This study was approved by the ethics committee of the First Affiliated Hospital of Chinese PLA General Hospital (Beijing, China). A total of 182 patients who had undergone radical surgical treatment for RCC at the Department of Urology, First Affiliated Hospital of Chinese PLA General Hospital were recruited between 1998 and 2003. All patients were informed of the content and agreed to join in the present study. All tumors were determined to be conventional clear-cell RCC by experienced pathologists who were blinded to the patient data. Tumor characteristics and stage were classified using the tumor-node-metastasis (TNM) classification system, and the nuclear grade was assessed according to the Fuhrman tumor grading system (31,32). The RCC samples and corresponding normal healthy renal tissue samples, located as far as possible from the tumor site, were surgically removed, fixed in formalin, dehydrated and paraffin-embedded. All samples were frozen in liquid nitrogen immediately following surgical resection and maintained at −90°C for protein and RNA extraction. Postoperative patients with RCC underwent routine checkups and follow-ups at the outpatient clinic in our hospital for 10 years. Blood biochemistry tests, ultrasonography, magnetic resonance imaging or computed tomography were performed during the follow up.

Four RCC cell lines, ACHN, Caki-1, NC 65 and A498 cells, were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in complete medium consisting of RPMI-1640 medium (Gibco, Bio-Cult Diagnostics Limited, Glasgow, UK), 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 1% nonessential amino acids purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). All cell lines were maintained as monolayers in a 10-cm plastic dish and incubated in a humidified atmosphere containing 5% CO₂ at 37°C.

BALB/C nude mice, aged 3 or 4 weeks, were purchased from CAMS Laboratories (Beijing, China) and randomly divided into control and FLOT1 vector groups containing 15 mice each. A total of 2×10⁷ RCC cells was injected into the back of each mouse. All mice were observed and tumor volumes were recorded once a week. After five weeks, all mice were sacrificed by decapitation under deep anesthesia and the final volume of each tumor was measured.

Exponentially growing RCC cells were harvested, seeded in a 96-well microtiter plate (Corning Inc., Corning, NY, USA) and placed in an incubator. At 24, 48 and 72 h, 10 μl WST-1 (Roche Diagnostics GmbH, Penzberg, Germany) was added to each well and the incubation was continued for an additional 2 h. The absorbance, reflected in the cell count in each well, was measured with a microculture plate reader (Immunoreader; Japan Intermed Co. Ltd., Tokyo, Japan) at 450 nm.

Paraffin sections (5 μm) were deparaffinized in xylene and rehydrated using graded alcohol. Endogenous peroxidase activity was inhibited with administration of 0.3% hydrogen peroxide for 15 min. The sections were blocked with 20% normal rabbit serum (Sigma, St. Louis, MO, USA) for 30 min prior to 1-h incubation with primary FLOT1 rabbit monoclonal antibody (Cell Signaling Technology, Inc., Danvers, MA, USA). The slides were washed twice in Tris-buffered saline and incubated with biotinylated rabbit anti-rabbit antibody (Dako, Glostrup, Denmark). Detection of the antibody reaction was performed using a standard streptavidin-biotin complex (Sigma). The immunohistochemistry results were obtained using a light microscope (Olympus BH2; Olympus, Tokyo, Japan) and FLOT1 immunostaining was conducted to semiquantitatively evaluate the slides for intensity (−, negative; +, weak; ++, moderate and +++, strong staining).

mRNA was isolated from normal and RCC tissue using the QuickPrep mRNA purification kit (GE Healthcare, Little Chalfont, UK) according to the manufacturer’s instructions. A first-strand cDNA synthesis kit (Amersham Biosciences, Amersham, UK) was used for reverse transcription. The PCR conditions were determined according to the manufacturer’s instructions and the PCR product was confirmed using agarose gel electrophoresis. qPCR was conducted with the LC FastStart DNA Master SYBR Green I (Roche Applied Science, Penzberg, Germany) and the products were quantified with a LightCycler (Roche Applied Science). The primers used were as follows: forward: 5′-CCCATCTCAGTCACTGGCATT-3′ and reverse: 5′-CCGCCAACATCTCCTTGTTC-3′ for FLOT1; and forward: 5′-ATCAAGAAGGTGGTGAAGCAGG-3′ and reverse: 5′-GTGGAGGAGTGGGTGTCGC-3′ for GAPDH.

