Identification of SHCBP1 as a novel downstream target gene of SS18-SSX1 and its functional analysis in progression of synovial sarcoma

The SS18-SSX1 fusion gene has been shown to play important roles in the development of synovial sarcoma (SS), but the underlying molecular mechanisms and its downstream target genes are still not clear. Here SHC SH2-domain binding protein 1 (SHCBP1) was identified and validated to be a novel downstream target gene of SS18-SSX1 by using microarray assay, quantitative real-time (qPCR) and western blot. Expression of SHCBP1 was firstly confirmed in SS cell line and SS tissues. The effects of SHCBP1 overexpression or knockdown on SS cell proliferation and tumorigenicity were then studied by cell proliferation, DNA replication, colony formation, flow cytometric assays, and its in vivo tumorigenesis was determined in the nude mice. Meanwhile, the related signaling pathways of SHCBP1 were also examined in SS cells. The results indicated that SHCBP1 was significantly increased in SS cells and SS tissues compared with adjacent noncancerous tissues. The expression of SHCBP1 was demonstrated to be positively correlated with the SS18-SSX1 level. Overexpression and ablation of SHCBP1 promoted and inhibited, respectively, the proliferation and tumorigenicity of SS cells in vitro. SHCBP1 knockdown also significantly inhibited SS cell growth in nude mice, and lowered the MAPK/ERK and PI3K/AKT/mTOR signaling pathways and cyclin D1 expression. Our findings disclose that SHCBP1 is a novel downstream target gene of SS18-SSX1, and demonstrate that the oncogene SS18-SSX1 promotes tumorigenesis by increasing the expression of SHCBP1, which normally acts as a tumor promoting factor.


INTRODUCTION
As an aggressive tumor of soft tissue, synovial sarcoma (SS) represents approximately ten percent in total soft tissue sarcomas, which affect predominantly children and young adults [1]. Monophasic and biphasic forms are the two major histological types of SS. Despite recent advances in therapies, the 5-year survival rate for SS remains only 36% and less than 10% for patients with metastasis [2,3].
Reciprocal t(X; 18) translocation that results in formation of a fusion protein product SS18-SSX (1-4) is a characterization of SS. This chromosomal translocation usually originates from the fusion of the SS18 gene that locates on chromosome 18 p11 to the SSX1 or SSX2, or occasionally the SSX4 gene that locates on chromosome Xq11 [4,5]. This SS18-SSX fusion is specifically expressed in more than 95% of cases [6]. Currently, molecular detection the transcripts of the SS18-SSX fusion represents the most specific and sensitive diagnostic method for SS [7,8].

