Overexpressed DEPDC1B contributes to the progression of hepatocellular carcinoma by CDK1

Background: Hepatocellular carcinoma (HCC) is the main type of primary liver cancer and shows a heavy burden worldwide. Its recurrence and mortality rate are still uncontrolled by the usage of present treatments. More attention has been focused on exploring specific genes that play important roles in HCC procession, and the function of DEP domain containing 1B (DEPDC1B) in HCC has not been researched. Methods: Immunohistochemical staining was used to detect the expression level of DEPDC1B in tumor tissues and adjacent normal tissues. After DEPDC1B and CDK1 knockdown in cell lines HEP3B2.1-7 and SK-HEP-1, MTT assay and colony formation assay was used to detect cell growth, flow cytometry assay was used to investigate cell apoptosis and cell cycle, wound-healing assay and Transwell assay were used to examine the tumor cell migration. Moreover, a xenograft model was constructed to research functions of DEPDC1B in tumor growth in vivo. Results: The results show that DEPDC1B knockdown inhibit the progression of HCC, through inhibiting cell proliferation, migration, colony formation, leading to G2 phase arrest, and promoting cell apoptosis in vitro, and CDK1 was selected for further mechanic research according to the results of Human GeneChip prime view. The results of recovery experiment displayed that the functions of DEPDC1B on HCC progression were mediated by CDK1. DEPDC1B knockdown can also inhibit tumor growth in vivo. Conclusions: The study confirmed that DEPDC1B knockdown restrains the tumor growth in vitro and vivo, and it can interact with CDK1 and rescued by CDK1. The study suggested that DEPDC1B was as a potential therapeutic target involved in HCC growth and progression.

AGING satisfy well the requirement of extending the life expectancy of them. Even if the surgical resection which is recommended in early-stage patients, the five year survival is 50%, the recurrence rate accounts for 70% [6]. For the purpose of overcoming the dilemma, more attention has been focused on the mechanism of HCC to find specific genes that play important roles in HCC procession [3,7,8].
DEPDC1B (Homo sapiens DEP domain containing 1B) is a human protein which is encoded by DEPDC1B gene located on chromosome 5 (5q12.1), and widely expressed throughout human tissue. However, the functions of DEPDC1B have not been investigated precisely. Study showed that DEPDC1B can accumulate in G2 phase to cooperate with de-adhesion events and cell-cycle progression at mitotic entry by inhibiting RhoA signaling, and it also can regulate cell proliferation in zebrafish embryogenesis [9]. Although the precise function of DEPDC1B is uncertain, it is reported that DEPDC1B knockdown can inhibit tumor growth in malignant melanoma and some other types of malignant tumors [10,11]. However, the functions of DEPDC1B in HCC have not been investigated. Therefore, the study aimed to discover the functions and general mechanism of DEPDC1B in HCC to instruct the therapy of HCC. If the functions of DEPEDC1B is studied in depth, the treatment methods and effects of HCC will be expanded and enhanced. To some degree, through further research, the study will solve the current problems in the treatment of HCC.

DEPDC1B was overexpressed in tumor tissues and related to advanced stage
The general information, tumor stage and T stage of these cases were collected in Table 1. The results of immunohistochemistry revealed that DEPDC1B was overexpressed in HCC tissues compared with paracarcinoma tissues ( Figure 1 and Table 2), and the expression level of DEPDC1B was significantly associated to the pathological stage of tumors and T stage (P <0. 05) ( Table 3), suggesting that DEPDC1B was overexpressed in tumor tissues and related to advanced tumor stage. The results of TCGA about lung adenocarcinoma showed that the expression level of DEPDC1B was significantly correlated with the pathologic T, pathologic N, tumor stage, and gender ( Table 4).

Establishment of DEPDC1B knockdown cell lines
The results of qRT-PCR showed that the expression level of DEPDC1B in HEP3B2.1-7, SK-HEP-1 and huh-7 cell lines was significantly higher, compared to HCCLM3 cell line (P<0.05) (Figure 2A). The results indicated that HEP3B2.1-7 and SK-HEP-1 cell lines were more appropriate to research the functions of DEPDC1B. For the purpose of studying the effects of DEPDC1B knockdown, LV-shDEPDC1B and LV-shCtrl were transfected into HEP3B2.1-7 and SK-HEP-1 cell lines. After infected for 72 h, the efficiencies of infection in all group were more than 80% under fluorescence microscope in the way of detecting the fluorescence of cells ( Figure 2B). The results of qRT-PCR displayed that the knockdown efficiencies of DEPDC1B of HEP3B2.1-7 and SK-HEP-1 cell lines in shDEPDC1B group were 71.35% (P<0.001) and 74.1% (P<0.001), compared with shCtrl group, respectively ( Figure 2C). The expression level of DEPDC1B in shDEPDC1B group were down-regulated obviously compared with DEPDC1B in shCtrl group by western blot ( Figure 2D). DEPDC1B knockdown cell models were constructed successfully according to the results of qRT-PCR and western blot.

