Nusap1是一种微管结合蛋白,这种蛋白在有丝分裂和癌症中起着至关重要的作用。以往的研究已经论证了NUSAP1 在多种恶性肿瘤中表达是相对升高的. 然而, NUSAP1 在肾细胞癌RCC) 中的生物学作用尚不清楚。在这次研究中,我们首先做了逆转录PCR(RT-PCR) 和western-blot分析来揭示NUSAP1 的表达在肾透明细胞癌(CCRCC)组织标本和此外,癌细胞株中是相对升高的。免疫组化分析显示NUSAP1 明显上调,fuhrman分级(P＜0.001), 肿瘤大小(P=0.016), 临床阶段(P＜0.001) ,远处转移(P=0.023)。此外, NUSAP1的高表达和透明细胞癌患者的总生存时间密切相关(P=0.006). 此外,我们研究肾癌细胞体外的生物学行为, 发现NUSAP1 消耗抑制RCC细胞迁移, 增殖和侵袭, 诱导细胞凋亡,阻滞细胞周期。在我们研究的基础上,NUSAP1被确定为一个潜在的预后指标和肾癌患者的新的治疗靶点。
肾细胞癌(RCC)是最常见的癌症,起源于肾实质, 约占所有成年肾恶性肿瘤的90%(1). 肾细胞癌的主要组织学亚型是肾透明细胞癌(RCC)约占肾细胞癌RCCs的70-80%, followed by papil-lary (10-20%), chromophobe (5%) and collecting duct (1%) subtypes (2). Globally, the incidence of kidney cancer is approximately 270,000 new cases and 116,000 deaths, annu-ally (3). Despite considerable improvements in the diagnosis and treatment of RCCs in recent years, nearly 20-30% of all patients present with metastasis at the time of initial diagnosis, and approximately 20% of RCC patients who undergo nephrec-tomy may suffer recurrence or metastasis of the disease (4).For these patients, the prognosis is extraordinarily poor and median survival is not more than one year (5). Therefore, it is of great necessity to investigate the underlying molecular mechanisms of the tumorigenesis and progression of RCC. Identifying more novel biomarkers is still required to promote early diagnosis, targeted therapy and prognosis evaluation.
Nucleolar and spindle-associated protein 1 (NUSAP1) is a recently identified protein with a molecular weight of 55 kDa that plays a crucial role in spindle microtubule organiza-tion (6). NUSAP1 exhibits a cell cycle-dependent localization and is selectively expressed in proliferating cells. Its mRNA and protein expression levels reach a peak at the transition of G2 to mitosis and then rapidly decline after cell division (7). The depletion and overexpression of NUSAP1 in cells result in abnormal chromosome segregation, aberrant spindle assembly,defective cytokinesis, G2/M arrest and microtubule bundling, respectively (7,8).
In addition to playing an essential role in mitosis, NUSAP1 has recently attracted broad attention for its involvement in cancers. Previous studies have shown that elevated expression of NUSAP1 is correlated with malignancies, including pancre-atic adenocarcinoma, melanoma, glioblastoma, hepatocellular carcinoma, and prostate cancer (9-13). NUSAP1 has also been associated with the aggressiveness of meningioma (11), high risk and poor outcome in breast cancers (14,15). In contrast, a study of childhood ALL revealed that the expression level of NUSAP1 was decreased in
patients presenting with a poor prognosis (16). These findings suggest a critical role ofNUSAP1 in the initiation and progression of human cancers. To date, rare research has been conducted on the expres-sion and clinical significance of NUSAP1 in RCCs. In the present study, the expression of NUSAP1 was firstly evaluated in both ccRCC tissues and RCC cell lines by reverse transcription‑polymerase chain reaction (RT- PCR), western blot (WB) assay and immunohistochemistry (IHC) assays. Then, we immunohistochemically analyzed its expression and correlation with clinicopathological characteristics of the ccRCC patients. The present study demonstrated that there was a close relationship between NUSAP1 expression and the prognosis of ccRCC patients. Furthermore, we investigated the biological behavior of human RCC cells after downregulation of NUSAP1 expression in vitro, which suggested that over-expression of NUSAP1 was associated with cell migration,proliferation and invasion of RCC.