Research Paper
Oncotarget 66823 www.impactjournals.com/oncotarget Although we knew little about the function of the SS18-SSX fusion, it was confirmed to be responsible for cell growth of the SS [9,10]. And the type of SS18-SSX fusion transcript was also correlated with the clinical behavior of SS [11,12]. Tumors with SS18-SSX1 fusion transcript present worse prognosis than those with SS18-SSX2 [12,13]. Our previous preliminary study suggested that the growth of SS cells could be significantly inhibited after SS18-SSX1 gene knockdown [14]. Takenaka S et al. also reported similar findings [15]. However, the molecular mechanisms underlying the coordinated regulation of SS18-SSX1 downstream target genes are largely unknown. To address this, the global gene expression patterns in SS cells after silencing SS18-SSX1 gene by RNAi were specially analyzed and compared by DNA microarray analysis. The comprehensive gene expression profile after SS18-SSX1 depletion revealed total 833 genes (including 510 upregulated and 323 downregulated genes) as possible mediators of SS18-SSX1 tumorigenic effects. Among them, the SHCBP1 gene was found to be a new downstream target of SS18-SSX1. As an evolutionarily conserved and ubiquitously expressed protein, SHCBP1 couples activated growth factor receptorsrelated signaling pathways [16], and plays important roles in cell growth and proliferation, differentiation, early embryonic survival and development, growth factor signaling pathway, and especially carcinogenesis [17][18][19][20]. SHCBP1, a new signaling pathway downstream of SHC adaptor proteins, is frequently found to be upregulated in several human malignancies including breast cancer, hepatocellular carcinoma, and certain leukemia/lymphoma [19][20][21][22][23]. Considering the relationship between SHCBP1 and SS18-SSX1, it is of great interest to investigate SHCBP1 expression and its biological function in SS.
For the first time, we confirmed the expression of SHCBP1 in SS cell line and SS specimens, and then the effects of SHCBP1 overexpression or knockdown on cell proliferation and tumorigenicity were further assessed in both vitro and vivo. SHCBP1 expression was found remarkably increased in SS cells and SS tissues. Furthermore, we showed that SHCBP1 can promote proliferation and tumorigenicity of SS cells in both vitro and vivo. Moreover, activation of the MAPK/ERK and PI3K/AKT/mTOR signaling pathways was demonstrated to be mechanistically related to the biologic behavior of SHCBP1. Our findings disclose that SHCBP1 is a novel downstream target gene of SS18-SSX1, and demonstrate that the oncogene SS18-SSX1 promotes tumorigenesis by enhancing the levels of SHCBP1, that normally serves as a cancer promoting factor.
To assess whether the expressions of 20 selected genes from the above results are important for the proliferation or survival of SS cells, we first constructed 20 lentivirus-mediated siRNAs (Table 1) to knock down the expression of the above 20 genes in the HS-SY-II cells. GFP-expressing cells were calculated for five continuous days by high content screening (HCS) assay after infection with the lentivirus containing the above mentioned 20 genes siRNAs or NC-siRNA. As shown in Figure 1B, we observed HS-SY-II cells with GFP in 5 days and assessed the cell proliferation at 5th day with fold changes of the targeted siRNA-treated groups relative to the negative control group. Those genes whose fold change was over 1.9 and the p-value was lower than 0.001 were considered to be differently expressed. Our results indicated that the growth of cells was significantly inhibited by interfering SHCBP1 (2.14-fold change), NID2 (2.02-fold change) and HOXC11 (1.95-fold change) (p < 0.001), respectively ( Figure 1B). Among the 20 downstream target genes of SS18-SSX1, SHCBP1 was identified to be one of the most significant.
To determine whether the expression of these genes was indeed decreased by SS18-SSX1-siRNA, gene expression was assayed by qPCR. We found the expression of SHCBP1, NID2 and HOXC11 was decreased in SS18-SSX1-siRNA cells ( Figure 1C). Similar results were obtained when we performed immunoblotting for these proteins ( Figure 1D).

SHCBP1 was overexpressed in SS
We first assessed the SHCBP1 gene expression in eight matched SS tissues and adjacent noncancerous tissues using qPCR, western blot analysis and immunohistochemistry (IHC). The results revealed that, when comparing with the adjacent noncancerous tissues, the relative mRNA and protein expression levels of SHCBP1 were markedly increased in SS tissues (Figure 2A and 2D). Furthermore, expression of SHCBP1 protein was also detected in eight matched SS tissues and adjacent noncancerous tissues by IHC ( Figure 2B). IHC analysis showed that the adjacent noncancerous tissues showed low levels of SHCBP1 staining, in contrast to SS, which exhibited strong SHCBP1 staining ( Figure 2B). The staining results showed that SHCBP1 protein is mainly located in the cytoplasm in SS cells ( Figure 2B). Moreover, we further confirmed the gene and protein expression of SHCBP1 in HS-SY-II cell line by qPCR (the average Ct value of GAPDH and SHCBP1 is 14.99 and 24.24, respectively) and immunocytochemistry (ICC) ( Figure 2C), respectively.