Knockdown of DEPDC1B inhibited HCC cell proliferation, migration and promoted HCC cell apoptosis and led to G2 phase arrest
MTT assay was performed to study the effects of DEPDC1B knockdown on HCC cell proliferation, and the results showed that the proliferation in shDEPDC1B group was slower than that in shCtrl group (P<0.001) ( Figure 3A). The results of flow cytometry assay exposed that the percentage of apoptotic cells in shDEPDC1B significantly increased compared with shCtrl (P<0.001) ( Figure 3B). Further study in cell cycle showed the cells in G2 phase had significant increase in shDEPDC1B group compared with shCtrl group after cultured for 5 days ( Figure 3C). Transwell assay and wound-healing assay were constructed to investigate the migration. Transwell assay showed that, compared to shCtrl group, the migration ability of HEP3B2.1-7 cells (48 h) and SK-HEP-1 cells (24 h) in shDEPDC1B were significantly decreased (P<0.001) ( Figure 3D). The results of wound-healing assay showed that the average migration rate of HEP3B2.1-7 cells (48 h) and SK-HEP-1 cells (24 h) in shDEPDC1B group were decreased by 68% (P<0.001) and 47% (P<0.001), compared with shCtrl group ( Figure 3E). Collectively, knockdown of DEPDC1B suppressed HCC progression by inhibiting cell proliferation and migration, promoting HCC cell apoptosis and leading to G2 phase arrest.

Exploration of potential mechanisms
Human apoptosis antibody array was used to search for the affected proteins in the SK-HEP-1 cells infected  I  6  4  2  II  78  56  22  III  94  44  50  T stage  0.003  T1  6  4  2  T2  80  56  24  T3  86  42  44  T4  6  2  4 with LV-shDEPDC1B and LV-shCtrl, because the specific function mechanism of DEPDC1B knockdown in cell growth was unclear. The expression of Caspase 3, p21, and IGFBP-6 was significantly up-regulated in shDEPDC1B compared with shCtrl (P<0. 05). The expression of Bcl-2, Bcl-w, HSP27, HSP70, Livin, sTNF-R2, TNF-α, and TNF-β was significantly downregulated in shDEPDC1B, compared to shCtrl (P <0.05) ( Figure 4A-4C). The results of western blot in SK-HEP-1 cells showed that the expression of P-Akt, CDK6, Cyclin D1, and PIK3CA was down-regulated in shDEPDC1B group, compared to shCtrl group ( Figure 4D).     Figure 5A, 5B). IPA pathway enrichment analysis of AGING DEGs showed that DEGs were mostly enriched in Salvage Pathways of Pyrimidine Ribonucleotides, NER Pathway, Estrogen-mediated S-phase Entry, and Cyclins and Cell Cycle Regulation pathway and the three pathways were significantly inhibited, which indicated that DEGs played important suppressive roles in these pathways ( Figure 5C). The IPA analysis about disease and function showed that cancer was one of the mostly enriched diseases ( Figure 5D), which suggested that the DEGs found in the experiment are mostly relevant to cancers. DEPDC1B could affect CCNA1, JUN, CCNB2, CCNE2, CDK1, CDK6, etc in Cyclins and Cell Cycle Regulation, and Wnt/β-catenin Signaling pathway through EED and FANCD2 ( Figure 5E). Further qRT-PCR showed that the expression levels of CCNA1, JUN, CCNB2, MYC, CDK1, and CDK6, were down-regulated in shDEPDC1B group, compared to shCtrl group (P<0.001) ( Figure 5F). The results of western blot showed that the expression of CCNE2, CDK1, CDK6, and E2F1 was significantly down-regulated in shDEPDC1B group, compared to shCtrl group ( Figure  5G). Collectively, the DEPDC1B knockdown suppressed the expression of CCNE2, CDK1, CDK6, and E2F1 at mRNA or protein level through Cyclins and Cell Cycle Regulation, and Wnt/β-catenin Signaling pathway. Among the several selected DEGs, CDK1 was found to be overexpressed in tumor tissues, compared with paracarcinoma tissues by IHC analysis ( Figure 5H).