Patients and tissue specimens. 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 AnhuiMedical University (Anhui, China) between March 2010 andAugust 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 verifica-tion 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 SecondAffiliated Hospital of Anhui Medical University and ZhujiangHospital of Southern Medical University.
Cell culture and transfection. Four human RCC cell lines(786 -O, A704, ACHN and A498) and an immortalized normal human proximal tubule epithelial cell line HK- 2 werepurchased 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 modi-fied 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'-GCACCAAGA
AGCUGAGAAUTTAUUCUCAGCUUCUUGGUGCTT-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.
Real -time quantitative RT-PCR. RNA isolation from the RCC cells and tissue samples was performed using TRIzol reagent (Invitrogen) in accordance with the manufacturer's instruc-tions. 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'-GCC CAATACGACCAAATCAGAG-3' (reverse) . The 2- Ct method (17) for calculating the relative levels of NUSAP1 mRNA was applied in the present study, and all experiments were accomplished in triplicate.
WB assay. 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 trans-ferred 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 poly-clonal 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 and staining analysis.IHC was performed to determine the protein level of NUSAP1 expression in the ccRCC andTable I. Relationship between NUSAP1 expression and clinicopathological characteristics of the ccRCC patients.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 multipli-cation 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 deter-mined, and scores of 0-4 were defined as low expression while scores >4 were considered as high expression. The immu-nohistochemical staining was evaluated by two independent observers blinded to the clinical outcomes.
Scratch migration assay. 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 5x105 cells/well and cultured overnight. Then, the monolayer cells were scratched with a sterile 10-µl pipette tip, washed thrice with phos-phate‑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.
Cell invasion assay. 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 (1x105 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 solu-tion and stained with 0.1% crystal violet. The invaded cells were observed under a light microscope and counted in five randomly chosen fields.
Cell proliferation assay.To determine cell proliferation, 786- O and A704 cells transfected with siRNA were plated in 96-well plates at a density of 3x103 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. 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.
Statistical analysis. 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 char-acteristics 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.
Factors influencing the overexpression of NUSAP1. 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.
Increased expression of NUSAP1 in ccRCC tissues and RCC cell lines. 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 linesincluding HK-2, A704, 786-O, ACHN and A498. The data revealed that NUSAP1 mRNA and protein levels were statisti-cally 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).,
Immunohistochemical analysis of NUSAP1 expression in ccRCC tissues and its correlation with clinicopathological characteristics. 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 expres-sion (Fig. 2B -D). The adjacent normal tissues exhibited either no or weak staining of NUSAP1 (Fig. 2A). The relationships between NUSAP1 expression and clinicopathological char-acteristics 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 metas-tasis (P>0.05).
NUSAP1 expression predicts the prognosis of ccRCC patients.The association between NUSAP1 expression and the prog-nosis 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 signifi-cantly longer overall survival (OS) time than those with high NUSAP1 expression
NUSAP1 downregulation suppresses the growth and aggressiveness of RCC cells in vitro. 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.
NUSAP1 downregulation induces apoptosis and cell cycle arrest ofRCC cells. 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.
Copy number alterations (CNAs) influence the expression of NUSAP1. 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 over- expressed 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).
Tremendous advances have been made over the last decade in ourunderstanding ofthe genetic basis and progress ofa targeted therapeutic armamentarium for RCC. As is known to all, various conventional agents that target the vascular endothelial growth factor (VEgF) pathway or mammalian target of rapamycin (mTOR) may benefit renal cell carcinoma (RCC) patients (19). However, despite more options and advances in treatment, most patients with advanced disease still exhibit a markedly poor outcome, and we have not yet elucidated the mechanism behind its development (20). Therefore, continued research to identify more genes which drive the initiation and progression of RCC is clearly warranted.
