For the RT-PCR analysis, 38 pairs of ccRCC tissue specimens and matched adjacent normal tissues were collected from patients who underwent radical nephrectomy at the Second Affiliated Hospital of Anhui Medical University (Anhui, China) between March 2010 and August 2012. In addition, we prepared 124 paraffin-embedded ccRCC samples for immunohistochemical analysis from Zhujiang Hospital of Southern Medical University (Guangzhou, China) between January 2006 and February 2010. Each case was histologically confirmed as ccRCC and with a verification of no preoperative chemotherapy or radiotherapy. The patients enrolled in the immunohistochemical analysis had been followed up until February 2015 with a median follow-up period of 51.5 months (5–60 months). Clinicopathological data of these patients including age, gender, tumor size, Fuhrman grade, clinical stage, lymphatic and distant metastasis (Table I) were gathered from well-documented medical records. Tumor stage and grade were classified according to the American Joint Commission on Cancer (AJCC) tumor, nodes and metastasis (TNM) system and Fuhrman criteria. The patients enrolled in the present study had given written informed consent, and the study was approved by the Ethics Committee of The Second Affiliated Hospital of Anhui Medical University and Zhujiang Hospital of Southern Medical University.

Four human RCC cell lines (786-O, A704, ACHN and A498) and an immortalized normal human proximal tubule epithelial cell line HK-2 were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and the American Type Culture Collection (ATCC; Rockville, MD, USA), respectively. HK-2 cells were cultured in K-SFM medium, while RCC cells were cultured in Dulbecco's modified Eagle's medium (DMEM), both supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (both from Gibco, Carlsbad, CA, USA). All cells were cultured in a sterile incubator under the condition of 5% CO₂ at 37°C. The small interfering RNA (siRNA) targeting NUSAP1 (si-NUSAP1) and scrambled siRNA (si-NC, as negative control) were purchased from GenePharma (Shanghai, China). The sequence designed for si-NUSAP1 was: 5′-GCACCAAGAAGCUGAGAAUTTAUUCUCAGCUUCUUGGUGCTT-3′. RCC cell lines 786-O and A704 were transfected with either si-NUSAP1 or si-NC using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Forty-eight hours after transfection, the mRNA and protein were harvested and analyzed.

RNA isolation from the RCC cells and tissue samples was performed using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instructions. The first-strand cDNA was synthesized by RNA using MMLV reverse transcriptase (Takara, Otsu, Japan) following the protocol provided. Real-time quantitative RT-PCR (qRT-PCR) was operated using SYBR Premix Ex Taq™ kit (Takara) on an ABI 7500 RT-PCR System (Applied Biosystems, Foster City, CA, USA). The primer sequences designed for cDNA amplification were as follows: NUSAP1, 5′-GAAGCTGAGAGACAGCCACT-3′ (forward), and 5′-TCTgTgAgTCAgggTCCACA-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a housekeeping gene), 5′-GAAAGCCTGCCGGTGACTAA-3′ (forward), and 5′-GCCCAATACGACCAAATCAGAG-3′ (reverse). The 2^−ΔΔCt method (17) for calculating the relative levels of NUSAP1 mRNA was applied in the present study, and all experiments were accomplished in triplicate.

Human RCC cells or tissue samples were lysed in RIPA buffer containing proteinase inhibitor cocktails. After centrifugation at 12,000 rpm for 20 min, the BCA protein assay kit (Sigma, St. Louis, MO, USA) was used to quantify the protein concentrations. Equivalent amounts of harvested proteins (50 µg) were separated using SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After being blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline and Tween-20 (TBST) for 2 h, the membranes were incubated overnight at 4°C with the following antibodies: rabbit polyclonal anti-NUSAP1 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and goat polyclonal anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, the membranes were washed and incubated with secondary antibodies at room temperature for 1 h. Proteins were finally visualized using ECL immunoblotting detection reagent (Biobox Biotech. Co., Ltd, Nanjing, China) according to the manufacturer's instructions.

IHC was performed to determine the protein level of NUSAP1 expression in the ccRCC and matched adjacent normal tissues using EliVision method for IHC staining. All formalin-fixed and paraffin-embedded samples were cut in 4-µm thick sections, deparaffinized and dehydrated. These sections were soaked in 3% H₂O₂ to block endogenous peroxides, then, immerged in effervescent citrate buffer (10 mM, pH 6.0) and incubated with 5% BSA for 30 min, respectively. Thereafter, the slides were incubated overnight at 4°C with primary rabbit polyclonal anti-NUSAP1 antibody (1:50; Thermo Fisher Scientific), and further incubated with the secondary antibody on the next day. The DAB kit (ZSGB-Bio, Beijing, China) was used to perform the visualization. The slides were washed in distilled water and counterstained with hematoxylin at the end of the staining process.