The impact of overexpression or knockdown of SHCBP1 on SS cell growth at an in vitro level
To further determine whether SHCBP1 affects the proliferation, HS-SY-II cells stably overexpressing SHCBP1 were established. The transfection efficiency was confirmed by qPCR (Supplementary Figure S2B) and western blotting (Supplementary Figure S2D). Then we performed in vitro MTT and colony formation assays. As shown in Figure 3B, the proliferation rate was significantly increased in SHCBP1-overexpressing HS-SY-II cells, as compared with control cells. These results were further confirmed by colony formation assay, and as shown in Figure 3D, SHCBP1-overexpressing cells displayed obviously more numerous and larger colonies compared with control cells.
To determine how SHCBP1 promotes cell viability, SHCBP1 expression was silenced using lentivirusmediated RNA interference technology. The transfection efficacy of the lentiviral vectors was viewed by checking GFP expression under a fluorescence microscope 72 h  Figure S2E). The promoting effects of SHCBP1 on proliferation of SS cells were assessed by HCS, MTT and colony formation assays. As shown in Figure 3A and 3C, compared with NC-siRNA lentivirus infected cells, the growth of SHCBP1-siRNA lentivirus infected cells was significantly suppressed. On day 5, OD 490 of SHCBP1-siRNA lentivirus infected cell was only 0.309 ± 0.02, while that of NC-siRNA lentivirus infected cells was 0.574 ± 0.052 ( Figure  3C). We then analyzed the effect of SHCBP1 on the clonogenicity of SS cells. As shown in Figure 3E, the size and the number of colonies in the SHCBP1-siRNA lentivirus infected cells were significantly reduced compared to NC-siRNA lentivirus infected cells. Collectively, our results suggest that SHCBP1 promotes the proliferation and tumorigenicity of SS cells in vitro.

SHCBP1 promotes the transition from G1 to S phase in SS cells
To elucidate the mechanism of the promoting effects of SHCBP1 on the proliferation capacity of SS cells, the BrdU incorporation and flow cytometric assays were conducted. As shown in Figure 4A, overexpression of SHCBP1 in HS-SY-II cells markedly increased the percentage of BrdU incorporated cells, in contrast, knockdown of SHCBP1 significantly decreased that ( Figure 4B). Accordingly, flow cytometry assay indicated the percentage of S-phase cells was significantly increased after overexpression of SHCBP1 and the percentage of G1/G0 phase cells was reduced ( Figure 4C), whereas knockdown of SHCBP1 led to opposite results ( Figure 4D). Taken together, the above results suggest SHCBP1 contributes to the transition from G1 to S phase in SS cells.

Silencing of SHCBP1 induced apoptosis of HS-SY-II cells
Annexin V-APC staining by FACS on HS-SY-II cells following lentivirus infection was further utilized to confirm the influence of SHCBP1 on cell apoptosis. Figure 6B showed the apoptotic rate in SHCBP1-siRNA lentivirus infected cells was significantly higher than that of NC-siRNA lentivirus infected cells (37.99 ± 0.99% and 9.33 ± 0.46%, respectively), suggesting that SHCBP1 knockdown promoted apoptosis of the SS cells. In the following, the effect of SHCBP1 overexpression on apoptosis of SS cells was also determined by flow cytometry assay. As expected, no significant difference in cell apoptosis was identified between the SHCBP1overexpressing and control cells (p > 0.05; Figure 6A). Altogether, SHCBP1 might play an oncogenic role in SS again.

