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AURKB Promotes the Metastasis of Gastric Cancer, Possibly by Inducing EMT

Authors Wang Z , Yu Z , Wang G, Zhou Y, Deng J, Feng Y, Chen J, Tian L

Received 17 March 2020

Accepted for publication 9 July 2020

Published 5 August 2020 Volume 2020:12 Pages 6947—6958

DOI https://doi.org/10.2147/CMAR.S254250

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Ahmet Emre Eşkazan



Zhen Wang ,* Zhu Yu ,* Gong-he Wang, Yi-ming Zhou, Jian-ping Deng, Yue Feng, Jun-qiang Chen, Lei Tian

Department of Gastrointestinal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Lei Tian; Jun-qiang Chen Email [email protected]; [email protected]

Aim: To investigate the function of Aurora kinase B (AURKB) in gastric cancer (GC).
Methods: Immunohistochemistry was used to assay the expression of AURKB in 50 pairs of GC and adjacent tissues, and qRT-PCR was conducted to test AURKB expression in normal gastric epithelial and GC cell lines. Two segments of small interference RNAs (siRNAs) targeting AURKB were synthesized and inserted into GV248 lentivirus vector. After transfected with LV-AURKB-RNAis, CCK8, wound healing, transwell and flow cytometric assays were performed to determine the influence of silencing AURKB on cell proliferation, invasion, migration, cell cycles and apoptosis of GC cells, and the expression of EMT (epithelial–mesenchymal transition)-related markers was demonstrated by Western blots (WB).
Results: AURKB was highly expressed in GC and closely associated with lymph node metastasis and advanced stages of GC. Down-regulating AURKB suppressed the proliferation and promoted the apoptosis of GC cells, arrested the cell cycle in G2/M phase, and inhibited the invasion and migration of GC cells. The expression levels of AKT1, mTOR, Myc, MMP2, and VEGFA were decreased, while the expression levels of OCLN and JUP were increased after knocking down of AURKB in both AGC and MKN45 cells.
Conclusion: AURKB is overexpressed in GC and closely associated with clinicopathologic characteristics of GC. It is likely that by inhibiting VEGFA/Akt/mTOR and Wnt/β-catenin/Myc pathways, silenced AURKB could inhibit the invasive and migratory abilities of GC cells. However, because of the small sample size and the absence of in-vivo experiments, these results should be verified by further studies.

Keywords: AURKB, gastric cancer, metastasis, EMT

Introduction

Although the incidence is declining recently, gastric cancer (GC) remains a major public health concern, ranking as the fourth most common cancer and the second most frequent cause of cancer-related death worldwide.1 The mortality rate of GC is highest in East Asia, including China. Approximately 60% of patients with GC are diagnosed in these regions.2,3 The prognosis of GC is improved as a result of the progress in comprehensive treatments, including surgery, chemotherapy, and targeted therapy.1 However, the five-year overall survival of GC remains very low.3 Metastasis is one of the main reasons for the poor prognosis of GC as well.1 Hence, it is urgent to explore the internal mechanisms of carcinogenesis and metastasis of GC.

Aurora kinase B (AURKB), which belongs to the mitotic protein kinase family, acts as a crucial regulator in mitosis. It serves as the enzymatic core of the chromosomal passenger complex (CPC),4 which also contains other three non-enzymatic components: Survivin, inner centromere protein (INCENP) and borealin.5 Several studies found that AURKB mainly functions on regulating chromatin modification and suppressing the procession of cytokinesis.6 Overexpression or amplification of AURKB is usually detected in human cancers, including breast cancer,7 ovarian cancer,8 gastric/gastrointestinal cancer and other tumors,9,10 and is proved to be related to poor prognosis.11 Therefore, AURKB has become a promising therapeutic target in tumors. Some researchers found that high expression of AURKB was associated with the improved overall survival in GC,12 however, others considered that inhibition of AURKB reduced GC cell viability and increased the sensitivity of resistant GC cell to cisplatin.13,14 Hence, the exact effect of AURKB in GC remains unclear.