Establishment of CDK1 knockdown and DEPDCB overexpression
As shown in the former article, CDK1 and CDK2 played important roles in perturbations of chromosomal stability, S phase and G2/M entry, which led to the tumorigenic events happen [12]. In order to further investigate the functions of CDK1 in cells lines and specific relationship between DEPDC1B and CDK1, CDK1 was chosen as target gene in further experiment.
The results of qRT-PCR showed that the expression level of CDK1 in SK-HEP-1, Huh-7, and BEL-7404 cell lines was significantly higher than in MHCC97-L cell line (P<0.05) (Supplementary Figure 1A), which indicated that HEP3B2.1-7 and SK-HEP-1 cell lines were more appropriate to research the functions of CDK1. The results of qRT-PCR showed that the knockdown efficiency of CDK1 in shCDK1-1 group is 99.7% compared with that in shCtrl group (P<0.01) (Supplementary Figure 1B). Therefore, CDK1-1 was chosen as effective interference target. In order to further verify the functions of DEPDC1B in HCC lines, investigate the functions of CDK1 knockdown in HCC  Figure 1C). According to the instructions, the efficiency and expression level were tested by qRT-PCR and Western blot, and the results indicated that these cell lines were constructed successfully (Supplementary Figure 1D, 1E).
(G) The results of western blot showed that CCNE2, CDK1, CDK6, and E2F1was significantly down-regulated in shDEPDC1B group, compared to shCtrl group. (H) The results of IHC showed that the expression level of CDK1 in cancer tissues was higher than in normal tissues (scale bar = 50 μm). AGING formation, CDK1 knockdown significantly inhibited cell colony formation, and to the contrast, DEPDC1B overexpression significantly promoted cell colony formation ( Figure 6B). CDK1 knockdown promoted cell apoptosis, and DEPDC1B overexpression inhibited cell apoptosis ( Figure 6C). The migration ability of cells was significantly increased due to DEPDC1B overexpression, on the contrary, CDK1 knockdown significantly inhibited the migration ability ( Figure 7A, 7B). The results of co-immunoprecipitation were displayed in Figure 7C, 7D and showed that DEPDC1B can interact with CDK1. According to the results above, CDK1 knockdown inhibited cell proliferation, colony formation, migration, and promoted cell apoptosis, which indicated CDK1 knockdown suppressed the progression of HCC cell lines. As shown in the results above, DEPDC1B knockdown inhibited the progression of HCC cell lines. When DEPDC1B was overexpressed, the progression in HCC lines was promoted, which suggested that DEPDC1B certainly played a part in HCC progression. At the same time, in DEPDC1B+shCDK1 group, the results AGING showed that the progression was inhibited in HCC cell lines, which suggested the promoting functions of DEPDC1B overexpression on HCC progression may be blocked after CDK1 knockdown. These results showed that CDK1 may be downstream gene of DEPDC1B, and the function of DEPDC1B on HCC progression was regulated by the expression of CDK1. Collectively, the results proved that DEPDC1B played a definite role in the HCC progression, and CDK1 played a key role in DEPDC1B regulating HCC progression. Based on the TCGA database, the correlation between CDK1 and DEPDC1B was analyzed with Pearson Correlation (R=0.72, P<0.001). The expression level of CDK1 was correlated with pathologic T, N, M, tumor stage, and gender in the analysis of TCGA on lung adenocarcinoma ( Table 5).

Effects of DEPDC1B knockdown on tumor growth in vivo
The mice xenograft model was built with BALB/c nude mice to detect the effects of DEPDC1B knockdown on the tumorigenicity of SK-HEP-1 cells.
After 51 days, the final weight and volume of tumors in mice was measured after the tumors were resected. The results showed that the volumes of tumors in shHEP3B2.1-7 group were smaller than that in shCtrl group ( Figure 8A), and the weight of tumors in shHEP3B2.1-7 group were significantly lighter than that in shCtrl group (P<0.01) ( Figure 8B). The data about fluorescence intensity was measured under small animal living imaging system. The bioluminescence intensity in shHEP3B2.1-7 group was significantly weaker, compared to shCtrl group (P<0. 01), which suggested the slower growth of tumors after DEPDC1B knockdown. (Figure 8C-8E). Collectively, the results above showed the slower growth of HCC cells in shDEPDC1B group compared with that in shCtrl group.