Silencing of SHCBP1 suppressed SS cell growth in vivo
To confirm whether silencing of SHCBP1 could inhibit the growth of SS cells in vivo, a subcutaneous human SS nude mouse xenograft model was established. The mouse group infected with SHCBP1-siRNA lentivirus had a lower proliferation rate, and formed evidently smaller tumors than the NC-siRNA lentivirus group, as shown in Figure 5A and 5B. The tumor size at the time of death in the SHCBP1-siRNA lentivirus group was 413 ± 69.6 mm 3 , which was significantly smaller than in the NC-siRNA group (1165 ± 160.3 mm 3 ). In addition, a western blotting assay was performed to determine whether SHCBP1 expression was suppressed by SHCBP1-siRNA in vivo, and we found that the expression of SHCBP1 was largely inhibited by SHCBP1-siRNA ( Figure 5C and 5D). These data suggest that SHCBP1-siRNA reduces tumor volume and growth rate of SS cells in vivo.
Silencing of SHCBP1 effectively promotes the inactivation of MAPK/ERK and PI3K/AKT/ mTOR signaling pathways and reduces the expression of cyclin D1 in SS cells SHC1, SHC (Src homology 2 domain containing) transforming protein 1, is important for normal and oncogenic signaling by epidermal growth factor receptor (EGFR) family receptor tyrosine kinases [24]. Phosphorylation of SHC1 by EGFR will stimulate the Mitogen-activated protein kinase (MAPK/ERK) and PI3K/AKT/mTOR signaling pathways [17], and control the growth of SS [25]. SHCBP1 binds with SHC1, which may be required for activation of MAPK/ERK and PI3K/ AKT/mTOR signaling pathways. Therefore, the effects of silencing of SHCBP1 on MAPK/ERK and PI3K/AKT/ mTOR signaling pathways in SS cells were assessed by western blotting. As shown in Figure 6C, the expression levels of the total MEK, ERK and AKT in HS-SY-II Oncotarget 66827 www.impactjournals.com/oncotarget cells were unchanged, but the phosphorylations of these proteins were significantly decreased after silencing of SHCBP1. These data indicate that SHCBP1 silencing effectively promotes the inactivation of MAPK/ERK and PI3K/AKT/mTOR signaling pathways.
As an important regulator of cell cycle progression, cyclin D1's deregulation is linked to the pathogenesis of SS [9,14]. To determine whether SHCBP1 silencing influences cyclin D1 expression, we detected the cyclin D1 level after SHCBP1 silencing by western blot analysis. As indicated in Figure 6C, knocking down SHCBP1 could significantly reduce the expression of cyclin D1.