Studies found that epithelial–mesenchymal transition (EMT) played important roles in the development of carcinomas, including gastric cancer. In the process of EMT, the various biochemical changes of epithelial cells can cause the loss of polarity and migratory capacity of cells so that it contributes to the changes of cell shape and promotes immobile epithelial cells transform to motile mesenchymal cells, eventually strengthen cell metastasis.15 It has been reported that EMT can lead to pathological migration and invasion in various cancer progressions, and EMT-induced carcinogenesis is the common cause for malignancies, including GC, neck squamous cell carcinoma, and ovarian cancer.1618 Recently, a study about breast cancer demonstrated that high expression of AURKB was associated with EMT activation and downregulated AURKB could inhibit EMT progression, which indicated that AURKB might induce EMT in breast cancer.19 Therefore, AURKB is possibly associated with EMT, which takes part in the metastasis of various carcinomas.

In order to evaluate the effects of AURKB in gastric cancer and explore the possible mechanisms, in this study we regulated the expression of AURKB in gastric cancer cell lines, tested the change of biological behaviors, and then detected the expression of EMT-related proteins in vitro.

Methods

Ethics Approval

All procedures performed in this study involving participants were approved by the Ethical Review Committee of the First Affiliated Hospital of Guangxi Medical University (Nanning, Guangxi, China), and written informed consent was obtained from all patients.

Patients

Fifty patients (38 males and 12 females) who were pathologically diagnosed with gastric cancer between 2016 and 2017 were enrolled in this study. All the patients received radical gastrectomy or laparotomy. And the following clinical and pathological characteristics were collected: age, gender, tumor size, lymph node status, distant metastasis, pathological type, and TNM stage.

Cell Culture

Gastric cancer cell lines MKN45 (Signet-ring cell carcinoma) and AGS (Gastric adenocarcinoma) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were incubated in RPMI‑1640 (Gibco, USA) with 10% fetal bovine serum (FBS) (Gibco, USA), 50 mg/mL penicillin, and 100 mg/mL streptomycin (Solarbio, Beijing, China). Cells were grown at 37°C in humidified air with 5% CO2.

PCR

Total RNA was extracted using TRIzol reagent (Pufei Biotech Co., Shanghai, China), and 1 ug of total RNA was used to amplify complementary DNA, using the QuantiTect SYBR Green PCR Kit and real-time PCR (TAKARA Bio., Japan) with custom Primetime qPCR Primers (Genechem Co., Ltd, Shanghai, China): human AURKB, 50-CAGAAGAGCTGCACATTTGACG-30 (forward) and 50-CCTTGAGCCCTAAGAGCAGATTT-30 (reverse); and human GAPDH, 50-TGACTTCAACAGCGACACCCA-30 (forward) and 50-CACCCTGTTGCTGTAGCCAAA-30 (reverse). Polymerase chain reaction (PCR) amplification was performed in 50 μL reaction volumes containing 0.2 mmol/L each dNTP, 0.1 RM of each oligonucleotide primer, and 1.25 U Tag polymerase in PCR buffer by using a thermal cycler (model C1000, Bio-Rad). cDNA was amplified on a PCR thermal controller with an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 20 s, 59°C for 30 sec, and 68°C for 30 s and a final extension step of 72°C for 10 min.

Western Blot

Cell lysates were harvested with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) and centrifuged at 15,000×g for 10 min at 4°C. Then, proteins were separated by SDS-PAGE and transferred to the PVDF membrane (Millipore, USA). Subsequently, the PVDF membranes were blocked with a mixture of 5% non-fat milk and Tris-buffered saline (TBS) for preventing non-specific binding. Specific primary antibodies against GAPDH (Santa Cruz, USA), AURKB (abcam, USA), MMP2 (CST, USA), AKT1 (CST, USA), mTOR (CST, USA), JUP (abcam, USA), Myc (abcam, USA), OCLN (abcam, USA), and VEGFA (abcam, USA) were utilized for incubating the membrane at 4°C overnight. Finally, the ECL Western Blotting Detection Kit (Thermo, USA) was applied to detect protein bands according to the manufacturer’s instructions.

Lentivirus‑Mediated RNA Interference (RNAi)

Two segments of small interference RNAs (siRNAs) targeting AURKB were designed. The sequences were presented as follows: 5′-CCAAACTGCTCAGGCATAA-3′ (KD1, 58,972–1) and 5′-CCTTTGAGAGTGCATCACA-3′ (KD2, 58,974–1). A negative sequence (5′-TTCTCCGAACGTGTCACGT-3′) served as a control. The siRNAs were synthesized and inserted into GV248 lentivirus vector (Shanghai Genechem Co., Ltd, China), and then verified by DNA sequencing (Sanger). The siRNAs packaged with lentivirus vector were transfected into cells at a multiplicity of infection of 10 PFU per cell (MOI = 10). Two GC cell lines (AGS and MKN45) were divided into four groups, respectively: GC cells in the control group without any treatment (CON), GC cells in the negative control group (NC) transfected with the negative control segment, GC cells in KD1 group transfected with 58,972–1, and GC cells in KD2 group transfected with 58,974–1.

