Forkhead Box P4 promotes the proliferation of cells in colorectal adenocarcinoma

Clinical data

The study subjects were randomly selected from patients diagnosed with CRAC and who were receiving radical surgery at Tianjin Hospital (China) from January 2017 to December 2022. Inclusion criteria: (1) specimens were subjected to postoperative pathological examination, sections were reviewed by two attending pathologists, and the diagnosis was performed in compliance with the criteria for pathological diagnosis; (2) clinical information was complete, including gender, age, degree of differentiation, tumor focus, tumor embolism, lymph node metastasis (LNMets), maximum tumor diameter (MTD), and depth of invasion (DOI). Exclusion criteria: (1) cases receiving preoperative radiochemotherapy; (2) cases with double or multiple primary cancers; (3) cases with Lynch syndrome; (4) cases with a history of gastrointestinal surgery. The study observed 64 cases, which included 38 males and 26 females aged 44 to 89 with a median of 70. Among them, cancer was found in the colon (ileocecal junction, ascending colon, transverse colon, descending colon, and sigmoid colon) in 37 cases and in the rectum in 27 cases. MTD was 1.5–10 cm, and the average was 5.0 ± 0.9 cm. A total of 29 cases were well differentiated, 24 were moderately differentiated, and 11 were poorly differentiated (including mucinous adenocarcinoma). LNMets was detected in 32 cases (at 1–15 nodes) and not in the 32 others. Tumor embolism was identified in 12 cases and not in the 52 others. Tumor tissue and normal mucosal tissue (located 5 cm+ away for tumor edges and confirmed through pathological examination) were sampled and embedded in paraffin. This study was approved by the Medical Ethics Committee of Tianjin Hospital pursuant to the criteria in the Helsinki Declaration, and it was registered in the ethics review document numbered 2022–150.

Evaluation method of sample size

Two independent proportions were compared with the computing formula as follows: n₁=n₂=1,641.4 × [(u_α + u_β)/(sin⁻¹ p 1  − sin⁻¹ p 2 )]. A total of 50 % of FOXP4 expression was found positive (5/10) in the tumor tissue (of 10 cases) for the pre-experiment, while 20 % was found positive (2/10) in the normal mucosal tissue (of 10 cases) for the pre-experiment. Thus, by setting p₁=0.5 and p₂=0.2, the calculation yields n₁=n₂≈64 cases.

Materials

The colon cancer cell lines SW480, HCT15, and SW620 and the normal colon cell line NCM460 were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The RPMI 1640 medium containing fetal bovine serum was purchased from Shanghai Zhaoyuan Biotechnology Co., Ltd. (Shanghai, China). Lipofectamine 3,000 was purchased from Shanghai Jin’an Biotechnology Center (Shanghai, China). CCK-8 (BA00208) was purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). RIPA lysis buffer (C1053) was purchased from Beijing Applygen Technologies Inc. (Beijing, China). siRNA-FOXP4 plasmids (sense strand AUCGUCAGGACUAAACUACUUAAAT and antisense strand UAUCUCGAGGGAUGGUCUAACUGUCAAT) were synthesized by Zhongshi Tontau Biotech Co., Ltd. (Tianjin, China). Rabbit anti-human FOXP4 (16772-1-AP) was procured from Wuhan proteintech (Wuhan, China), and rabbit anti-human proliferating cell nuclear antigen (PCNA) (A00125) was procured from Wuhan Boster Biological Technology (Wuhan, China). Secondary antibody and 3,3′-diaminobenzidine (DAB) were acquired from Beijing ZSGB-BIO (Beijing, China).

Cell culture

Three human colon cancer cell lines (SW480, HCT15, and SW620) and one normal human colon cell line (NCM460) collected from liquid nitrogen were immediately placed into a water wash preheated to 37 °C, which was then shaken until fully melted. The cryogenic vial was sterilized by wiping it with alcohol, and the solution was poured into a microfuge tube containing culture medium of 10 mL. The RPMI 1640 medium containing fetal bovine serum and both antibodies were cultured at 37 °C with 5 % CO₂ 95 % of cells grew well in the monolayer attached to the surface.

Bioinformatics analysis

The GEPIA (Gene Expression Profiling Interactive Analysis) database (http://gepia.cancer-pku.cn) was used to analyze FOXP4 expression in colon adenocarcinoma (COAD), especially the FOXP4 expression in the tumor tissue of 275 cases and the normal mucosa of 349 cases from the projects of TCGA (The Cancer Genome Atlas) and GTEx (Genotype-Tissue Expression).