DISCUSSION
Recently accumulating data showed that SS18-SSX plays critical roles in oncogenesis and development of SS [26,27]. Our previous study indicated that inhibition of SS18-SSX by siRNA could prevent the proliferation of SS cells [14]. However, the downstream molecular mechanisms involved in SS18-SSX1-mediated oncogenesis are still poorly understood.
To identify new downstream targets of SS18-SSX1, a cancer-related gene expression panel with samples from SS18-SSX1-siRNA SS cells and control cells was carried out in our research. Twenty target genes including the following: SHCBP1, NID2, HOXC11, MRPL35, CCBE1, CEBPG, ALDH1A3, HAUS6, FAM54A, HOXC10, DLX1, ZADH2, CARD8, RYBP, DLX2, SERTAD4, CENPN, BCL2, E2F8, and DCP2 were identified. To investigate which of them is associated with cellular proliferation. The effect of 20 target genes knockdown on cell proliferation was firstly assessed by HCS assay. Three genes, including SHCBP1, NID2, and HOXC11, were identified as SS18-SSX1 downstream target genes. Individual siRNA-mediated knockdown of them was sufficient to inhibit the proliferation of SS cells. Among them, SHCBP1 which demonstrated the most significant fold changes (2.14-fold change) was identified as one of the most significantly target gene, whose expression was upregulated by SS18-SSX1 overexpression, and downregulated by SS18-SSX1 inhibition. To our knowledge, this is the first report that SHCBP1 is a downstream target gene of SS18-SSX1.
Three overlapping proteins encoded by the SHC gene share a common carboxy terminal SH2 domain [28][29][30], which plays an important role in the signal transduction pathways. Studies have also shown that SHC proteins mediate cell proliferation as well as cell survival via tyrosine phosphorylation signal pathway [31,32]. The direct interaction of SHCBP1 with the SH2 domain of SHC is independent of tyrosine phosphorylation. SHCBP1, located on chromosome 16q11.2, also modulated the fibroblast growth factor signaling pathway in neural progenitor cells [33]. Studies have demonstrated that SHCBP1 is correlated with cell growth and proliferation, differentiation, early embryonic survival and development, growth factor signaling Oncotarget 66828 www.impactjournals.com/oncotarget pathway, and especially carcinogenesis [17][18][19][20]. In breast cancer, SHCBP1 has been shown highly expressed in tumor samples and is correlated with metastatic potential, advanced stage, and poor prognosis [19]. Additionally, SHCBP1 was demonstrated significantly overexpressed in human hepatocellular carcinomas (HCC), and the proliferation and colony formation of HCC cells were markedly reduced after SHCBP1 inhibition [20]. Although SHCBP1 is indicated to be overexpressed in several kind of cancers, it has not been associated with SS. Here we found the expression of SHCBP1 is higher in SS cell line HS-SY-II. SHCBP1 gene and protein are significantly elevated in SS tissues compared with the adjacent noncancerous tissues. Meanwhile, in agreement with previous studies [17,20], we found that staining of SHCBP1 is also mainly localized in the cytoplasm. These results indicated that SHCBP1 overexpression may take part in the oncogenic process of SS. The relationship between SHCBP1 expression and the clinical features of SS is definitely worth further exploration.
Functional investigations were mainly focused on the impact of overexpression or knockdown of SHCBP1 on the growth and apoptosis of SS cells. The results disclosed that cell proliferation, colony formation and DNA replication could be promoted in SHCBP1 overexpressed SS cells. In contrast, targeting SHCBP1 produced the opposite effects. Moreover, silencing of SHCBP1 led to remarkable apoptosis of HS-SY-II cells. Most importantly, the in vivo data demonstrated that silencing of SHCBP1 could significantly prohibit xenograft tumor growth in mouse model. These findings indicate that SHCBP1 is involved in carcinogenesis of SS, and thus it may be considered as one of the novel potential therapeutic targets in SS treatment. These findings reported here are consistent with a previous report in HCC cells [20].
Flow cytometry also showed that overexpression of SHCBP1 accelerated the G1-S-phase transition, whereas silencing of SHCBP1 induced G1-S-phase arrest. In addition, we found that silencing of SHCBP1 effectively inhibited the expression of cyclin D1 in SS cells. Thus, we showed that the mechanism of SHCBP1-mediated proliferation was linked to alternations of the expression of cyclin D1. Taken together, these results reveal that Flow cytometric analysis of cell cycle in SHCBP1-overexpressing (C) and SHCBP1-silenced (D) cells. All values are mean ± SD of 3 independent experiments; *p < 0.05, **p < 0.01. www.impactjournals.com/oncotarget SHCBP1 involves in cell cycle regulation of SS and is critical for expression of cyclin D1. These findings are in accordance with previous reports [20,34].
To investigate the underlying mechanisms by which SHCBP1 promotes proliferation of SS cells, we explored the effects of SHCBP1 silencing on the activity of MAPK/ ERK and PI3K/AKT/mTOR signaling pathways which are important in the pathogenesis of various tumors [35][36][37] including SS [38,39]. We speculated that SHCBP1 may cross talk with the MAPK/ERK and PI3K/AKT/mTOR  Oncotarget 66830 www.impactjournals.com/oncotarget signaling pathways in SS progression. The MAPK/ERK and PI3K/AKT/mTOR signaling pathways may be first stimulated by the elevated expression of SHCBP1, which leads to increased tumor growth potential. Our present data showed that the MAPK/ERK and PI3K/AKT/ mTOR signaling pathways were inactivated by silencing of SHCBP1 in HS-SY-II cells via reducing the levels of phosphorylated MEK, ERK and AKT. Therefore, the result explains, at least in part, why SHCBP1 silencing inhibited the cell proliferation and induced the apoptosis in HS-SY-II cells, which is consistent with these reports [20,33]. Molecular mechanism underlying SHCBP1 inactivating the MAPK/ERK and PI3K/AKT/mTOR signaling pathways in SS cells is under investigation in our laboratory currently.
Collectively, SHCBP1 was identified as a novel downstream target gene of SS18-SSX1 for the first time. SS18-SSX1 functions as an oncoprotein by promoting tumorigenesis via increasing the expression of SHCBP1 in SS, and then activating the MAPK/ERK and PI3K/ AKT/mTOR signaling pathways. The precise underlying mechanism through which SS18-SSX1 increases the expression of SHCBP1 is still underway in our lab.

Ethics statement
Investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the institutional review board of the Second Hospital of Shandong University. For tissue specimens, patients were informed that the resected specimens were stored by the hospital and potentially used for scientific research, and that their privacy would be maintained. Informed consent has been obtained. For animal research, all experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by our institutional ethical guidelines for animal experiments.