DISCUSSION
In 2012, 782500 new liver cancer cases and 745500 deaths occurred in the world and 50% of the total cases happened in China. Presently, five means are used in HCC to achieve the best outcome (surgical resection, transplantation, local ablation, transarterial embolization and radiotherapy (TACE), and systemic pharmacological treatment), in which, surgical resection, transplantation, and local ablation are treated as potentially curative treatment for only early-stage HCC patients and widely used in the world [4,7,13]. TACE can be chosen as an effective treatment option in intermediate-stage HCC patients [14]. In systemic pharmacological treatment, sorafenib (inhibitor of VEGFR1, VEGFR2 and VEGFR3) is the first-line systemic treatment of patients with advanced-stage HCC, and results of phase III randomized controlled trials have proved that the function of sorafenib in prolonging the survival of advanced-stage HCC patients [4,13]. More and more researchers pay more attention to the occurrence, progression, and recurrence, strive to find specific genes and mechanism to cure HCC. More and more chemotherapy and molecular targeted drugs were found effective in advanced-stage HCC patients: regorafenib (similar to sorafenib) [15,16]; lenvatinib (inhibitor of VEGFR1-3, FGFR1-4, PDGFRα, RET, and KIT) [4]; cabozantinib (inhibitor of MET, VEGFR2, AXL, RET, and KIT) [17]; ramucirumab (monoclonal antibody targets against VEGFR2) [18]; and nivolumab (inhibitor of programmed cell death protein-1 immune checkpoint) [19]. However, the recurrence and mortality rate remained high. Therefore, further research about the molecular mechanism is important for finding the effective treatment of HCC.
The functions of DEPDC1B were investigated in this study. DEPDC1B is localized to the adhesion sites between cells and contains two structural domains: a DEP domain and a RhoGAP domain [11,20]. DEPDC1B can be treated as a negative regulator in RhoA/ROCK signaling pathway, due to the decreased expression of RhoA and MLC2 (main target of ROCK) in DEPDC1B knockdown cells [21]. After DEPDC1B silencing, the delay in mitosis was occurred, especially at the point from G2 to M, and probably caused by defects in de-adhesion. The delay could be rescued by RhoA silencing or inhibiting ROCK [21]. DEP domain is often found in proteins which participate in signal transduction and interact with various membrane partners (phospholipids and membrane receptors), which is necessary for Wnt signaling [22,23]. It plays important roles in mediating membrane localization, and regulating wide range cell functions [24,25]. RhoGAP domain can interact with GTPase and small GTPase, responsible for Rho GTPase signaling [26]. Yang Y, et al [27] showed that DEPDC1B can tumor cell migration and invasion through activating Wnt/β-catenin signaling pathway in non-small cell lung cancer. Ahuja P and Singh K [28] showed that DEPDC1B promoted oral cancer cell growth, invasion, and anchorage independent growth mediated by the interaction between DEPDC1B and Rac1. However, the functions of DEPDC1B in HCC has never been investigated. The study is the first research displaying the functions and mechanism of DEPDC1B in HCC. The results of experiments showed that DEPDC1B knockdown of DEPDC1B inhibited cell proliferation and migration, which is consistent with other reports involving functions of DEPDC1B in other cancers [10]. In IPA analysis, DEPDC1B can affect various genes in Cyclins and Cell Cycle Regulation and Wnt/β-catenin Signaling pathway through EED and FANCD2. In above genes, the expression of CCNE2, CDK1, CDK6, and E2F1 were down-regulated after DEPDC1B knockdown. The results of gene recovery experiments showed that promotion effect of DEPDC1B on cell proliferation and migration in HCC cells could be rescued by CDK1 knockdown. The results of Co-IP indicated the interaction between DEPDC1B protein and CDK1 protein in SK-HEP-1 cells. The results of human apoptosis antibody array showed that the expression of Caspase3, p21, and IGFBP-6 was upregulated, and Bcl-2, Bcl-w, HSP27, HSP70, Livin, sTNF-R2, TNF-α, and TNF-β was down-regulated. CDK1 is a member of the Ser/Thr protein kinase family and known as M-phase promoting factor (MPF), which plays important roles in eukaryotic cell G1/S and G2/M phase transitions. It was thought that the activation of CDK1 by A-type cyclins at the end of interphase promoted mitotic entry [29]. CDK1 can cooperate with cyclins A and B to promote S, G2, and M phase progression, CHK1 and WEE1 can inhibit CDK1 through inhibitory phosphorylation and CDC25 family [12,30,31], therefore the down-regulated CDK1 and CCNB coordinated to lead G2/M phase in the study. In summary, these results in the study proved that DEPDC1B can regulated HCC cell progression based on the CDK1. If CDK1 was knockdown, the pathway of DEPDC1B would be inhibited or blocked, which was consistent the results in other cancers. Caspase3 is a member of the caspase family, which was thought essential regulators in programmed cell apoptosis [32,33]. The cells in hepatoma and various phosphorylated forms can secret IGFBP-6. IGFBP-6 has stronger affinity for IGF-II than IGF-II, and the inhibiting cell function was thought to achieve through IGF binding [34]. p21 is an inhibitor of CKI and inhibitor of all cyclin/CDK complexes. p21 can inhibit G1/S at the mitotic entry, and it can be activated by p53 to link DNA damage to cell cycle arrest [35]. Study has shown that p21 was related to cell differentiation, tumor growth, and metastatic potential in melanoma [36]. Bcl-2 and Bclw are members of BCL-2 family, which plays an important role in programmed cell apoptosis as inhibitor of cell apoptosis and cell cycle entry, and regulator of anti-apoptotic intracellular signals [37,38]. HSP27 is a partner of sHSP, and it is highly expressed in many cancers, which displays the AGING functions of promoting cell migration, invasion, and inhibiting cell apoptosis [39,40]. At the same time, Hsp70 has the functions of binding to unfolded protein, trafficking protein, and regulating enzyme activity, functions as regulators of tumor proliferation, migration, cell apoptosis [40][41][42]. Livin (known as BIC7) is a member of IAP family, which plays an important role in the sensitivity of chemotherapy to cancers, and the expression level was related to the tumor progression [43]. TNF-α and TNF-β are members of TNF family, the functions of TNF consist of cell proliferation, differentiation, and apoptosis. Collectively, DEPDC1B knockdown induces cell apoptosis, leads G2 phase arrest, and inhibit growth and migration in tumor mediated by CDK1.

