Tissue sample collection
The present work gained approval from the Medical Ethics Committee of Yijishan Hospital of Wannan Medical College (Wuhu, China). Each case provided the informed consent for participation. A total of 108 paraffin sections were collected from the Pathology Department, Yijishan Hospital Affiliated to Wannan Medical College, and 22 CRC tissue samples with matched adjacent non-carcinoma tissue samples (around 5 cm apart from cancer edge) were acquired and frozen within liquid nitrogen at once.
Cell culture and transfection
Various types of CRC cells (SW480, HCT116, SW620, HT-29) were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China); meanwhile, the colon epithelial cells(NCM460) were obtained from the Affiliated Comprehensive Cancer Center of Drum-Tower Hospital of Medical School of Nanjing University. The CRC cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) within the incubator under 5% CO2 and 37 °C conditions at the Roswell Park Memorial Institute. Then, the Lipofectamine 3000 Kit (Invitrogen, Carlsbad, CA, USA) was used to transfect cells in accordance with manufacturer instructions. The small interfering RNA (siRNA) cyclin-dependent kinase 1 (CDK1), together with the corresponding negative control (NC), was prepared by Guangzhou Ribo Biotechnology Co. Ltd (Guangzhou, China). In addition, the CDK1 overexpression plasmid as well as the empty plasmid was provided by GenePharma Co. Ltd (Shanghai, China), whereas the miR-378a-5p inhibitor, miR-378a-5p mimic, together with corresponding NCs were provided by Guangzhou Ribo Biotechnology Co. Ltd. (Guangzhou, China).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
The Trizol reagent (Invitrogen, CA, USA) was used for extracting the total tissue and cellular RNA in accordance with specific protocols. Total RNA was reversely transcribed to complementary DNA following the protocols of the RT Kit (Takara, Dalian, China). Real-time fluorescence PCR was carried out by the qPCR Kit (Takara, Dalian, China).
Cell counting kit-8 (CCK-8) assay
Cell viability was assayed using Cell Proliferation Kit (Keygen Biotechnology Co. Ltd. (Nanjing, China). CRC cells were detached and inoculated into the 96-well plates at 2 × 104 cells/well, and the adhering cells after 12-h cell culture were transfected. At 12, 24, 36, 48, and 60 h of transfection, every group was added with 10 mL reagent to detect cell proliferation, then cells were incubated for another 2 h, and the absorbance (OD) was detected at 450 nm.
5-Ethynyl-2’-deoxyuridine (EdU) assay
CRC cells (1 × 105/ well) at logarithmic stage were seeded into the 96-well plates to culture until the normal growth stage. Thereafter, cells were labeled with EdU, stained with Apollo and DNA as per the manufacturer’s instructions (Guangzhou Ribo Biotechnology Co. Ltd., Guangzhou, China); at last, the confocal laser scanning microscope was utilized to observe cells.
Colony formation assay
CRC cells (200 cells/well) after transfection were grown into the 6-well plates and further incubated for 2 weeks under 37 °C. At last, 4% paraformaldehyde was used to fix cells, while 0.1% (w/v) crystal violet was used to stain cells. The Imag-Pro Plus 5.0 software was employed for counting cell colonies.
Wound healing assay
The single straight wound was made using a sterilized 10 µL pipette tip at the bottom of 6-well plates full of transfected CRC cells with cell debris removed by washes with phosphate buffer saline (PBS). Then, every well was added with RPMI-1640 containing 2% FBS, followed by further cell culture under 5% CO2 and 37 °C conditions. The width of the scratch was measured with ImageJ software and an inverted optical microscope. The scratch width measured initially was assigned to be the original scratch width, and the scratch width measured again after 48 h was used as final scratch width. Relative scratch width was calculated by the ratio of final scratch width to the original one.
Transwell assay
At 24 h after transfection of CRC cells, 100 µL of the cells diluted to 1 × 105 with serum-free RPMI-1640 medium was plated at apical side of a Transwell chamber, whereas 600 µL of 20% RPMI-1640 medium was put at basolateral side. After 24 h of cell incubation under 37 °C and 5% CO2 conditions, the 4% paraformaldehyde was used to fix the chamber, while 0.1% crystal violet was used for staining. Thereafter, a cotton swab was used to remove upper chamber cells. Finally, migrating cells were photographed and counted with an inverted optical microscope.