Tissue specimens and cell lines
Eight matched SS and adjacent noncancerous tissues were collected and were fixed with 10% neutral-buffered formalin and embedded in paraffin; 4 μm-thick sections were prepared for IHC. All cases of SS and adjacent noncancerous tissues were diagnosed clinically and pathologically.

Construction of plasmids and transfection
The SS18-SSX1 expression construct or SHCBP1 expression construct were generated by subcloning PCR-amplified full-length human SS18-SSX1 cDNA or full-length human SHCBP1 cDNA into the pcDNA.1(+) plasmid (Genechem), respectively. Cells were then transduced with the recombinant plasmid carrying the human SS18-SSX1 or SHCBP1 gene using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer's advised procedure. The effects of SS18-SSX1 overexpression on expression of SHCBP1 in Saos-2 cells or cultured HS-SY-II cells were determined by qPCR and western blot assays. Empty plasmid-transfected cells were used as control. Primer sequences for vectors construction are listed in Supplementary Table S1.

Quantitative real-time PCR
Total RNA was purified with by Trizol reagent (Invitrogen, Carlsbad, CA, USA), and reversely transcripted to cDNA with M-MLV reverse transcriptase Oncotarget 66831 www.impactjournals.com/oncotarget kit (Promega, USA) following the manufacturer's instructions. qPCR was performed with the SYBR Green Real-Time PCR assay kit (TAKARA, Otsu, Japan) on an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, Foster City, California, USA). The 20 μl PCR reaction mixture was: 10 μl 2 × SYBR premix ex taq, 0.5 μl each primer (2.5 μM), 1 μl cDNA and 8 μl ddH 2 O. Cycling conditions were as follows: initial denaturation at 95ᵒC for 15 s; denaturation 95°C for 5 s; annealing extension of 60°C for 30 s (a total of 45 cycles). The absorbance values were read at the extension stage. Fold changes in expression were calculated using the 2 -ΔΔCt method [41]. Experiments were performed at least three times. Primer sequences are listed in Supplementary Table S1.

Immunohistochemistry and immunocytochemistry analysis
For IHC, tissue sections (4 μm) were placed on glass slides, heated at 70°C for 30 min, and then deparaffinized with xylene and ethanol. For antigen retrieval, the deparaffinized and rehydrated slides were heated in 10 mM citrate buffer for 20 min in a microwave oven and allowed to cool for 20 min at room temperature. Slides were incubated with 3% H 2 O 2 in methanol for 15 min at room temperature to eliminate endogenous peroxidase activity. Then slides were incubated at 4°C overnight with antibody against the rabbit polyclonal anti-SHCBP1 antibody (1:50, Sigma-Aldrich, St Louis, MO, USA). After incubation with the biotinylated secondary antibody at room temperature for 20 min, the slides were incubated with a streptavidin-peroxidase complex at room temperature for 20 min. IHC staining was developed using 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich) and counterstained with haematoxylin.
For ICC, cells were plated on 2.5 cm coverslips at a density of 5 × 10 5 cells/well in 6-well plates. After 24 h of attaching, coverslips were fixed in 4% paraformaldehyde in PBS for 30 min and incubated with 3% H 2 O 2 in methanol for 10 min. Coverslips were incubated with 10% goat serum at room temperature for 10 min to block nonspecific bindings, and then immunostained with the anti-SHCBP1 antibody with the same protocol as IHC.

Microarray assay
After 72 h infection with recombinant SS18-SSX1-siRNA lentivirus, total RNAs were extracted using the RNeasy Mini Kit (Qiagen, Basel, Switzerland). Then RNA quality was analyzed using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Analysis of gene expression was performed using GeneChip ® PrimeView™ Human Gene Expression Array (Affymetrix, USA; catalog # 901838) which contains probes for 36000 genes as described previously [42]. Labeling and hybridization were performed following Affymetrix protocols. Primary array processing was conducted using the Affymetrix GeneChip ® Command Console ® Software (AGCC, version 1.1) and subsequent analysis was performed using the Affymetrix Expression Console (EC, version 1.1).

