High expression of IQGAP3 indicates poor prognosis in colorectal cancer patients

CRC tissue and paired normal adjacent tissue samples were acquired from patients undergoing a surgical procedure at the Third Affiliated Hospital of Soochow University. Both the tumor and normal tissues were sent for histological analysis and diagnostic confirmation. Written consent was obtained from all patients, and all protocols concerning the use of patient samples in this study were approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University. Tissue samples were immediately frozen in liquid nitrogen at the time of surgery and stored at −80°C.

Human CRC high-density tissue microarray from Shanghai Outdo Biotech Co. Ltd contains 100 primary tumor specimens with clinical follow-up information included for the analysis of IQGAP3 expression and survival analysis. The patient baseline characteristics are listed in Table 1. Of note, some of the patients’ clinical information is not available and they were secluded from the statistical analysis.

The human CRC cell lines LoVo, HT-29, SW620 and the human intestinal epithelial cell line FHs 74 Int were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The human CRC cell line colo205 was a gift from Shanghai Ji Man Biotechnology Co. Ltd. The cells were cultured in RPMI 1640 or DMEM (GIBCO-BRL) medium supplemented with 10% fetal bovine serum (FBS, 10%), 100 U/mL penicillin and 100 mg/mL streptomycin in humidified air at 37°C with 5% CO₂. The IQGAP3 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CRC cells were transfected with siRNA oligonucleotides with plasmids using Lipofectamine 2000 (Invitrogen Life Technologies, Shanghai, China), according to the manufacturer’s protocol. After 48 h of transfection, the cells were harvested for further study.

Trizol reagent (Thermo Fisher Scientific) was used to isolate total RNA. RNA quantity and quality were determined by a NanoDrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). RNA was then reverse-transcribed to cDNA using a Reverse Transcriptase Kit (Funeng). Polymerase chain reaction (PCR) parameters were as follows: 95°C for 3 min, 40 cycles of 95°C for 10 s, and 58°C for 30 s. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. The primer sequences were as follows: IQGAP3 (sense: 5′- ACTGGCTTACATTTCCGTCACAC-3′, antisense: 5′- GTTCTTTTTGTCATAGATGTCCGTG-3′), GAPDH (forward): 5′- GGGAGCCAAAAGGGTCAT-3′ and GAPDH (reverse): 5′-GAGTCCTTCCACGATACCAA-3′. All RT-qPCR data were calculated and expressed relative to threshold cycle (shown as ΔCT) values and then converted to fold changes.

Western blot was performed as Li et al. described.¹² Total cell lysates were prepared with a detergent lysis buffer (Beyotime, Shanghai, China). Total Protein concentrations were measured by the Bradford assay (BioRad, Hercules, CA, USA). Equal amounts of proteins (20 μg each lane) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Richmond, CA, USA). Then the membranes were blocked in 5% skim milk for 1 h at room temperature, followed incubated with specific primary antibodies overnight at 4°C as follows: anti-IQGAP3 and anti-GAPDH (all antibody from Santa Cruz Biotechnology Inc., California, USA; all diluted 1:1000). After extensive washing, they were incubated with secondary horseradish peroxidase (HRP)-conjugated antimouse or anti-rabbit antibodies (Santa Cruz Biotechnology) at room temperature for 2 h. Protein bands were visualized using chemiluminescent detection system (ECL, Thermo Scientific, Rockford, IL, USA). Each blot was repeated three times. The intensity of the protein bands was analyzed by densitometry, after normalization to the corresponding protein controls.

As described in our previous article,¹³ immunohistochemical (IHC) analysis of IQGAP3 was performed using a standard streptavidin–biotin-peroxidase complex method. For antigen retrieval, paraffin tissue slides were microwave-treated for 2 h and boiled in a 0.01 M citrate buffer (pH = 6.0) for 18 min. The slides were the treated with 0.5% Triton for 20 min to break the cell membranes. The slides were incubated with mouse mAb against human IQGAP3 (Millipore, 1:200 dilution) overnight at 4°C and then incubated with goat anti-mouse IgG for 2 h. Negative control slides were probed with goat serum followed by the secondary antibody under the same conditions. Two pathologists with no knowledge of the patients’ information examined the stained sections independently. The IQGAP3 immunostaining densities were assessed according to the H-score method described by Chen et al.¹³ H-score = (% of tumor cells unstained × 0) + (% of tumor cells stained weak × 1) + (% of tumor cells stained moderate × 2) + (% of tumor cells stained strong × 3). The H-scores ranged from 0 (100% negative tumor cells) to 300 (100% strong staining tumor cells). Each tissue sample was scored based on H scores. Results from the two pathologists were averaged and used for statistical analysis. A receiver operating characteristic (ROC) curve was used to select the optimal cutoff value for the H score of the immunohistochemical sample, which divided the expression level of IQGAP3 into “low” and “high.”

To investigate the expression of IQGAP3 in human CRC tissues and normal colorectal tissues, we retrieved CRC data on The Cancer Genome Atlas (TCGA; https://tcga-data.nci.nih.gov/tcga/). IQGAP3 gene expression was compared in CRC tissues and normal colorectal tissues.