Immunohistochemistry (IHC) staining
CDK1 expression was determined with the CDK1 antibody (1: 100, Abcam, USA). Following deparaffinization/rehydration, antigen retrieval, and staining, each tissue section was rated using the light microscope based on the staining level (0, 1, 2, 3 suggested negative, yellowish, light brown, dark brown staining, separately) and the extent of positivity (1, 2, 3, and 4 represented 0–25%, 26–50%, 51–75%, and 76–100%, respectively). Finally, the scores can be added for comparison. All IHC sections were evaluated independently by two expert pathologists.
Dual-luciferase reporter gene assay
Luciferase reporter gene assay was performed to assess the direct binding of miR-378a-5p with CDK1. The CDK1 3’-untranslated region (UTR) that may bind to miR-378a-5p was mutated from GTCAGGAA to CAGTCCTT, and a mutant fragment and an unmutated fragment of the CDK1 3’-UTR were transfected to pmirGLO reporter plasmid, respectively. 293T cells were then inoculated in the 24-well plates, followed by co-transfection with equivalent unmutated or mutated pmirGLO and miR-378a-5p mimic or NC-mimic. Renilla luciferase was adopted to be endogenous control. After 24 h of cell culture, the Dual-luciferase Reporter Gene Assay Kit (Shanghai Beyotime Biotechnology Co. Ltd., Shanghai, China) was applied in detection.
Cell apoptosis detection
Flow Cytometric Kit (BD Biosciences, CA, USA) was utilized to measure cell apoptosis. In brief, cells after transfection were inoculated into the 12-well plates to further culture for another 12 h. Thereafter, cells were cultivated with serum-free medium for another 24 h for apoptosis induction; later, 0.25% trypsin was used to detach CRC cells, followed by 5–10 min centrifugation at 2000 rpm under ambient temperature. Thereafter, cells were harvested to suspend in the pre-chilled 1 × PBS (4 °C), followed by 5–10 min of centrifugation at 2000 rpm. Then, cells were washed and resuspended into 300 µL 1 × binding buffer, sufficiently blended using 5 µL Annexin-V-fluorescein isothiocyanate (FITC), followed by 15 min of incubation in dark under room temperature. Afterward, the cells were subjected to 5 min of staining using 5 µL propidium iodide (PI) solution before placing on a flow cytometer, and 200 µL 1 × binding buffer added. Finally, flow cytometry (BD Biosciences, CA, USA) was adopted to detect cell apoptosis.
Cell cycle detection
The cell cycle was determined using Flow Cytometry kit (Keygen Biotechnology Co., Ltd., Nanjing, China) according to specific protocols. In brief, after 24 h transfection, cells were rinsed by PBS, followed by 5 min of centrifugation at 2000 rpm. Then, cells were collected and the cell density was regulated at 1 × 106/mL. Afterwards, 1 mL single-cell suspension was collected for centrifugation, and the supernatant was fixed with 500 µL of the 70% pre-chilled ethanol overnight at 4 °C. After PBS washes, 500 µL of PI/RNaseA staining solution was added to further incubate in dark for 1 h, and cell cycle was directly examined by flow cytometry (BD Biosciences, CA, USA).
Tumor xenograft mouse models
4 weeks old male nude mice reared in SPF animal room(Spf Laboratory Animal Room of Wannan Medical College) were injected with 2*10^6 HT-29 cells in the left armpit. After observing the size of the tumor every day for 15 days, the nude mice were killed by cervical dislocation, and then the tumor was taken out and recorded.
Western blotting analysis
The radioimmunoprecipitation (RIPA) lysis buffer (Thermo Fisher Scientific, MA, USA) was utilized for extracting total cellular protein in CRC cells transfected for 48 h. Bicinchoninic Acid (BCA) Protein Detection Kit (Thermo Fisher Scientific, MA, USA) was adopted for measuring the protein content. 10% SDS-PAGE (Bio-Rad Laboratories, Hercules, CA, USA) was adopted to separate proteins, and CDK1 (Abcam, USA) was determined later, with β-actin being the internal reference (Cell Signaling Technologies, Beverly, MA, USA).
Statistical methods
Prism (GraphPad Prism 8) was utilized for statistical analysis. The results were expressed as mean ± SD and examined by t-test. A difference of p < 0.05 was deemed to be statistically significant, * p < 0.05, ** p < 0.01, and *** p < 0.001.