Colony Formation, CCK8, Cell Cycle and Apoptosis Assays

Gastric cancer cells were transfected with the lentiviral vectors containing siRNA sequence targeting AURKB or negative control sequence. Two weeks later, the colonies were fixed in ice-cold methanol for 10 min and stained with 1% crystal violetin methanol for 15 min. Finally, the colonies were rinsed with water and then counted.

Cell counting Kit-8 (CCK-8, Sigma, US) was conducted to verify cell viability. 2×103 cells/well were seeded into 96-well plates and cultivated for 4 h. Briefly, adding 10 uL CCK-8, the absorbance of each well was evaluated at 450 nm. The growth curve was delineated with time as the horizontal axis and the absorbance as the vertical axis.

Gastric cancer cells were collected and trypsinized. After using ice-cold 70% ethanol to fix cells at 4°C overnight, those cells were incubated with RNase A (50 μg/mL) and stained by propidium iodide (50 μg/mL, Sigma, USA). Then, cell cycle distribution was analyzed by flow cytometry measurement of DNA content using a flow cytometer (Millipore, USA). The percentage of each population was analyzed using the InCyte software (Millipore, USA).

Apoptosis was performed using the Annexin V‑FITC Apoptosis Detection kit (eBioscience, USA) based on the manufacturer’s protocol. After being harvested and added to 200 μL 1X binding buffer, gastric cancer cells were incubated in 10 μL Annexin V ‑FITC for 10 min in dark. Apoptotic cells were quantified by a flow cytometer (Millipore, USA) using the InCyte software (Millipore, USA).

Matrigel Invasion and Wound-Healing Assays

Matrigel invasion assay was conducted using polycarbonate membrane transwell (Corning, United States) coated with the matrigel (BD Biosciences, San Jose, CA, United States). The transfected cells AGS (2.0 × 105 cells/mL) and MKN45 (1.5 × 105 cells/mL) were seeded into the upper chambers, respectively, and then cultured in a humidified atmosphere with 5% CO2 for 24 h at 37°C. The bottom chamber included medium with 5% FBS. The remaining cells on the upper surface were mechanically removed. Then, membranes were washed, fixed, and stained by Methyl Violet (Medion Diagnostics, Germany). Finally, the number of cells that had migrated to the lower side of the filter was counted under a bright field with a microscope (Olympus, Japan) to determine the invasive ability of cells.

Migration ability was tested by wound-healing assay. Stably transfected GC cells were cultured in 6-well plates and fused to approximately 90% (5×103 cells/well). The next day, a straight cell-free wound was created by a sterile pipette tip when ~100% of the surface was occupied. Washed twice by PBS, next, the detached cells were reincubated in RPMI 1640 medium with 1% FBS for 24 h at 37°C. The wound images were obtained using a fluorescence inverted microscope at 0 and 24 h, respectively. The wound distance was measured as follows:

Immunohistochemistry

Paraffin-embedded GC and para-carcinoma tissue blocks were subjected to immunohistochemistry. The tissues were deparaffinized, hydrated, and permeabilized with 0.5% Triton X-100/1 PBS for 10 min, then hybridized with the rabbit anti-human AURKB (abcam, USA) antibodies (1:200 dilution) for 2 h at room temperature. After that, an HRP-conjugated goat anti-rabbit antibody functioned as the secondary antibody. Then, the sections were immersed with the substrate-chromogenic solution and counterstained with hematoxylin. Pictures were acquired by microscopy.

Statistical Analysis

Quantitative data were presented as mean ± standard deviation (SD). All experiments were repeated in triplicate. All analyses were calculated using Student’s t-test or one-way ANOVA of GraphPad 6 (GraphPad Software, Inc). P<0.05 was considered statistically significant.

Results

AURKB Was Highly Expressed in Gastric Cancer and Associated with Clinicopathologic Characteristics of GC

In total, we tested the expression level of AURKB by using IHC in 50 pairs of tissues from GC and para-carcinoma, respectively. As a result, we found that AURKB was highly expressed in GC tissues compared with para-carcinoma (P=0.0068, Figure 1A and B).

