Levels of human replication factor C4, a clamp loader, correlate with tumor progression and predict the prognosis for colorectal cancer

The expression of RFC4 mRNA in CRC tissues was obtained by The Cancer Genome Atlas (TCGA), Gene Expression Omnibus databases (GEO) and BioGPS database (Biogps.org). For TCGA analysis, we queried The Cancer Genome Atlas [http://​tcga-data.​nci.​nih.​gov/​] for colon cancer patients. “Level 3 of Exp-Gene” files from COAD Data Matrix Datasets were downloaded and used to extract mRNA expression for RFC4. For GEO data analysis, we downloaded and analyzed data publicly available from the GEO [http://​www.​ncbi.​nlm.​nih.​gov/​geo/​; 82 samples from GSE9348 and 105 samples from GSE5206 and 64 samples from GSE8671]. “Series Matrix” Files were used to extract mRNA expression for RFC4. The genes amplification folds were analyzed and visualized by Graph Prism6 (GraphPad software Inc., San Diego, CA).

First, to detect the expression dynamics of RFC4 in CRC, 30 paired freshly frozen specimens and 49 paired formalin-fixed, paraffin-embedded (FFPE) specimens from primary CRC tissues and patient-matched normal colonic tissues were obtained from the 6^th Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). Each normal colonic tissue was acquired from a distance of at least 10 cm from the tumor margin. Second, to determine the clinical relevance of RFC4 in CRC, 331 FFPE CRC tumor specimens resected between January 2000 and December 2006 were obtained from the pathology archives at the 1^st Affiliated Hospital of Sun Yat-sen University (Guangzhou, China) for tissue microarray analyses. Among the 331 patients, 91 patients (27.5%) had been censored as death and 97 patients (29.3%) had developed distant metastasis or local recurrence after a median follow-up of 73.0 months (range 1-122). None of the patients had received neoadjuvant chemotherapy or radiotherapy, and the status of all samples was confirmed by pathologists after resection. Tumor tissues were staged according to the 7^th edition of the Union for International Cancer Control Tumor-Node-Metastasis (TNM) staging system. Written informed consents for using tissue samples for research purposes were obtained from all patients. This study was approved by the Institutional Review Board of the 1^st and 6^th Affiliated Hospital of Sun Yat-Sen University and all clinical and pathological data of the enrolled patients were collected from the Institutional Review Board approved CRC database, which was maintained by specialists.

The human CRC cell lines, SW480 and DLD1, were obtained from the Culture Collection of the Chinese Academy of Science (Shanghai, China) and cultured in RPMI 1640 media. All media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. The cells were cultured at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

All RNA extractions were performed using the Trizol Reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocols. For first-strand complementary DNA synthesis, total RNA was reverse-transcribed with an oligo-dT primer using the RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Burlington, Canada). Quantitative PCR (qPCR) reactions were performed with an ABI PRISM® 7500 Fast Real-time PCR System (Applied Biosystems, Foster City, CA, USA) and a SYBR Green qPCR Mix Kit (Takara, Japan). β-actin expression was used as the normalization control. The following temperature profiles were used: initial heating at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15’s, annealing at 60°C for 60’s, and extension at 95°C for 15’s. The primers used were:

Data were analyzed using the 2^-ΔΔCt method.

Total cellular proteins were extracted from tissues or cells, separated by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (Pall, New York, USA). Membranes were blocked with 5% nonfat milk in 1% Tween-PBS (PBST) and then probed overnight with anti-RFC4 rabbit polyclonal antibody (1:1000, Epitomics, Burlingame, CA, USA) or anti-ACTB antibody (1:1000, Proteintech, Chicago, IL, USA). After three washing steps of 10 min in PBST, membranes were incubated with species-appropriate fluorescently-conjugated secondary antibodies (1:10000 in PBST, LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. The immunoreactive signals were detected using the two-color fluorescent western blotting Odyssey infrared imaging system (LI-COR Biosciences).

To validate the overexpression of RFC4 and to define the clinical roles of RFC4 in CRC, two TMAs were constructed. The first contained tissue cores of 49 paired specimens of primary CRC tissues and matched normal colonic tissues. The second contained additional spots of 331 resected CRC specimens. For each case, two cores (1 mm diameter) were taken from the selected tumor area, and two additional cores (1 mm diameter) were taken from histologically confirmed normal adjacent colorectal mucosa to construct the TMAs using Tissue Array (Alphelys, MINIPORE, Plaisir, France). The TMAs were performed as we described previously [14].

