Patients and tumor specimens
Eighty specimens from the Department of Gastroenterology and General Surgery of Beijing Tiantan Hospital affiliated with Capital Medical University from June 2013 to March 2017 were collected and confirmed by pathology as colorectal adenocarcinoma. There were 49 males and 31 females; aged 24–73 years, mean age (54.88 ± 10.32) years old, 35 cases less than 65 years old, 45 cases older than 65 years; 26 cases of rectal adenocarcinoma, 54 cases of colon adenocarcinoma; 51 cases with diameter ≤ 5 cm, 29 cases with diameter > 5 cm; 55 cases with infiltration into serosal layer, 25 cases without infiltration into the serosal layer; 25 cases with highly differentiated adenocarcinoma, 22 cases with moderately differentiated adenocarcinoma, 33 cases with low differentiated adenocarcinoma; 28 cases without lymph node metastasis, 52 cases with lymph node metastasis; 9 cases with distant metastasis, 71 cases with no distant metastasis; Dukes staged 32 cases in A + B stage and 48 cases in C + D stage. All cases occurred with no other tumors, no tumor bleeding, intestinal perforation, intestinal obstruction, acute or chronic infection. In the control group, 20 normal tissues and 40 benign colon adenomas were selected. The appropriate paraffin tissue specimens were selected and serially sectioned 4 μm thick for immunohistochemical staining, screened by two pathologists, and the final results were confirmed.
Immunohistochemistry staining
Wnt7a immunohistochemical analysis was performed on 80 CRC specimens. Firstly, the paraffin-embedded and formalin-fixed tissues were cut into 4-mm sections and dried at 70°C for 2 hours. After dewaxing and hydration, the antigen was rinsed with phosphoric acid buffer saline (PBS) and soaked in 3% hydrogen peroxide for 10 min. The antigen was extracted from the citrate buffer (pH 6.0). Finally, after incubation with secondary antibodies for 30 min, staining with 3, 3-diaminobenzidine (DAB) for 10 min, slight back-dyeing with 10% Mayer’s hematoxylin, dehydration, and pilling.
Immunohistochemical evaluation
The staining was assessed by a semi-quantitative analysis and a protein level score which is equal to the positive cell proportion score added to the cell staining score. A well-stained area was selected and observed in 10 high-power fields continuously, and more than 50 cells per field were observed. The procedure outlined in Wang et al. [16] was then followed to calculate the proportion of stained cells. If the proportion of positive cells < 10%, the score is 0; if the proportion of positive cells is between 10–40% the score is 1; if the proportion of positive cells is between 40–70% the score is 2; if the proportion of positive cells is ≥ 70% the score is 3. The scores designate: no coloration (0 points); yellow staining (1 point); brownish yellow staining (2 points); yellowish brown staining (3 points). Final protein level scores are as follows: 0 is negative, 1–3 is +, and 4–6 is ++. Two pathologists analyzed the stained tissue sections and they were blinded to the patient's clinical parameters.
Cell culture
Colorectal cancer cell lines HT-29 and HCT-116 were cultured in a constant temperature and humidity incubator containing 5% CO2 at 37°C using complete medium (containing McCoys5A medium, 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin). in. According to the cell growth condition, the medium was cultured once every 2–3 days, and when the cells covered most of the surface of the bottom wall of the bottle, the cells were passaged or collected.
Plasmid construction
Expression of Wnt7a was inhibited by small hairpin RNA (shRNA) technology constructing a lentiviral vector. According to the design principle of shRNA target sequence screening, combined with the gene sequence of human Wnt7a and website analysis, a specific shRNA target sequence was selected and determined. Using the NCBI database to perform Blast analysis on the identified target sequences, it is shown that there is no homology with other known genes, and then the sense and antisense strands of the synthetic oligonucleotide are designed to be annealed, that is, they are separately dissolved in double steaming. In water, the equimolar number was mixed and heated at 95°C for 5 min, and naturally cooled to 37°C to form a double-stranded oligonucleotide. The annealed shRNA sequence was inserted into the digested pSIH1-H1-Puro vector, and the ligated product was transformed into E. coli DH5α competent cells, and monoclonal sequencing was performed.