IHC staining results were measured based on the multiplication of staining intensity and density scores. The intensity score was calculated according to the average intensity of positive NUSAP1-staining cells (0, none; 1, weak; 2, moderate; 3, strong). The density score was calculated from the results of the percentage of positive-staining cells (1, <5%; 2, 5–25%; 3, >25–50%; 4, >50%). The overall score was finally determined, and scores of 0–4 were defined as low expression while scores >4 were considered as high expression. The immunohistochemical staining was evaluated by two independent observers blinded to the clinical outcomes.

Cell migration ability was determined by scratch migration assay. 786-O and A704 cells transfected with the negative control (si-NC) or si-NUSAP1 were seeded into 6-well plates at a concentration of 5×10⁵ cells/well and cultured overnight. Then, the monolayer cells were scratched with a sterile 10-µl pipette tip, washed thrice with phosphate-buffered saline (PBS) and incubated at 37°C in 5% CO₂. The wound closure was observed and photographed by a inversion fluorescence microscope (Olympus, Tokyo, Japan) after 24 h. Each experiment was performed in triplicate.

Transwell invasion assay was conducted on a 24-well Transwell chamber plates with a pore size of 8 µm. The Transwell filter inserts were precoated with 40 µl Matrigel (dilution at 1:3; BD Biosciences) at 37°C for 5 h. 786-O and A704 cells (1×10⁵ cells/well) were seeded in serum-free DMEM in the upper chamber, and the lower chamber contained DMEM with 10% FBS. After incubation at 37°C for 24 h, the non-invaded cells were scraped off and the invaded cells were fixed with 4% paraformaldehyde solution and stained with 0.1% crystal violet. The invaded cells were observed under a light microscope and counted in five randomly chosen fields.

To determine cell proliferation, 786-O and A704 cells transfected with siRNA were plated in 96-well plates at a density of 3×10³ cells/well. Cell Counting Kit-8 (CCK-8; Ding Guo Biotech. Co,. Ltd, Guangzhou, China) was applied to quantify the cell proliferation at 0, 12, 24 and 48 h. After CCK-8 solution (10 µl) was added to each well, the cells were incubated for 2 h at 37°C. The absorbance that represented proliferating cell numbers was detected at 450 nm using a microplate reader (Thermo). Each experiment was performed in triplicate and independently repeated three times.

Flow cytometric analysis was performed to determine cell apoptosis and cell cycle distribution. The Annexin V-FITC apoptosis detection kit (Beyotime-Bio, Shanghai, China) and propidium iodide (PI) was used for apoptosis assay. 786-O and A704 cells transfected with siRNA were harvested after incubation for 48 h, washed twice by PBS and suspended in 195 µl binding buffer with 5 µl Annexin V-FITC, and incubated for 10 min at room temperature in the dark. Then, the cells were re-suspended in 190 µl binding buffer with 10 µl PI, incubated in ice water and immediately analyzed. For cell cycle analysis, 786-O and A704 cells were collected and washed twice with PBS, fixed in 75% ice-cold ethanol overnight. After that, the cells were suspended in 300 µl PBS containing 20 µl RNase and incubated at 37°C for 30 min, and then 400 µl PI was added in the cell suspension, mixed and incubated at 4°C for 30 min. The results were analyzed by flow cytometry (BD Biosciences) and each experiment was performed in triplicate.

All statistical analyses were analyzed using SPSS 20 statistical software (IBM, Chicago, IL, USA). Continuous data expressed as mean ± SD were determined using the two-tailed paired Student's t-test. The Pearson's Chi-square test was performed to analyze the correlations between NUSAP1 expression and clinicopathological characteristics of the ccRCC patients. The overall survival curves were drawn according to the Kaplan-Meier method and compared using log-rank test. P-value <0.05 was considered to indicate a statistically significant difference.

Recently, we completed reduced representation bisulfite sequencing (RRBS) and transcriptome sequencing of 34 pairs of RCC tissues and adjacent normal controls. In addition, whole-genome sequencing (WGS) was performed for another 61 paired RCC and adjacent normal tissues. All the analytical processes were referred to in our previous study (18). To date, studies related to these data have not yet been published.

To investigate the expression of NUSAP1 at the mRNA and protein levels in ccRCC tissues, qRT-PCR and WB assay were applied in 38 pairs of ccRCC and matched adjacent normal tissues. Our results revealed that NUSAP1 was over-expressed in ccRCC tissues, when compared with levels in the matched normal tissues (Fig. 1A; P<0.001). In line with the mRNA data, the protein level of NUSAP1 in representative cancer tissues was also clearly higher than that in the adjacent normal tissues (Fig. 1B).