The TMAs were deparaffinized in xylene and rehydrated with graded ethanol. Sections were then heated in antigen retrieval solution (EDTA, pH 9.0) for 20 min and incubated with 3% H₂O₂ for 10 min. The sections were then incubated with anti-RFC4 rabbit polyclonal antibody (1:800, Epitomics) at 4°C overnight. The sections were then treated with the secondary antibody for 15 min at room temperature and stained with 3, 3′-diaminobenzidine until brown granules appeared in the membrane, cytoplasm, or nucleus (Dako, EnvisionSystem/DAB-chromogen, Glostrup, Denmark). The sections were counterstained with hematoxylin for 2 min at room temperature. A negative control was employed by exchanging the specific primary antibody with non-immune serum immunoglobulins at a 1:200 dilution.

The immunoreaction was semi-quantitatively scored by assessing the extent and intensity of nuclear staining. Weak cytoplasmic staining was identified in some cases. The intensity of nuclear staining was scored as follows: negative (0), weak (1), moderate (2), and strong (3). The extent of nuclear staining was graded into quartiles according to the percentage of stained cells in each field: 0-25% (1), 26-50% (2), 51-75% (3) and 76-100% (4). The merged overall score (staining intensity plus staining extent) was then calculated. IHC staining was scored independently by two blinded pathologists to the clinical data (Huang and Fan).

To generate a logical cutoff score for further analysis, the nuclear RFC4 expression scores of 49 paired CRC and matched normal colonic tissues were subjected to the receiver operating characteristic curve (ROC) analysis. The score localized closest to the point at both maximum sensitivity and specificity (0.0, 1.0) was selected as the cutoff score leading to the greatest number of specimens which were correctly classified as tumor or normal.

To knockdown endogenous RFC4 expression, cell lines were transfected with 20 nM of duplexed siRNA (RiboBio, Guangzhou, China) using Lipofectamine® RNAiMAX Reagent (Life Technologies). Duplex siRNAs included the following: RFC4 siRNA #1 (sense 5′-CAACUCAGCUCGUCAAUCAdTdT-3, antisense 3′-dTdTGUUGAGUCGAGCAGUUAGU-5); RFC4 siRNA #2 (sense 5′-GACCAAGGAUCGAGGAGUAdTdT-3, antisense 3′-dTdTCUGGUUCCUAGCUCCUCAU-5); RFC4 siRNA #3 (sense 5′-GAAAGUGAUUACAGACAUUdTdT-3, antisense 3′-dTdTCUUUCACUAAUGUCUGUAA-5). Duplexes of siRNA with non-specific sequences were used as scrambled siRNA. SW480 and DLD1 cells were transfected with RFC4 siRNA or scrambled siRNA according to the manufacturer’s instructions.

Cells were plated at equal densities in 96-well plates for 24 h and then transiently transfected with the indicated siRNAs (6 biological replicates per condition). Cell proliferation was assessed using the Vybrant MTT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s instructions. Absorbance was measured at 490 nm.

The effect of RFC4 on proliferation was also tested by the 5-ethynyl-2′-deoxyuridine (EdU) cell proliferation assay using the Cell-Light™ EdU DNA Cell Proliferation Kit (RiboBio, Guangzhou, China). Briefly, cells (1 × 10⁴) were seeded in each well of 96-well plates for transfection with RFC4 siRNA or scrambled siRNA. After incubation at 37°C under 5% CO₂ for 96 h, 50 μM EdU was added and the cells were incubated for another 2 h. Cells were then fixed with 4% paraformaldehyde and stained with Apollo® Dye Solution for proliferating cells. Nucleic acids in all cells were stained with Hoechst 33342. The extent of cell proliferation was calculated according to the manufacturer’s instructions. Images were taken using a fluorescence microscope (Olympus FSX100). All experiments were performed in triplicate.

Cells were trypsinized, washed with cold PBS, and fixed overnight in 70% ethanol pre-chilled to -20°C. For the measurement of DNA content, cells were stained with a propidium iodide solution (50 mg/mL propidium iodide, 100 mg/mL RNase A, 0.05% Triton X-100 in PBS) and incubated at 37°C in the dark for 30 min. DNA content was assessed using a BD FACSCanto II flow cytometer (BD Biosciences) with ModFit LT software (Verity Software House, Topsham, ME).