CONCLUSIONS
In conclusion, this is the first study concerning about DEPDC1B in HCC development. The study confirmed that DEPDC1B is overexpressed in hepatic carcinoma tissues, and the development of HCC were regulated by DEPDC1B through controlling cell apoptosis, inducing G2 phase arrest, migration, and proliferation. It can interact with CDK1, and played a key role in DEPDC1B regulating HCC progression. The study has proved that DEPDC1B play an important role in the progression of HCC and can be treated as a novel gene target to instruct the treatment of HCC. Thus, further studies may accelerate our understanding about the functional role of DEPDC1B in HCC.

Patients and tissue samples
A total of 178 cases (collect 2 slides from each tissue and weed out 2 off slides) diagnosed as HCC by preoperative image examinations and postoperative immunohistochemistry in The First Affiliated Hospital of Zhengzhou University were gathered in the study. This study had got approval from Ethics committees of the First Affiliated Hospital of Zhengzhou University and written informed consent was acquired from all participants in the study.

Immunohistochemistry
The immunohistochemistry assay was conducted to detect the expression level of DEPDC1B and CDK1 in hepatocellular carcinoma tissues and para-carcinoma tissues according to the manufacturer's instructions. The slides were made from tissues according to the routine slides preparation procedure and then incubated with primary antibody (1:200, Cat. No. bs-14278R, BIOSS, Beijing, China) against DEPDC1B at 4° C overnight. The secondary antibody was added to interact with the anti-DEPDC1B antibody at 37° C for 1 h. The 3, 3'-diaminobenzidine (DAB) was used as developer and then the expression levels of DEPDC1B were detected with the assistance of inverted microscope (Olympus IX73). The positive cells score was classified on the basis of the proportion of positive cells: 0 (0%), 1 (10-25%), 2 (25-50%), 3 (50-75%), and 4 (75-100%). Staining intensity score was graded as 0 (No color), 1 (Buff), 2 (Brownish yellow), and 3 (Dark brown) according to the color of cytoplasm, membrane, nucleus, and stroma. And the score of expression levels were: 0, negative; 1-4, positive; 5-8, strong positive; and 9-12, stronger positive, based on the sum of positive cells score and staining intensity score.

Establishment of cell lines and Celigo cell counting assay
Cell lines which expressed more DEPDC1B and CDK1 were selected among HEP3B2.