Immunohistochemical test and result determination

The paraffin-embedded tissue was assayed for the expression of FOXP4 and PCNA. The best-diluted concentration for development as proven in the pre-experiment (1:250 for FOXP4 and 1:200 for PCNA) was used for the formal experiment. Sections of 4 μm were cut and assayed using the EnVision immunohistochemical technique strictly in compliance with the study protocol [9]. DAB staining was performed with proper quality control [10]. Positive FOXP4 and PCNA expression levels were both located at the nucleus of epithelial cells, and cells stained into light yellow, yellow, or brown were regarded as positive. The experimental results were determined by two attending pathologists after double-blind review of sections. The screening was first performed under low magnification (×100). Then, five fields that were densely populated by epithelial cells and most visually stained were selected under high magnification (×200) for observation and counting with reference to a two-dimensional assessment method [11]. Percentage of stained cells: <5 %, 5–25 %, 26–50 %, 51–75 %, and >75 % were scored 0, 1, 2, 3, and 4 points, respectively; color intensity: no color, light yellow, yellow, and brown were scored 0, 1, 2, and 3 points, respectively. The final score from the product of the two ranged from 0 to 12, and 0–5 points were negative and 6–12 were positive. Then, the rate of positive expression was computed.

siRNA-FOXP4 transfected SW480 and HCT15 cell lines

The SW480 and HCT15 cell lines were seeded into a 6-well plate at the density of 3 × 10⁵ per well after thawing. Serum-free culture medium of 100 μL was used to dilute siRNA-FOXP4 plasmid of 2 μg for transfection, which was mixed with transfection reagent Lipofectamine 3000 of 25 μL that had been diluted with a serum-free culture medium of 100 μL. Then, the solution was left to stand at room temperature for 30 min. The cells were cultured up to about 80 % confluent and washed twice with a serum-free medium. Serum-free medium of 1 mL was added into each well, and the transfection solution was added drip by drip. Thereafter, the solution was blended and incubated at 37 °C for 24 h. Next, the solution was replaced with a complete medium, and incubation was continued for 3 days to ensure transfection of the SW480 and HCT15 cell lines with si-FOXP4. Meanwhile, the empty vector transfection (NC-FOXP4) and no-treatment control (NC) groups were created. Western blot was applied to test the efficiency of transfection with FOXP4.

Western blot test and result determination

The SW480 cell lines in si-FOXP4, NC-FOXP4, and NC groups were assayed with a Western blot to determine the expression of FOXP4 (1:2,500) and PCNA (1:2,000). β-actin was used as an internal control. Total protein was first extracted for concentration measurement. Then, the assay was continued step by step as follows: gel preparation, sample preparation, sample loading and electrophoresis, gel transfer, blocking, primary antibody incubation, wash in TBST, secondary antibody incubation, wash in TBST, and ECL exposure. The ratio of target protein to β-actin was used for analysis. Grayscale analysis was conducted using ImageJ V1.80. The assay was performed three times.

CCK-8 assay

The cell lines SW480 and HCT15 in si-FOXP4, NC-FOXP4, and NC groups were used to prepare cells in evenly distributed suspension. The cell density was computed and adjusted to 3.0 × 10⁴ cells/mL. Cell suspension of 100 μL was seeded into a 96-well plate at the density of 3.0 × 10³ cells per well. In each group, 6 replicate wells were used for the seeding. CCK-8 reagent of 10 μL was added at 24, 48, 72, and 96 h separately. After 1 h, the optical density was determined at 450 nm for each well with an MK3 microplate reader (Thermo, USA) under lightproof conditions. The assay was performed three times.

Statistical analysis

The statistical software SAS 6.12 was used for data processing with normally distributed quantitative data expressed as mean ± SE. The homogeneity of variance test and normality test were performed. Comparison involving multiple groups was conducted using ANOVA, while pairwise comparison was conducted with the SNK method. Enumerated data were expressed in percentages, and χ ² test was used for intergroup comparisons. Linear regression analysis was conducted with p<0.05 regarded as statistically significant.

Bioinformatics analysis results

We analyzed FOXP4 expression trends on 275 tumors and 349 normal mucosal tissues by the GEPIA database (http://gepia.cancer-pku.cn). A statistically significant trend is found for FOXP4, which suggests that FOXP4 expression may be elevated in colon cancer (COAD).

Expression of FOXP4 is significantly higher in CRAC than in normal mucosa

Expression of FOXP4 is significantly higher in CRAC tissue than in normal mucosa (p<0.05, Table 1 and Figure 1).

Figure 1:

Expression of FOXP4 in CRAC and normal mucosa (IHC EnVision method). A–F: Expression of FOXP4 in CRAC. G, H: Expression of FOXP4 in normal mucosa.

A & B: FOXP4 staining is located in the nuclei of tumor cells in yellow (scored 2 points) with a positive rate of 10 % (scored 1 point), which results in a total of 2 points (B is a magnified version of A); C & D: FOXP4 staining is located in the nuclei of tumor cells in yellow (scored 2 points) with a positive rate of 40 % (scored 2 points), which results in a total of 4 points (D is a magnified version of C); E & F: FOXP4 staining is located in the nuclei of tumor cells in brown (scored 3 points) with a positive rate of 90 % (scored 4 points), which results in a total of 12 points (F is a magnified version of E).

