2.1 Tissue Specimens
The tumor tissues and normal paracancerous tissues of 51 male patients who underwent radical resection of CRC at Linyi Central Hospital from April 2017 to April 2018 were randomly collected. None of patients received chemotherapy or radiotherapy before surgery. The control group specimens were resected from normal paracancerous tissues of the same patient (at least 2 cm from the surgical margin), and no cancer cells were found during pathological examination. The collection and use of patient tissue samples were approved by the Linyi Central Hospital Ethics Committee. All patients involved gave informed consent to the study and signed a written consent form. Tissue samples were surgically removed and immediately stored at -196 °C in liquid nitrogen until use.
2.2 Cell culture
Human CRC cell line (HCT-116, HT29, SW480, SW620, DLD-1 cells) and normal colonic epithelial cell line CCD841 were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 100 U/mL penicillin/streptomycin (Life Technologies, USA) in a constant temperature incubator at 37 °C in 5% CO2. The solution was changed every 3 d, and the cells were passaged when the bottom of the culture flask was covered with the cells. Cells in the logarithmic growth were selected for assays.
2.3 Cell transfection
The cells in the logarithmic growth phase were inoculated into a 6-well plate with 2.5 × 105 cells per well and the cells were transfected according to the instructions of the Lipofectamine® 3000 (Invitrogen; ThermoFisherScientific, Inc., USA). TTTY15 shRNA, TTTY15 overexpression plasmid, miR-29a-3p mimics, miR-29a-3p inhibitor, empty vectors, microRNA mimics control were transfected into cells respectively. After 48 h of culture, RNA was extracted to verify the transfection efficiency.
2.4 RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted and purified from tissues and cell lines according to the instructions of TRIzol reagent (Invitrogen, Carlsbad, USA). RNA purity and concentration were measured using the NanoDrop ND-2000 spectrophotometer (NanoDrop Wilmington DE). M-MLV Reverse Transcriptase (Thermo Fisher Scientific, Inc., Rockford, IL, USA) containing 5 μg of total RNA was used to reverse transcribed to cDNA. qRT-PCR was performed on the ABI 7300 machine (Applied Biosystems, Foster City, CA, USA) with SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.). The qRT-PCR conditions were as follows: pre-denaturation at 95 °C for 10 min, 95 °C for 15 s, 60 °C for 15 s, 45 cycles, and the fluorescence signal temperature was obtained at 60 ° C. The relative expression of TTTY15, miR-29a-3p and DVL3 was calculated by the 2-ΔΔCt method using GAPDH and U6 as internal reference. Primers were listed in Table 1.
2.5 Cell counting kit-8 (CCK-8) assay
The cell proliferation curve was determined using CCK-8 assay. HCT-116 and SW480 cells (3000 cells per well) were seeded in 96-well plates and incubated for 1, 2, 3, and 4 d, respectively. Subsequently, 10 μL of enhanced Cell Counting Kit-8 (Beyotime, Beijing, China) was added and the cells were incubated at 37 °C for 1 h. Then then culture was terminated and the absorbance value (OD value) of each well was measured at 450 nm on a microplate reader, and the relative OD ratio was used to indicate the cell proliferation.
2.6 Plate colony formation assay
Cells in the logarithmic growth phase were trypsinized with 0.25% trypsin and dispersed into single cells and counted. The cell concentration was adjusted to 1000 cells per dish, and the cell suspension was inoculated separately into a dish containing 3 mL of 37 °C pre-warmed culture solution and gently rotated to evenly disperse the cells. Subsequently, the cells were cultured in an environment of 37 °C, 5% CO2 and saturated humidity for 2 to 3 weeks. The number of cells contained in each cell clone was counted, and the cell colony formation rate was calculated and photographed. Clonal formation rate = number of clones/number of cells inoculated * 100%.
2.7 Tranwell assay
Transwell assay was used to detect the migration and invasion. Matrigel was used in invasion assay, but not in migration assay. The cells in each group transfected for 48 h were taken, trypsinized, resuspended with serum-free medium, counted and seeded into the upper chamber, which was not used in the migration assay. RPMI-1640 medium containing 10% FBS was added to the lower chamber. The cells were placed in a 37 °C, 5% CO2 incubator and cultured for 24 h. After that, the cells that failed to migrate or invade were removed from the upper chamber. After being fixed for 4 minutes with 4% paraformaldehyde, the migrated or invaded cells were stained with 0.5% crystal violet. After being rinsed off by the tap water, the cells were observed under an inverted microscope, and cell counts were performed using five fields of view, and the mean value was taken.
2.8 Flow cytometry
Cellular apoptosis was detected and analyzed by flow cytometry. Cells in the logarithmic growth phase were washed twice with PBS, fixed in 70% ethanol, and stored at 4 °C overnight. Then the cells were washed once with PBS, and the cell density was adjusted to 1 × 106 cells/mL. The cells were then double-stained with AnnexinV-FITC and Propidium iodide and incubated for 15 minutes in the dark at room temperature. Finally, the stained cells were analyzed using flow cytometry (BD Biosciences).
2.9 Dual-luciferase reporter gene assay
Luciferase reporter gene assay was performed using the dual-luciferase reporter assay system (Promega, Madison, WI, USA). The target fragment of wild type TTTY15 and mutant TTTY15 was constructed and integrated into pGL3 vector (Promega, Madison, WI, USA) to construct pGL3-TTTY15-wild type (TTTY15-wt and pGL3-TTTY15-mutant (TTTY15-mut) reporter vector. Then TTTY15-wt or TTTY15-mut was co-transfected with miR-29a-3p mimics or control microRNA mimics. After 48 h of transfection, luciferase activity was determined in accordance with the manufacturer's instructions. All experiments were performed in triplicate and repeated three times. The same method was used to construct DVL3-wt and DVL3-mut, and to detect the targeted binding relationship between miR-29a-3p and the 3’UTR of DVL3.
2.10 Western Blot
The RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) containing PMSF was used to extract the protein sample. Protein samples were added for SDS-PAGE and transferred to the nitrocellulose membrane. After being blocked with 5% fat-free milk, the membrane was incubated with primary antibody anti-DVL3 (dilution 1:1000, Cell Signaling Technology) and anti-GAPDH (dilution 1:2000, Santa Cruz) antibody at room temperature for 8 h. After being washed, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody (1:2000, Santa Cruz Biotechnology) for 1 h at room temperature. Ultimately, The membrane was placed on an automatic developing device (ChemiDocXRS imaging system) to calculate the gray value.
2.11 Statistical analysis
Data analysis was performed using SPSS18.0 statistical software. All data were expressed as mean ± SD. Statistical analysis was performed by student’s t-test. Pearson assay was employed to analyze the correlation among TTTY15, miR-29a-3p and DVL3 expression. P < 0.05 was statistically significant.