The protein was extracted and protein concentration assessed according to the manufacturer’s instructions, and SDS polyacrylamide gel electrophoresis was performed. FLOT1 antibody (dilution 1:1,000) and secondary HRP conjugated antibody (dilution 1:2,000) were was purchased from Cell Signaling Technology, Inc.. Anti-β-actin monoclonal antibody (Abcam, Cambridge, UK) served as a loading control. The immune complexes were examined using electrochemiluminescence (Amersham, Aylesbury, UK).

siRNA oligonucleotide sequences were designed with siDirect software (University of Tokyo, Tokyo, Japan). RCC cells were seeded in plastic culture dishes in complete medium without antibiotics until 40 to 50% confluence was achieved. The cells were then transfected with siRNA oligonucleotides using Lipofectamine 2000 (Invitrogen Life Technologies). After 48 h incubation, gene expression was confirmed with western blotting. The coding sequence of human FLOT1 was cloned using RT-PCR with HK-2 normal kidney cell line (purchased from ATCC, Manassas, VA, USA) cDNA as a substrate, and the products were subcloned into pcDEF3, a mammalian expression vector (Invitrogen Life Technologies). The RCC cell lines were stably transfected with Lipofectamine 2000. Using this expression vector, which contained full-length FLOT1 cDNA, monoclonal antibodies were selected using G418 aminoglycoside antibiotics (Calbiochem, Bad Soden, Germany) and gene expression was confirmed by western blotting.

SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA) was used to perform statistical calculations. All experiments were performed in triplicate and the results expressed as the mean ± standard deviation. Statistical significance was determined using Student’s t-test, and the χ² test was used to evaluate the association between FLOT1 expression levels and clinicopathological parameters. Survival curves were plotted using Kaplan-Meier analysis. P≤0.05 was considered to indicate a statistically significant difference.

Subjects comprised 99 male and 83 female patients, aged 42–86 years (mean age, 64 years). The RCC tumor sizes were between 2 and 13 cm (average, 4.5 cm). A total of 98 patients had stage I cancer, 47 had stage II, 23 had stage III and 14 had stage IV. With regards to Fuhrman tumor grade, 97 patients had grade 1, 60 had grade 2 and 25 had grade 3. The presenting symptoms included flank pain (21 patients), hematuria (19 patients) and detection of a palpable mass (15 patients).

Malignancy was an incidental finding on routine examination in 98 patients. Laboratory examination at diagnosis revealed an elevated sedimentation rate in 32 patients, while anemia, thrombocytopenia and erythrocytosis were each identified in two patients. A total of 37 patients suffered from one or more comorbidities, including diabetes mellitus, urolithiasis, angina pectoris and valvular heart disease. Five patients had previously been treated with a radical nephrectomy on the other side and 12 patients exhibited metastatic disease at diagnosis.

FLOT1 protein expression levels in human RCC and normal healthy renal tissue were determined using immunohistochemistry. FLOT1 expression was upregulated in RCC tissue compared with in the corresponding normal renal tissue (Fig. 1). FLOT1 expression was observed in 163 of 182 RCC tumors (89.6%) but in only 29 of 182 (15.9%) normal renal tissue samples. χ² analysis revealed significant associations between increased FLOT1 protein expression levels and advanced tumor stage, high histological grade and large tumor size (all P<0.05). However, no significant differences were detected between gender and age variables and FLOT1 protein expression levels (Table I). The results from this sample analysis suggest that FLOT1 is involved in tumorigenesis and RCC progression.

To confirm the results demonstrating upregulated FLOT1 expression in RCC through immunohistochemical analysis, RT-qPCR and western blotting were used to measure FLOT1 expression levels in human RCC and normal healthy renal tissue. The levels of FLOT1 expression were determined relative to an internal control. The results indicated that FLOT1 expression was upregulated to a significantly greater extent in RCC than in the corresponding normal renal tissue (P=0.001) and that FLOT1 expression levels were similar to those detected using immunohistochemistry in all samples. The results of four pairs of samples are shown in Fig. 2.

An expression vector containing full-length FLOT1 cDNA was stably transfected into ACHN, Caki-1, NC 65 and A498 cell lines, and siRNA was used to inhibit FLOT1 expression. All transfections were confirmed by western blotting. FLOT1 expression was significantly downregulated by siRNA and upregulated by the FLOT1 vector insertion (Fig. 3A). The effect of FLOT1 on RCC cell proliferative ability in vitro was analyzed using a WST-1 assay, which revealed that RCC cells expressing high levels of FLOT1 exhibited significantly higher proliferative ability than control cells (P=0.001). By contrast, RCC cells with low FLOT1 expression levels, due to siRNA administration, exhibited lower proliferative ability (P=0.002; Fig. 3B). Similar results were observed in vivo in the xenograft experiment with BALB/C nude mice (P=0.001; Fig. 3C).

As FLOT1 expression levels, and RCC stage and grade were correlated, the possibilities for FLOT1 as a prognostic marker in human RCC were investigated. Kaplan-Meier analysis was used to calculate the association between the FLOT1 expression levels detected by immunohistochemistry (classified as low, − or +; or high, ++ or +++) and patient survival times. A total of nine patients succumbed to myocardial infarction and five patients succumbed to disseminated malignant disease. The average survival time was significantly greater in the group with low FLOT1 expression levels compared with the group with high FLOT1 expression levels (P<0.05; Fig. 4). After 10 years follow-up, 51 of 79 (64.6%) patients with low FLOT1 expression levels were alive and disease-free, compared with 35 of 103 (34%) patients with high FLOT1 expression levels. Therefore, FLOT1 expression levels were an independent prognostic marker.