G & H: FOXP4 staining is located in the nuclei of normal mucosal epithelial cells in light yellow (scored 1 point) with a positive rate of 70 % (scored 3 points), which results in a total of 3 points (H is a magnified version of G).

Comparison of FOXP4 expression on clinicopathological characteristics in CRAC

The differences in FOXP4 expression are found to be statistically significant with regard to MTD and DOI (p<0.05). However, the differences are statistically insignificant in terms of gender, age, degree of differentiation, tumor focus, tumor embolism, or LNMets (p>0.05, Table 2).

Correlation analysis of FOXP4 and PCNA in CRAC

IHC assay was performed to assess the expression of PCNA in CRAC with a scale ranging from 2 to 12 points. Linear correlation analysis indicates a positive correlation between FOXP4 and PCNA (the formula is Y=0.273X + 4.780, r=0.68, p=0.0169). This positive correlation suggests positive cooperativity between FOXP4 and PCNA expression levels (Figures 2 and 3).

Figure 2:

Correlation of FOXP4 and PCNA in CRAC.

Figure 3:

Expression of PCNA in CRAC (IHC EnVision method).

A & B: PCNA is located in the nuclei of tumor cells in yellow (scored 2 points) with a positive rate of 80 % (scored 4 points), which results in a total of 8 points (B is a magnified version of A).

Expression of FOXP4 is significantly higher in colon cancer cell lines than in normal colon cell line

Western blot shows that the FOXP4 expression is significantly higher in colon cancer cell lines SW480, HCT15, and SW620 than in normal colon cell line NCM460 (p<0.05). The highest expression is found in cell line SW480, followed by that in HCT15 (Table 3 and Figure 4).

Figure 4:

Expression of FOXP4 in cell lines of colon cancer and normal colon (Western blot).

Validation of transfection effect

Given that the FOXP4 expression is higher in SW480 and HCT15 than in SW620, the colon cancer cell lines SW480 and HCT15 were established for the si-FOXP4, NC-FOXP4, and NC groups separately. Western blot was utilized to assess the transfection effect of the FOXP4 protein. The results show that FOXP4 expression is significantly lower in si-FOXP4 group (0.75 ± 0.09) than in NC-FOXP4 group (1.30 ± 0.12) and NC group (1.34 ± 0.10) (F=6.32, p<0.05) in cell line SW480. FOXP4 expression is significantly lower in si-FOXP4 group (0.67 ± 0.08) than in NC-FOXP4 group (1.35 ± 0.10) and NC group (1.31 ± 0.09) (F=8.35, p<0.05) in cell line HCT15. This finding proves that the transfection is successful (Figure 5).

Figure 5:

Transfection effect validation.

Proliferative activity is significantly lower in si-FOXP4 group than in NC-FOXP4 and NC groups by CCK-8 assay

CCK-8 assay was performed to assess the proliferative activity of cell lines SW480 and HCT15 in si-FOXP4, NC-FOXP4, and NC groups. The results show that the proliferative activity of cells in si-FOXP4 group (0.67 ± 0.06) (F=18.78, p=0.0026) is significantly decreased at 72 h compared with those in NC group (1.13 ± 0.13) and NC-FOXP4 group (0.98 ± 0.08) of SW480. This condition persists until 96 h in cell line SW480 (1.67 ± 0.07 vs. 1.57 ± 0.03 vs. 1.23 ± 0.33) (F=109.15, p<0.0001). The proliferative activity of cells in si-FOXP4 group (0.71 ± 0.09) (F=15.74, p=0.0075) is significantly decreased at 72 h compared with those in NC group (1.07 ± 0.12) and NC-FOXP4 group (0.99 ± 0.10) of HCT15. This condition persists until 96 h in cell line HCT15 (1.73 ± 0.11 vs. 1.62 ± 0.07 vs. 1.11 ± 0.20) (F=34.10, p<0.0001). This finding suggests a significant inhibiting effect of siRNA-FOXP4 transfection on cell proliferation activity (Figure 6).

Figure 6:

Comparison of cell proliferation activity at different points in time in si-FOXP4, NC-FOXP4, and NC Groups.

Expression of PCNA is significantly lower in si-FOXP4 group than in NC-FOXP4 and NC groups

Western blot was used to assess PCNA expression in si-FOXP4, NC-FOXP4, and NC groups. The results show that the expression of PCNA is significantly lower in si-FOXP4 group (0.82 ± 0.11) than in NC-FOXP4 group (1.50 ± 0.10) and NC group in cell line SW480 (1.56 ± 0.13) (F=5.22, p=0.008). The expression of PCNA is significantly lower in si-FOXP4 group (0.80 ± 0.08) than in NC-FOXP4 group (1.65 ± 0.08) and NC group in cell line HCT15 (1.59 ± 0.10) (F=6.52, p=0.001). This result suggests that siRNA-FOXP4 transfection has a significant inhibiting effect on the proliferation marker PCNA (Figure 7).

Figure 7:

Comparison of PCNA expression in SW480 in si-FOXP4, NC-FOXP4, and NC Groups.