High content screening assay
Cell growth was evaluated by counting the viable cell number with Cellomics Array-ScanTM VTI HCS Reader (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. Briefly, HS-SY-II cells at 10 days post-transfection with candidate genes siRNA or NC-siRNA lentivirus were seeded at 2000 cells per well on 96-well plates, and then incubated at 37°C with 5% CO 2 . From the second day, cells with GFP were taken photos and counted each day by Cellomics machine. Cell growth was observed continuously for 5 days, and cell growth curves were drawn.

BrdU incorporation assay
Cells were seeded in triplicate in 96-well plates at a density of 2 × 10 3 cells/well. A 5-bromo-2-deoxyuridine (BrdU) incorporation assay was detected on 1 and 4 days after seeding using the BrdU Cell Proliferation ELISA kit (Roche, USA) as previously described [43]. Briefly, 10 ml of 1/100 diluted BrdU was added to each well. After incubation for 8 h, the medium was carefully aspirated out and FixDenat solution (200 μl/well) was added into each well. Cells were then incubated at room temperature in the dark for 30 min, blocked in 5% bovine serum albumin (BSA) at room temperature in the dark for a further 30 min. Then 100 μl/well diluted Anti-BrdU-POD working solution (1:100) was added into each well, and incubated at room temperature for 90 min. The plates were washed three times, and then 100 μl substrate solution was added into each well. The cells were incubated at room temperature for 5 to 30 min until the solution was a deep blue, 50 μl 10% H 2 SO 4 was added into each well, and the plates were read at 450 nm in an ELISA reader (Biotek Elx800, USA).

Colony formation assay
Cells were plated at 500 cells per well in 6-well plates and continually cultured in DMEM containing 10% FBS for 10 days. Then, the supernatants were discarded and cells were washed twice with PBS, and fixed with methanol for 15 min. The cells were stained with 0.1% crystal violet for 10 min, subsequently, washed with PBS till the plates were clear. The plates were dried at room temperature and the colony numbers were photographed and counted. The experiments were performed in triplicate.

Flow cytometry analysis of the cell cycle and apoptosis
Flow cytometry was used to analyze the effect of SHCBP1 on cell cycle. Cells were cultured in 6-well plates and were harvested by trypsinization and centrifugation at 1200 rmp for 5 min, washed once with ice cold PBS, and fixed in 70% alcohol for 1 h. Then, fixed cells were resuspended in PBS containing RNase (100 μg/ml) on ice, and stained with propidium iodide (PI, 50 μg/ml) (Sigma-Aldrich). Cells were analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA), according to the manufacturer's protocol. For Lentivirusmediated RNAi knockdown of SHCBP1 experiments, cell apoptosis was determined by staining with Annexin V-APC Apoptosis Detection Kit (eBioscience, San Diego, CA, USA) and detected by FACS. For cells with overexpression of SHCBP1, cell apoptosis was assayed by staining with Annexin V-FITC (ebioscience) and PI following manufacturer's instructions and detected by FACS. Briefly, cells were collected and washed with cold PBS. They were then resuspended in 1 ml 1 × staining buffer. Then 5 μl Annexin V-APC and 5 ul PI was added into 100 μl of the above cell suspension (about 1 × 10 6 -1 × 10 7 cells), and incubated for 15 min at room temperature in the dark. After incubation, cells were analyzed by flow cytometer in one hour. All experiments were repeated three times.

SHCBP1 knock-down in a nude mouse tumor model
For the SS xenograft model, 4-week-old BALB/c nude mice (Vitalriver, Beijing, China) were housed in a temperature-controlled, pathogen-free environment and used for experimentation. Medium (200 µl) containing 2 × 10 6 HS-SY-II cells at the end of SHCBP1-siRNA or NC-siRNA lentivirus infection for 48 h was injected subcutaneously into the right flank of nude mice. Tumor length (L), width (W) and diameter were measured every week from week 1 to week 6; tumor volume (mm 3 ) was calculated using the formula L × W 2 /2 [44]. After 6 week, mice were sacrificed, and tumors were harvested.

Statistical analysis
The data shown are presented as the mean ± standard deviation (SD) of three independent experiments. Statistical significance was determined with Student's t test using GraphPad Prism 5 software (GraphPad Software Inc., San Diego CA, USA). P < 0.05 was considered statistically significant.