Figure 1 AURKB is highly expressed in gastric cancer tissues and cell lines. (A) The images of AURKB expression tested by IHC in gastric cancer and para-carcinoma tissues (X100 and X400, respectively). (B) The quantification of IHC in gastric cancer and para-carcinoma tissues of fifty-one patients (C) The mRNA level of AURKB in normal gastric epithelial cell lines and GC cell lines. (*P < 0.05). Abbreviations: C, cancer tissues; N, normal tissues.

Besides, the association between the expression level of AURKB and clinicopathologic characteristics of GC patients was evaluated. By using univariate analysis, we found that high AURKB expression was closely related to lymph node metastasis (P=0.008) and advanced stages (P=0.016) of GC (Table 1).

Table 1 The Association Between the Expression Level of AURKB and Clinical Pathological Characteristics of GC Patients

To confirm the association of AURKB and gastric cancer, we tested the AURKB mRNA level of the normal gastric epithelial cell line (GES-1) and GC cell lines. As a result, we found that the AURKB mRNA level of GC cell lines was much higher than that of GES-1 (P <0.05, Figure 1C).

Silencing of AURKB Inhibited the Proliferation and Promoted the Apoptosis of GC Cells

In order to evaluate the biological roles of AURKB in GC, we constructed two siRNAs targeted AURKB and packaged them into a lentiviral vector, which was then transfected into AGS and MKN45 cells, respectively. As shown in Figure 2A, the two siRNA segments were verified by DNA sequencing. After transfection, the transfection efficiency was observed under microscopy, and the expression level of AURKB was tested by qPCR. As shown in Figure 2BD, AURKB was significantly down-regulated after RNA interference (P <0.05).

Figure 2 The construction and confirmation of lentivirus-mediated RNA interference plasmid targeting AURKB. (A) Two siRNAs targeted AURKB were constructed and confirmed by RNA-sequence, a: PSC58972-1, b: PSC58974-1. (B) Transfection efficiency of GC cell line AGS and MKN45 according to GFP signal. C, (D) The mRNA level of AURKB in CON, NC, and AURKB -KD group, (C) AGS, (D) MKN45. (*P < 0.05). Abbreviations: CON, control group; NC, negative control group; KD, knockdown group.

To evaluate the role of AURKB on the proliferation and apoptosis of GC, CCK8 assay, colony formation, and flow cytometry were used. As presented in Figures 3AE and 4A and B, proliferation was significantly inhibited and apoptosis was significantly promoted by down-regulating AURKB in GC cell line AGS and MKN45 (P <0.05).

Figure 3 Proliferation (colony formation and CCK8 assay) of gastric cancer cells after silencing AURKB. (AC) Colony formation assay showed that the ability of cell proliferation decreased significantly after knockdown of AURKB in AGS cells and MKN45 cells. After transfection, GC cells were then allowed to form colonies for approximately 6 days (A). Corresponding graphs show the mean number of colonies in AGS cells (B) and MKN45 cells (C). (DE) CCK8 assay showed that the proliferation rate significantly decreased at each time point after silencing AURKB in AGS cells (D) and MKN45 cells (E). (*P < 0.05). Abbreviations: CON, control group; NC, negative control group; KD, knockdown group.

Figure 4 Cell cycle distribution and apoptosis (tested by flow cytometry) of gastric cancer cells after silencing AURKB. (A, B) Flow cytometry showed the apoptosis rate increased significantly after silencing of AURKB in AGS cells and MKN45 cells. (CE) The proportion of cells in S phase and G2/M phase after silencing of AURKB increased significantly, but that of cells in G1 phase decreased markedly. (*P < 0.05). Abbreviations: CON, control group; NC, negative control group; KD, knockdown group.

Silencing of AURKB Arrested the Cell Cycle of GC Cells in G2/M Phase

To detect the effect of down-regulated AURKB on the cell cycle of GC, flow cytometry assay was conducted. As shown in Figure 4CE, the cell counts in G1 phase in KD1 and KD2 groups decreased, and those in G2/M phase increased, compared with those in CON and NC groups (P <0.05). These indicated that down-regulating AURKB could arrest GC cells in G2/M phase so that it could prevent the proliferation of GC cells.