Colony formation assay
The cells in NC (OE+KD) group, DEPDC1B+NC(KD) group, shCDK1+NC(OE) group, and DEPDC1B+ shCDK1 group were trypsinized and resuspended in logarithmic growth phase, and cells number was counted. 500 cells/well in each group were reseeded into six-well plates in triplicate. Cells were incubated for 8 days, and the medium was displaced every 3 days. 1 mL paraformaldehyde was added into well for 50 min to fix cells. 500 μL GIEMSA staining was added into each well for 20 min after cells were washed with PBS. Finally, the photos of colony cells were taken by microscope equipped with digital camera.

Flow cytometry assay
The cell apoptosis was induced by apoptosis kit (Cat.

Cell cycle analysis
The analysis of cell cycle was detected under flow cytometry system (FACSCalibur, Becton, Dickinson and Company, New Jersey, USA

Human GeneChip PrimeView
The total RNA of was extracted from SK-HEP-1-shCtrl group and SK-HEP-1-shDEPDC1B group in three times for subsequent study. Human GeneChip PrimeView was operated to find DEGs (differentially expressed genes) according to the manufacturer's instructions. The GeneChip was reacted with moderate Pre-Hybridization Mix for 10 min at 45° C. Then, Hybridization Master Mix containing fragmented and labeled cRNA was reacted with the GeneChip for 16 h at 45° C 60rpm. Finally, the GeneChip was scanned under GeneChip Scanner, and analyzed by matched GeneChip software. Quality evaluation of chip data was measured by signal histogram, relative log expression plot, correlation analysis, and principal component analysis. The significant P-value was calculated by linear model based on empirical Bayesian distribution and corrected to obtain FDR. |Fold Change|≥ 1.5 and FDR< 0.05 between two groups were regarded as criteria to screen DEGs. DEGs were analyzed with volcano plot and hierarchical clustering plot. Ingenuity Pathway Analysis (IPA) was used in classic pathways analysis, upstream regulation analysis, disease and function analysis, and interaction network analysis among the DEGs.

Mice xenograft model
The mice xenograft model was created with 4-weeks nude mice (BALB/c, female, Charles River, Beijing, China) according to the manual instructions and the experimental procedure continued for 51 days. A total of 20 mice were randomly divided into two groups before mice were injected cells into their front legs. 4×10^6 SK-HEP-1 cells/mouse transfected with LV-shDEPDC1B and LV-shCtrl were injected into the mice in shDEPDC1B group and shCtrl group, respectively. The length, width, and fluorescence intensity of tumors were detected with small animal living imaging system after mice had been anesthetized with intraperitoneal injection of 0.7% sodium pentobarbital. At the final of experiment, tumors in mice were resected to detect the weight and calculate volume (based on the formula: volume = length × width^2 × 3.14/6) after being sacrificed with excessive sodium pentobarbital followed by cervical dislocation method. All the animal experimental protocols were approved by the Ethics Committee at the First Affiliated Hospital of Zhengzhou University.

Statistical analysis
Statistical analysis of data was performed with SPSS 22.0 software. These experimental procedures were operated in triple. Student's t-test, Mann-Whitney U test, and Pearson Correlation were performed to analyze continuous and categorical variables from the experiment. All values in the text and figures were expressed as the mean ± standard deviation. All statistical tests were two-tailed, and P value <0. 05 was considered statistically significant.

Availability of data and materials
The main datasets used and analyzed in the present study are available from the corresponding authors on reasonable request.

Ethics approval and consent to participate
Written informed consent was gained from all patients whose tissue samples were used in this study and this study was approved by Ethics committees of the First Affiliated Hospital of Zhengzhou University. All the animal experimental protocols were approved by the Ethics committee at the First Affiliated Hospital of Zhengzhou University.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest. The results of Western blot show that, compared to NC group: DEPDC1B protein level of DEPDC1B+NC(KD) group increased obviously, CDK1 protein level had no significant change, and DEPDC1B protein level of shCDK1+NC(OE) group increased obviously, but CDK1 protein level decreased obviously. Compared with DEPDC1B+NC(KD) group, DEPDC1B and CDK1 protein level of DEPDC1B+shCDK1 group decreased obviously. Compared to shCDK1+NC(OE) group, DEPDC1B and CDK1 protein level of DEPDC1B+shCDK1 group decreased obviously. *: P <0.05. **: P <0.01. ***: P <0.001.