In addition, we determined the expression of NUSAP1 according to the above methods in five types of cell lines, including HK-2, A704, 786-O, ACHN and A498. The data revealed that NUSAP1 mRNA and protein levels were statistically elevated in three RCC cell lines (A704, 786-O and ACHN) compared with that in the HK-2 cell line (Fig. 1C and D; P<0.001).

We analyzed the protein level of NUSAP1 in 124 pieces of ccRCC sections by immunohistochemical staining. A total of 68 (54.8%) cases showed high expression of NUSAP1 while 56 cases (45.2%) revealed low expression (Fig. 2B–D). The adjacent normal tissues exhibited either no or weak staining of NUSAP1 (Fig. 2A). The relationships between NUSAP1 expression and clinicopathological characteristics of the ccRCC patients are summarized in Table I. Our data demonstrated that the expression of NUSAP1 was significantly correlated with Fuhrman grade (P<0.001), tumor size (P=0.016), clinical stage (P<0.001) and distant metastasis (P=0.023), while no significant correlation was found between NUSAP1 expression and gender, age and lymph node metastasis (P>0.05).

The association between NUSAP1 expression and the prognosis of ccRCC patients was analyzed by Kaplan-Meier method. The 5-year survival rate in the group of patients with high NUSAP1 expression was 61.8%, whereas it was increased to 78.6% for these patients with low NUSAP1 expression (Fig. 2E). The log-rank test was used to compare the correlation between survival and NUSAP1 expression in the low and high expression groups. Our results suggested that the patients with low NUSAP1 expression had a significantly longer overall survival (OS) time than those with high NUSAP1 expression (P=0.006).

As shown in Fig. 1C and D, three RCC cell lines (A704, 786-O and ACHN) exhibited an obviously increased expression of NUSAP1. We thus chose A704 and 786-O cell lines to determine the biological behaviors of human RCC cell lines following the downregulation of NUSAP1 expression in vitro. After transfection with siRNA targeting NUSAP1 (si-NUSAP1), cells exhibited a significant decrease in mRNA and protein levels of NUSAP1 compared with those transfected with scrambled siRNA (si-NC) (Fig. 3A and B; P<0.001). Our results suggested that the NUSAP1 expression was efficiently downregulated in the human RCC cells in vitro.

Moreover, we performed scratch migration, Transwell invasion and CCK-8 assays to investigate the effects of the downregulation of NUSAP1 on cell migration, invasion and proliferation in the two RCC cell lines (A704 and 786-O), respectively. The results showed that downregulation of NUSAP1 by siRNA caused a significant inhibition of cell migration of the 786-O (P<0.05) and A704 (P<0.001) cell lines (Fig. 4A). In accordance with this result, downregulation of NUSAP1 also resulted in a clear decrease in cell invasive ability (Fig. 4B; P<0.001). In the CCK-8 assay, we found that the proliferation rate of the cells transfected with si-NUSAP1 was significantly decreased compared with the rate in the cells treated with si-NC/blank (Fig. 4C; P<0.05, P<0.001). In summary, these results indicated that NUSAP1 expression was closely associated with cell growth and aggressiveness of RCC.

To further investigate the mechanism of cell growth inhibition by downregulation of NUSAP1 expression in RCC cells, the cell cycle progression of 786-O and A704 cells was analyzed using flow cytometry. As shown in Fig. 5B, after transfection with si-NUSAP1, a higher rate of G₂/M phase arrest when compared with that in the si-NC group (mean rate, 20.37 vs. 13.69%; P<0.05) was identified in the 786-O cells, as well as in the A704 cells (mean rate, 19.11 vs. 13.59%; P<0.05). Moreover, flow cytometric analysis showed that there was a higher percentage of apoptotic cells in the si-NUSAP1-transfected RCC cells, when compared with this percentage in cells treated with si-NC (Fig. 5A; P<0.001). Therefore, our results suggest that downregulation of NUSAP1 expression could induce apoptosis and cell cycle arrest of RCC cells.

We analyzed the RBBS-seq data, and no methylation variation of the promoter region of NUSAP1 (0/34, 0%) was identified by quantitative analysis. However, when analyzing the transcriptome data, we identified that NUSAP1 was overexpressed in 30 of the 34 paired RCCs relative to the adjacent normal controls (Fig. 6). Intriguingly, we identified that CNAs of the NUSAP1 region existed in all of the 61 paired RCCs (61/61, 100%) (Fig. 7 and Table II). Thus, we assumed that the overexpression of NUSAP1 in RCC was mostly due to CNAs. All of the analysis pipelines were referred to in our previous study (18).