Silencing of AURKB Inhibited the Invasion and Migration of GC Cells

As we know, epithelial–mesenchymal transition (EMT) was closely associated with the invasion and migration of cancers. Therefore, we evaluated the role of AURKB on the invasion and migration of GC cells by using matrigel invasion and wound-healing assays. As a result, we found that the invasion and migration abilities of GC cells in KD1 group were significantly impeded compared with those in CON and NC groups (Figures 5AC and 6AD, P <0.05), but not in KD2 group (P >0.05). These showed that the silencing of AURKB could inhibit the invasion and migration of GC cells.

Figure 5 Invasion ability (matrigel invasion assay) of GC cell lines after silencing of AURKB. (A) The number of AGS cells and MKN45 cells crossing the basement membrane markedly decreased after silencing of AURKB. (B, C) Quantification of GC cells crossing the basement membrane after silencing of AURKB, (B) AGS, (C) MKN45. (*P < 0.05).

Figure 6 Migration ability (wound-healing assay) of GC cell lines after silencing of AURKB. (A, C) The migration potential of stably transfected GC cells was checked by wound-healing assays. Images were captured at a time interval between 0 and 24 h, (A) AGS cells, (C) MKN45 cells. (B, D) Percentage of migration was statistically analyzed, (B) AGS cells, (D) MKN45 cells. (*P < 0.05). Abbreviations: CON, control group; NC, negative control group; KD, knockdown group.

The Effect of AURKB on Metastasis of GC is Possibly Through Inducing EMT

As is well known, the aberrant activation of an epithelial–mesenchymal transition (EMT) promotes tumor cell invasion and dissemination. EMT is characterized by loss of epithelial markers and higher expression of mesenchymal markers. Therefore, we investigated the potential mechanisms that down-regulation of AURKB inhibited invasion and migration of GC cells. After silencing ARUKB in GC cells, we detected the expression levels of EMT markers by WB, including AKT1, mTOR, JUP, MMP2, OCLN, Myc, and VEGFA. As performed in Figure 7AD, the expression levels of AKT1, mTOR, Myc, MMP2, and VEGFA were all decreased after silencing AURKB in both AGC and MKN45 cells, and the changes were all statistically significant (P <0.05) except that of MMP2 (P >0.05). While the expression levels of OCLN and JUP were increased after knocking down AURKB in both AGC and MKN45 cells, although the change of OCLN was not statistically significant in AGS cells (P >0.05). Therefore, we considered that inhibition of invasion and migration by silencing of AURKB was possibly induced by EMT in GC.

Figure 7 EMT relative protein expressions in GC cells after silencing of AURKB. (A, B) Western blot was used to test the expression of EMT relative protein in AGC cells (A) and MKN45 cells (B) after silencing of AURKB. (C, D) Quantification of EMT relative protein in AGC cells (C) and MKN45 cells (D) after silencing of AURKB according to Western blot. (*P < 0.05). Abbreviations: CON, control group; NC, negative control group; KD, knockdown group.

Discussion

As a member of aurora kinase family, AURKB has been reported to be highly expressed in several cancer types and mainly plays critical roles in cancer cell mitosis.20 AURKB mainly exerts its functions on suppressing the completion of cytokinesis and regulating chromatin modification. Studies have indicated that AURKB can promote the growth and metastasis of malignant tumors such as breast cancer,7 ovarian cancer,8 non-small cell lung cancer,10 and osteosarcoma.21 In fact, the effect of AURKB on GC was already studied, but the results remained controversial. Enjoji et al found that overexpression of AURKB was related to a favorable prognosis of GC.12 However, Xiao Han et al reported that upregulated AURKB led to metastasis of GC.14 Therefore, we conducted this study to assess the exact function of AURKB in GC.

By analyzing the expression level of AURKB in 50 GC patients, we found that AURKB was significantly high expressed and closely related to lymph node metastasis (P=0.008) and advanced stages (P=0.016) of GC. Then, in-vitro experiments were performed after silencing AURKB. As a result, we found that the down-regulation of AURKB inhibited the proliferation and promoted the apoptosis of GC cancer cells, arrested the cell cycle of GC cells in G2/M phase, and inhibited the invasion and migration of GC cells. Our results were similar to previous studies, which found that AURKB was closely correlated with the prognosis, invasiveness, chemoresistance of various tumors.711 Based on these results, we considered that AURKB could function as a novel oncogene and become a potential therapeutic target in GC.

