcam) to bind the miR-615 gene promoter. Then, the cells were added with Protein A Agarose/Salmon Sperm DNA (Merck Millipore, Billerica, MA, USA) to bind to the promoter complex and precipitate the complex. The precipitated complex was cleaned to remove some nonspecific binding. After elution, the enriched miR-615 promoter complex was obtained and then crosslinked. The promoter fragment of enriched miR-615 was purified for qPCR.
Methylated DNA immunoprecipitation (meDIP)
meDIP was performed using MeDIP kit (MSK Biotechnology Co., Ltd., Wuhan, Hubei, China). Briefly, genomic DNA was extracted from HCT-8 cells and purified using standard procedures. Genomic DNA was cut by ultrasound to produce 200–1000 BP random fragments. The DNA fragment was denatured at 95 °C to obtain single stranded DNA fragment, which was then incubated with 5-mC antibody (ab214727, Abcam) to precipitate the DNA containing 5-mC. The 5-mC was captured by magnetic beads. The 5-mC antibody pull-down DNA was extracted and purified by phenol/chloroform for real-time fluorescent quantitative PCR.
Subcutaneous xenograft tumor in nude mice
Eighteen specific pathogen-free male BALB/c nude mice (5 weeks, 15–18 g) purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China) were randomly assigned into control group, 125I group, 125I + miR-615 antagomir group, with 6 mice in each group. HCT-8 cells transfected with miR-615 antagomir were prepared into cell suspension (1 × 107 cells/mL). The prepared cell suspension was injected into the left axillary skin of nude mice with a 1 mL syringe to establish the subcutaneous xenograft tumor model. When the tumor diameter of nude mice was about 1 cm, the 125I seeds were implanted into the tumor center. The mice in the 125I group were implanted with a 125I seed [0.8 mCi (29.97 MBq)] into the center of the tumor with a No. 18 implant needle. The mice in the 125I + miR-615 antagomir group were injected with miR-615 antagomir and implanted with 0.8 mCi 125I at a dose of 10 nmol/mouse. The mice were killed by cervical dislocation after 28 days of observation. The tumor tissues were dissected, photographed, weighed and measured. Some of the tumor tissues were used for DNA extraction and MSP.
Immunohistochemistry
The tissues were fixed with 10% formaldehyde (Aladdin biochemistry), embedded in paraffin and sliced (4 μm). The tissue sections were dried in an oven at 60 °C for 1 h, dewaxed with conventional xylene (Aladdin biochemistry), then dehydrated with gradient alcohol, incubated in 3% H2O2 at 37 °C (Sigma-Aldrich) for 30 min, washed with PBS, boiled in 0.01 M citric acid buffer at 95 °C for 20 min, cooled to room temperature, and washed with PBS. The sections were blocked with normal goat serum working solution (Biolab Technology Co., Ltd., Beijing, China) at 37 °C for 10 min, and added with rabbit anti-Ki67 (1:500, ab15580, Abcam) at 4 °C for 12 h. After PBS washing, the sections were cultured with biotin-labeled goat anti-rabbit secondary antibody for 10 min. After washing, the sections were cultured with horseradish peroxidase-labeled streptomyces ovalbumin working solution (S-A/HRP) for 10 min. The sections were developed with 2,4-diaminobutyric acid (DAB) and stored in the dark for 8 min. Afterwards, the sections were washed with tap water, stained with hematoxylin violet, dehydrated, cleared, sealed, and observed under the light microscope. The positive cells were counted by Nikon image analysis software (Tokyo, Japan). Three visual fields (×200) were selected from each section to calculate the number of positive cells.
TUNEL staining
The sections were dewaxed and treated with 50 μL 1% protease K (ST535, Beyotime Biotechnology Co., Ltd., Shanghai, China) diluent at 37 °C for 30 min. The sections were incubated with 0.3% H2O2 methanol solution at 37 °C for 30 min to eliminate endogenous peroxidase (POD) activity. Then the sections were incubated with TUNEL reaction solution (C1098, Beyotime) at 37 °C in the dark for 1 h and treated with 50 μL Converter-POD (C1098, Beyotime) at 37 °C for 30 min. Thereafter, the sections were developed with 2% DAB solution at room temperature for 15 min, followed by observation under the microscope. The reaction was terminated by adding distilled water after the brownish yellow nucleus appeared. The sections were counterstained with hematoxylin and the reaction was terminated by distilled water. The sections were dehydrated with 50, 70, 90 and 100% ethanol, cleared with xylene, sealed with neutral gum and observed under the microscope. Ten visual fields were randomly selected from each section. The cells with brown nuclei were apoptotic positive cells, and the blue nuclei were normal cells.
Bioinformatics analysis
The enrichment of the KEGG pathway was analyzed using the KOBAS3.0 database (http://kobas.cbi.pku.edu.cn/kobas3/help/). The differential expressions of candidate target genes in colon and rectal cancer included in TCGA and GTEx were searched. The target genes of miR-615 were predicted using the TargetScan database (http://www.targetscan.org/vert_71/) and StarBase database.
Statistical analysis
Data analysis was analyzed and introduced using SPSS 21.0 (IBM Corp., Armonk, NY, USA). Data are expressed as mean ± standard deviation. The t test was adopted for comparison between two groups. One-way analysis of variance (ANOVA) was employed for the comparisons among multiple groups, followed by Tukey’s multiple comparisons test. The p value was obtained from a two-tailed test, and the p < 0.05 meant a statistically significance.