Overall survival (OS) time was measured from the date of surgery to the date of death due to any cause, or the date of the last clinical follow-up. Disease-free survival (DFS) time was defined as the time from surgery to the date confirmed local failure, distant metastases, or death due to disease or treatment, whichever occurred first. Kaplan-Meier survival curves were evaluated by the log-rank test. Univariate Cox regression analyses were performed to determine the prognostic significance of individual clinicopathologic variables. Multivariate analysis to test statistical independence and the significance of multiple predictors was performed by stepwise Cox analysis with backward selection using the Akaike Information Criterion. Wilcoxon matched-pairs, signed-rank test were used to assess IHC score within groups (cancer and matched normal tissues). Chi-square tests were used to compare dichotomized RFC4 groups and baseline clinicopathologic factors. Mann-Whitney U test were used to compare continuous variables and categorical variables. The level of significance for all tests was defined as P <0.05. All statistics were performed using SPSS v. 17.0 (SPSS, Inc., Chicago, IL).

First, we analyzed the data from the TCGA and GEO for mRNA expression in CRC patients. RFC4 expression was higher in colon cancer than normal colon tissue (Figure 1A). Furthermore, by using the BioGPS Gene Expression Atlas, we found that RFC4 expression was highly expressed in colon cancer (Additional file 1: Figure S1).

To validate our finding, RNA was extracted from 30 paired CRC and adjacent normal tissues, and qPCR was done to measure the levels of RFC4 mRNA. RFC4 mRNA was significantly elevated in 21 of 30 CRC tissues, compared with patient-matched normal tissues (Figure 1B). Western blot analysis also revealed overexpression of RFC4 in 12 of 16 cancer tissues that had analyzed by qPCR, compared with normal colonic tissues (Figure 1C). Immunohistochemistry on TMA contained an independent set of 49 paired CRC and matched-normal tissues showed CRC tend to exhibit a higher level of RFC4 expression than normal colonic tissue (P <0.05) (Figure 1D). The clinical features of these patients were summarized in Table 1. Together, these data confirm the up-regulation of RFC4 in CRC.

For further survival analysis, ROC curve analysis of 49 paired specimens was performed to determine a reasonable cutoff point for RFC4 overexpression (Figure 1E). A score of 4.5, which maximizes the sum of sensitivity and specificity according to the ROC curves, was determined as the cutoff point.

The potential significance of RFC4 protein levels in CRC was examined by IHC on the TMA consisting of tissues from 331 CRC tumors, which were divided into a normal expression group and an overexpression group based on the above cutoff value. Dichotomized groups were evaluated in relation to clinicopathologic factors and overall survival. The RFC4 IHC staining results are summarized in Table 2 and Figure 2. RFC4 expression positively correlated with differentiation and TNM stage (P <0.05; Table 2). Patients with poorly differentiated or advanced TNM stage CRC were frequently identified with RFC4 overexpression. No significant associations were noted between RFC4 expression and other clinicopathologic variables. In relation to clinical outcomes, high levels of RFC4 expression were significantly associated with poor OS and DFS by Kaplan-Meier analysis (Figure 3). The 5-year OS and 5-year DFS were 81% and 78%, respectively for normal RFC4 expression patients, while the high RFC4 expressing patients had 5-year OS and 5-year DFS of 67% and 65%, respectively. In addition, univariate analysis revealed that the extent of differentiation, CEA level, CA19-9 level, pN stage, pM stage, and RFC4 expression were risk factors for death in CRC. Further Multivariate analysis was performed using the Cox proportional hazards model for all of the significant variables in the univariate analysis, including differentiation, preoperative CEA level, preoperative CA19-9 level, TNM stage and RFC4 expression Table 3. The results showed that RFC4 expression was not an independent predictor of worse overall survival, which might attribute to that there were more stage III/IV patients (60.9%) having high expression levels of RFC4.

Each of three distinct RFC4 siRNAs sharply reduced the expression of RFC4 mRNA and protein by 70% to 85% in both SW480 and DLD1 cells (Figure 4A, B, C and D). The number of viable SW480 and DLD1 cells 120 h after RFC4-siRNA transfection was significantly reduced (as assessed by the MTT assay) compared to that of the scrambled siRNA or non-siRNA groups (P <0.05) (Figure 4E and F). DNA synthesis in RFC4 knockdown tumor cells was significantly inhibited as compared to that in control tumor cells (P <0.05) (Figure 4G).

To determine whether the inhibition of tumor cell growth was related to cell cycle regulation, the effects of RFC4 knockdown on cell cycle progression was investigated. Notably, the percentages of both SW480 and DLD1 cells in the S phase of the cell cycle were substantially increased following RFC4 knockdown (Figure 5). These data indicate that depletion of RFC4 induces S-phase cell cycle arrest, which might contribute to the suppression of cell proliferation.