Epithelial–mesenchymal transition (EMT), which is a process of cell remodeling critical during embryonic development and organogenesis, has been implicated in carcinogenesis and confers metastatic properties upon cancer cells by enhancing invasion and mobility. Studies found that several molecular mechanisms are known to promote the process of EMT, including regulation of the levels of specific cell-surface proteins, cell-extracellular matrix-degrading enzymes, and altering the expression of certain transcription factors.22 In order to explore the possible mechanisms of AURKB in regulating the invasion and migration of GC, we tested the expression level of several biological markers, including AKT1, mTOR, JUP, MMP2, OCLN, Myc, and VEGFA, which were all reported to be closely associated with EMT.2224 As a result, we found that the expression levels of AKT1, mTOR, Myc, MMP2, and VEGFA were all decreased after silencing AURKB in both AGC and MKN45 cells, and the changes were all statistically significant except that of MMP2. While the expression levels of OCLN and JUP were increased after knocking down AURKB in both AGC and MKN45 cells, although the change of OCLN was not statistically significant in AGS cells (P >0.05). Based on these results, we considered that AURKB possibly promoted the invasion and migration of GC by inducing EMT.

AKT, a Serine/Threonine kinase, is a key component in numerous processes. Recent studies found that AKT activation could phosphorylate and activate the EMT transcription factors, and AKT/mTOR pathway was one of the key signals regulating EMT in different cancer cells, including oral cancer, thyroid cancer and melanoma.2527 Vascular endothelial growth factor A (VEGFA), exerting an important role in endothelial cell proliferation and vascular remodeling by activating its tyrosine kinase receptor, has been reported to be dysregulated and promote tumor progression in various carcinomas.28,29 Recently, studies found that VEGFA could induce EMT and promote metastasis of various tumors by playing roles in the response to angiogenesis during tumorigenesis,30 and this process was through activating PI3K/Akt/mTOR signaling pathway.31 Matrix metalloproteinases (MMPs) are well known to degrade E-cadherin, which is an important epithelial marker.32 It has been shown that EMT extension was associated with the level of MMP-2 activity and MMP-9 secretion,33 and PI3K/AKT pathway has been shown to positively regulate MMPs in various cancers, including gastric cancer.34 In our study, AKT, mTOR, VEGFA, and MMP2 (although statistical significance was not achieved) were all downregulated after silencing of AURKB, suggesting that AURKB might promote invasion and migration of GC cells by activating the VEGFA/AKT/mTOR pathway. And this was consistent with previous studies.

It is reported that Wnt/β-catenin signaling pathway has an important role in EMT during carcinoma progression.35 During the process of EMT, the loss of E-cadherin may abnormally activate Wnt/β-catenin signaling pathway. Then, the degradation of β-catenin is inhibited, and β-catenin enters into the nucleus to promote the expression of downstream target genes such as Myc, which finally promotes the invasion and migration of tumor cells.36 Both occludin (OCLN) and plakoglobin (JUP) are epithelial markers, which are often decreased during the process of EMT.37,38 Studies found that decreased expression of OCLN and JUP was associated with malignant progression and poor prognosis of cancers.39,40 These were verified in our studies, which found that the expression levels of OCLN and JUP were increased, while the expression level of Myc was decreased after silencing AURKB, suggesting that knockdown of AURKB could regain the expression of epithelial markers and inhibit Wnt/β-catenin pathway.

In conclusion, AURKB was highly expressed in GC, and closely associated with lymph node metastasis and advanced stages of GC. Down-regulating AURKB inhibited the proliferation and promoted the apoptosis of GC cells, arrested the cell cycle in G2/M phase, and inhibited the invasion and migration of GC cells. The process was possibly through inhibiting VEGFA/Akt/mTOR and Wnt/β-catenin/Myc pathways. However, owing to the small sample size and the lack of in-vivo experiments, these results should be verified by more studies in the future.

Ethics and Consent

All procedures performed in this study involving participants were approved by the Ethical Review Committee of the First Affiliated Hospital of Guangxi Medical University, Guangxi, China, and written informed consent was obtained from all patients.

Author Contributions

All authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

National Natural Science Foundation of China 10.13039/501100001809 Natural Science Foundation of Guangxi This manuscript was supported by the National Natural Science Foundation of China (81660134) and the Natural Science Foundation of Guangxi (2016GXNSFAA380162, 2017GXNSFAA198051 and 2018GXNSFBA281159).

Disclosure

The authors have no conflict of interest to declare for this work.

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