Patients and cell lines
The research cohort recruited at Xi'an First Hospital from September 2018 to mid-September 2019. These were colorectal cancer patients who were treated not received radiotherapy and chemotherapy before the removal of tissue samples. Before the operation, the patients signed and agreed to participate in the study by signing informed consent forms distributed by the ethics committee of this hospital. After tissue samples were removed, some portions were stored in liquid nitrogen, and some were embedded in wax blocks for later use. The colorectal cancer cell lines and colorectal normal mucosal tissue cell lines selected in this experimental group were purchased from Shanghai Cell Bank.
Transfection
APC2 interfering RNA was purchased from Shanghai Shenggong. We used control and interfering RNA to silence the APC2 gene in the HT29 cell line, and then used it for protein and cell cycle experiments. Subsequently we referred to the lines as Sil-control and Sil-APC2. APC2 plasmids and its control group were purchased from Origen, and we later used them to up-regulate APC2 gene expression in HCT8 and SW480 cell lines. PCMV6 and PC-APC2 were used to refer to the up-regulated HCT8 cell lines. PCMV6-SW480 and PC-APC2-SW480 refer to the up-regulated SW480 cell lines. The transfection reagent lipo3000 was purchased from Thermo Scientific.
Real Time PCR
TRIzol reagent (Takara Bio, Shiga, Japan) was used to isolate total RNA from frozen cancer tissues. To do this, we added 1 ml of TRIzol reagent and 300 µl of chloroform to the tissue, mixed, and then centrifuged at 10,000 rpm for 15 minutes. Next, an equal volume of isopropanol was added to the supernatant, and then the sample was centrifuged again at 10,000 rpm for 15 minutes. The supernatant was discarded and the precipitate was washed with 1 ml of 75% ethanol. Next, we followed the manufacturer's instructions (Takara) to reverse transcribe the extracted RNA using the kit. Then, we designed the amplification primers for the messenger APC2 based on the specific sequence (Shanghai, China). The expression of mature APC2 was measured using the thermal cycler Dice real-time system II under the following thermal cycling conditions: 95 °C for 30 s, 45 cycles, 95 °C for 5 s, 60 °C for 60 s, and then conducting melting curve analysis. The relative expression of APC2 was normalized to the expression of glyceraldehyde 3-phosphate dehydrogenase mRNA, and the fold-change in expression was calculated using the 2ΔΔCt method. Compared with adjacent non-tumor tissues, we defined negative values as relatively low expression levels of APC2, and positive values as relatively high expression levels of CRC of APC2.
Immunohistochemistry
From the resected tumors of patients with colorectal cancer, thin paraffin sections were prepared, baked in a 70-degree oven for 2 hours, and then dewaxed (xylene-pure ethanol-75% ethanol for 30 minutes each). According to the instructions of Maixin reagent immunohistochemistry, we added solution A (catalase) dropwise to eliminate non-atopic staining (30 minutes), rinsed with PBS three times and in incubated solution B (Ultra V Block) for about 30 minutes, then added primary antibody, and stored at 4 °C overnight. On the second day, we rinsed with PBS solution 3 times, incubated with solution C (Primary Antibody Enhancer) for about 15 minutes, rinsed with PBS solution 3 times, and incubated with solution D (enzyme labeled secondary antibody) for about 15 minutes. DAB chromogenic solution was stained for 5 minutes, then concentrated hydrochloric acid was added for 6 seconds, hematoxylin was stained for 5 minutes, and alkaline water turned blue for 3 minutes. After the final dehydration (75% ethanol-pure ethanol-xylene for 30 minutes each), the seals are retained.
Scoring: We observed the stained sections under a microscope. After selecting the field of view, according to the staining intensity, very strong = 3 points, strong = 2 points, weak = 1 point, and none = 0 points. Evaluation of the range of dyeing: 100–75%: 4 points, 74–50%: 3 points, 49–25%: 2 points, 24–0%: 1 point. Those with a total score equal to or greater than 6 points were judged to have strong expression.
Cell cycle
After 48 hours, the cells transfected with iRNA or plasmids were trypsinized into centrifuge tubes, rinsed 3 times with PBS solution, and transferred to the cell cycle staining solution (PI), protected from light at 37 degrees for 30 minutes, and then placed in a bleed cytometer for detection, and the results obtained are analyzed and detected by software. Cell cycle reagent stains were purchased from Thermo Fisher Scientific.
MTT
In the MTT experiment, after 24 hours of transfection, the cell line was eluted, and it was seeded into 96-well plates at 2500 cells per well, cultured for 5 days, and medium was changed daily (5 plates repeat one of the plates). After cleaning the medium, we added DMSO to the microplate reader for detection. The experiment was performed three times, and the data analysis is presented below.
Western blot and co-immunoprecipitation
After culturing cells for 48 hours in PBS, we washed them three times and incubated them in RIPA buffer for 30 minutes on ice. After centrifugation, we removed the supernatants and tested on a microplate reader to quantify with standard protein. To the same concentration for use, after configuring the electrophoresis gel, we dropped the sample into wells of the gel plate in the same volume (30 µl) one-by-one and started the electrophoresis process to the loading mark to the bottom of the gel plate, then cut the gel according to MARKER and transferred onto PVDF membranes. Membranes were then washed 3 times with TBST solution (Thermo Fisher Scientific), and primary antibody was incubated overnight at 4 degrees. After washing 3 times with TBST solution, the rabbit secondary antibody was incubated and incubated at room temperature for 2 hours. After washing thoroughly, we used luminescent solution (Thermo Fisher Scientific) in an ECL luminometer (Bio-Rad) for detection.
Statistical analysis
Patient tumor-related information and immunohistochemical scores were evaluated using χ2 tests, and expression levels of related mRNA and protein were measured using the independent sample T-test after obtaining quantitative data. with p values less than 0.05 were considered significant. All data analyses were performed using SPSS 17.0.