Hundred microliter complete 1640 medium including 5 × 10³ HT-29 or colo205 cells were seeded in 96-well plate and cultured for 24 and 48 h. At the exact time, 10 microliter cell counting CCK-8 solution (Dojindo Molecular Technology Inc, Shanghai, China) was added to each well and incubated at 37°C for 1.5 h. Using an ELX- 800 spectrometer reader (Bio-Tek Instruments, Winooski, USA), the absorbance was measured at 450 nm.

Migration assays were performed in a 24-well Chemotaxis chamber containing an 8-μm pore membrane. Cells were added to the upper chamber (2 × 10⁴ cells/well), and 0.5 mL RPMI-1640 plus 20% FBS was added to the lower compartment. Cells in the upper chamber were removed using a cotton swab after 24 h of incubation. The membrane was washed three times with PBS and stained by crystal violet. The degree of migration was expressed as the average number of cells in five fields per chamber.

Statistical analyses were performed by SPSS 17.0 (SPSS Inc.; Chicago, USA). The Chi-square test was used to analyze the relationship between IQGAP3 expression and clinicopathological features. The survival rate was evaluated by Kaplan–Meier method and differences between survival curves were tested by the log-rank test. Univariate and multivariate analysis were done according to the Cox proportional hazards model. Data are presented as median and range or mean ± SD where appropriate and analyzed using the Student’s t-test for comparisons between groups. P<0.05 was considered statistically significant.

Table 1 summarizes the patient clinicopathological characteristics of 97 primary CRC specimens in the high-density tissue microarray. A total of 56 men (57.7%) and 40 women (41.2%) with CRC were included in the analysis, 70 (72.2%) patients were older than 60 years, and 26 (26.8%) were not. There were 3 with stage I, 45 with stage II, 44 with stage III, and 5 with stage IV CRC tissues on the high-density tissue microarray. The median follow-up period was 51 (range, 1–87) months.

Data from the TCGA was queried, yielding 380 CRC tissues and 50 unmatched normal adjacent tissues as well as yielding 30 paired CRC tissues and matched normal adjacent tissues. We found that IQGAP3 expression was always higher in the CRC tissues (P<0.001) (Figure 1(a)). And then, expression pf IQGAP3 mRNA was measured by RT-qPCR in 12 paired CRC tissues and normal adjacent tissues. IQGAP3 showed a higher expression in the CRC tissues (P<0.01) (Figure 1(b)). IHC was applied to compare the IQGAP3 protein expression levels in CRC tissues and normal adjacent tissues, and IQGAP3 protein expression was higher in CRC tissues (Figure 1(c)). Moreover, IQGAP3 protein and mRNA expression were evaluated in CRC cell lines LoVo, HT-29, SW620, colo205, and the human intestinal epithelial cell line FHs 74 Int by western blot and RT-qPCR (Figure 1(d)). CRC cell lines showed a higher IQGAP3 expression than the human intestinal epithelial cell line FHs 74 Int. HT-29 and colo205 cell lines displayed the higher expression of IQGAP3 than the other CRC cell lines and selected for the downstream studies.

The relationships between IQGAP3 and the clinicopathological characteristics of the 97 patients are shown in Table 1. Higher expression levels of IQGAP3 were associated with higher tumor node metastasis (TNM) stage (P = 0.005) and the presence of lymph node metastasis (P = 0.004).

We next analyzed the relationship between IQGAP3 expression and CRC patients’ survival based on 97 clinical samples. Based on the Kaplan–Meier survival analysis, the overall survival (OS) rates were significantly lower in the high IQGAP3 expression group compared with the low group (P = 0.022) (Figure 2(b)). In addition, among lymph node positive CRC patients, high levels of IQGAP3 patients had a significant (P = 0.015) worse survival rate than that of patients with low levels of IQGAP3 (Figure 2(c)). These outcomes indicate that IQGAP3 expression is significantly related to the survival rate of CRC patients.

The results of the univariate and multivariate analysis are shown in Table 2. The univariate Cox regression analysis showed that TNM stage and IQGAP3 expression were associated with the OS of CRC patients. Next, the multivariate Cox regression analysis was preformed to test the effect of IQGAP3 expression, together with the clinical parameters (age, sex, pathological classification, tumor size, TNM stage, and lymph node metastasis). Our data revealed that IQGAP3 expression was an independent and unfavorable prognostic factor for CRC patients (hazard ratio (HR) 2.881; 95% confidence interval (CI) 1.307, 6.350; P = 0.009).

To test the roles of IQGAP3 expression in CRC cell growth, we knocked down IQGAP3 in HT-29 and colo205 cells (Figure 3(a)), respectively. The results from MTT assay showed that IQGAP3 knockdown significantly inhibited cell viability all in the two CRC cell lines compared with control cells (Figure 3(b)).

We then applied cell migration assay to assess the effect of IQGAP3 on the migration power of colon cancer cells. The results showed that IQGAP3 silencing caused a significant decline in cell motility in the two CRC cell lines compared with control cells (Figure 3(c)).