The function of NUSAP1 has been investigated in several recent studies, which demonstrate its crucial role in cell mitosis and tumorigenesis (6). Although, the overexpression of NUSAP1 at the mRNA level has been found in several types of cancers, there is limited research concentrating on the expression and clinical significance of NUSAP1 in RCC. Our previous transcriptome sequencing data indicated that the expression level of NUSAP1 was significantly higher in RCC tissues than that in matched adjacent normal tissues. Methylation of high-density CpG regions known as CpG islands (CGIs) has been widely described as a mechanism associated with gene expression regulation (21), whereas no methylation change of NUSAP1 (0/34, 0%) was identified in the present study. In addition, in a previous study using both transcriptional profile data and CNA, they identified that genes with differential expression may be caused by CNAs (22). To our surprise, CNAs of NUSAP1 existed in all of the 61 paired RCCs, which suggest that CNAs could be the primary cause for the overexpression of NUSAP1 in RCC.
Next, we determined the mRNA and protein levels of NUSAP1 expression in 38 pairs of ccRCC and matched normal tissues and five cell lines (including four RCC cell lines and the Hk-2 cell line). The qRT-PCR and western blot analyses showed that NUSAP1 was relatively overexpressed in the RCC tissues, as well as in three RCC cell lines (A704, 786-O and ACHN). Our data demonstrated that an elevated expression of NUSAP1 at the transcription and translation levels may closely correlate with the tumorigenesis of RCC, and NUSAP1 may be a potential indicator for RCC patients. In addition, immunohistochemical analysis was performed to determine the association between NUSAP1 expression and clinicopathological characteristics of the ccRCC patients. The results showed that NUSAP1 expression was significantly associated with the level of malignancy of ccRCC. Namely, upregulation of NUSAP1 was associated with aggressive features of ccRCC, such as Fuhrman grade, tumor size, clinical stage and metastasis. In various recent studies, NUSAP1 expression was found to be associated with the poor prognosis of patients with melanoma (10), and NUSAP1 was also identified as part of a malignancy‑risk gene signature for breast cancer (14). Consistent with these studies, our survival analysis similarly revealed that overexpression of NUSAP1 was obviously correlated with a shorter overall survival time of ccRCC patients. These results suggest that NUSAP1 could play an important role in the progression of RCC.
In attempting to determine the biological role of NUSAP1 in RCC, we performed a series of functional experiments after downregulation of NUSAP1 expression in vitro. In the si-NUSAP1-transfected cell lines, the migration, prolif- eration and invasion of RCC cells were significantly inhibited compared with the control groups. A study by Gordon et al (23) demonstrated that knockdown of NUSAP1 by siRNA reduced the proliferation and invasion in prostate cancer cell lines, suggested that NUSAP1 could influence tumor cell growth and aggressiveness. Furthermore, Nie et al (24) found that NUSAP1 depletion blocked the migration
of neural crest cells in zebrafish embryos, which indicates that its overexpression may promote cancer cell migration. As we mentioned earlier, NUSAP1 is a microtubule-binding protein that is selectively expressed in proliferating cells. Its depletion causes G2/M arrest in cell cycle progression. In the present study, we also discovered that downregulation ofNUSAP1 induced apoptosis and G2/M arrest of RCC cells. In agreement with our results,knockdown of NUSAP1 by siRNA in HeLa cells also led to mitotic arrest and abnormal chromosome condensation (7). In addition, Vanden Bosch et al (8) found the rapid disintegration and small cellular fragments were present in reduced growth of NUSAP1-null mice embryos, and presumed that lack of NUSAP1 may result in apoptosis. Based on its crucial role in mitosis, and NUSAP1 depletion suppressed cell growth and induced apoptosis, we thus assumed that it could represent a novel therapeutic target for RCC patients.
In conclusion, we demonstrated that NUSAP1 overexpres- sion was closely related to the clinicopathological features of RCC and predicted an unfavorable prognosis for RCC patients. Downregulation of NUSAP1 induced cell apoptosis and inhibited cell migration, proliferation and invasion. NUSAP1 may thus serve as a potential prognostic indicator and a novel therapeutic target for RCC patients. However, our research is only a preliminary discussion on the expression and biological function of NUSAP1 in RCC. Further studies are necessary to confirm these findings and uncover the mechanisms of these processes.
The present study was supported by grants from the National Natural Science Foundation of China (nos. 81301740 and 81402336).
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