Plasmid transient transfection
The cells were cultured in DMEM medium containing 10% newborn calf serum and double antibody, and HT-29 cells and HCT-116 were seeded in 12-well plates in DMEM medium containing no double antibody and 10% fetal bovine serum. A cell cell density of 80% during dyeing, and transfection after 24 hours of culture was achieved. A total of 2 μg of the plasmid with 50 μL serum-free and antibiotic-free DMEM medium were mixed, and then added to 1 μL Vigorous with 50 μL serum-free antibiotic-free DMEM medium and left to stand for 15 min at room temperature. The cells were harvested in a 12-well plate at 37°C 5% CO2 for 48h. Wnt7a shRNA lentiviral particles were obtained, and intestinal cancer cells HT29 cells (i.e., cells infected with Wnt7a shRNA, HT29-shWnt7a-b, HT29-shWnt7a-c) were obtained, and intestinal cancer HT29 cells and HCT-116 were infected with eGFP shRNA lentivirus as control. Blank reagents were used.
Western blot
Total protein was extracted and lysed with RIPA buffer containing protease inhibitor cocktail (Roche). The proteins were separated by SDS-PAGE electrophoresis and transferred to membrane. Membranes were probed with specific primary antibodies against β-actin (Santa Cruz), Wnt7a (abcam), and detected by horseradish peroxidase-conjugated secondary antibodies.
qRT-PCR
Colon cancer cells were harvested 48 h after transfection, and Wnt7a total RNA was extracted according to the Trizol instructions. qRT-PCR analysis was performed using GoScriptTM Reverse Transcription System and GoTaq® qPCR Master Mix to detect Wnt7a expression levels, and the results were normalized by the expression level of phosphoglycerate dehydrogenase (GAPDH). The Wnt7a primer sequence was: upstream primer: 5'-CCTGGGCCACCTCTTTCTCAG-3', downstream primer: 5'-TCCAGCTTCATGTTCTCCTCCAG-3'. The product size was 573 bp. Using GAPDH as an internal reference, the upstream primer: 5'-GCATCCTGGGCTACACTGAGC-3', the downstream primer 5'-GGTACATGACAAGGTGCGGC-3', and the length of the PCR product fragment was 368 bp. The results of qRT-PCR were analyzed and their values relative to the number of critical cycles were calculated and then converted to fold changes relative to GAPDH, normalized using the 2−ΔΔCt method.
Plate clone formation assay
The cells in the logarithmic growth phase were digested with 0.25% trypsin and blown into individual cells, and the cells were suspended in DMEM medium containing 10% fetal bovine serum for use. The cell suspension was diluted as a gradient. Each group of cells was inoculated with 10 mL of 37°C pre-warmed culture medium at a gradient density of 50, 100, and 200 cells per dish, and gently rotated to disperse the cells evenly. It was then incubated in a cell culture incubator at 37°C 5% CO2 and saturated humidity for 2 to 3 weeks. When macroscopic clones appear in the culture dish, the culture was terminated. The supernatant was discarded and carefully immersed twice in PBS. Then, 5 mL 4% paraformaldehyde was added to fix the cells, after which the appropriate amount of Giemsa staining solution was applied for 10 to 30 minutes, and slowly washed away with running water and air dried. The plate was inverted and a grid of transparencies was overlaid. The clones were counted directly. Finally, the clone formation rate was calculated. Clonal formation rate = (number of clones / number of cells inoculated) × 100%.
Sphere formation assay
The colorectal cancer cells (HT-29 HCT-116) were digested with typsine and filtered with a 40-um strainer. The cells were grown in serum-free DMEM/F-12 (Gibco) supplemented with 10 ng/mL human recombinant bFGF (basic fibroblast growth factor; R&D), 10 ng/mL epidermal growth factor (EGF; Gibco) and B27 (Gibco). The cells were cultured in 2 mL medium in each well of a 6-well ultralow attachment plate and medium was added every 3 days. The size of tumorsphere were evaluated after 7 days of culture. Each experiment was performed in triplicate and measured by two investigators.
Statistical analysis
SPSS 19.0 was used for statistical analysis. Measurement data that conform to the normal distribution are expressed as mean ± standard deviation (x ± s), independent sample t-test or one-way ANOVA were used for comparison between groups, and non-normal distribution is expressed by median and interquartile range; In terms of percentage, the comparison between the positive rates was performed by χ2 test; the correlation analysis of the two variables was performed by χ2 test or Spearman correlation analysis, the test level was 0.05, and the two-sided test was used. P < 0.05 was statistically significant.