Silencing CDCA8 inhibits the proliferation and invasion of gastric cancer cells and induces apoptosis by blocking the Akt pathway


 
 
 
Purpose: To investigate the effect of cell division cycle associated 8 (CDCA8) on malignant progression of gastric cancer (GC) cells. 
Methods: Short hairpin RNAs (shRNA) were transfected into two gastric cell lines to knock down expression of CDCA8. Transfection efficiency was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Then, Cell Counting Kit 8 and colony formation and Transwell assays were utilized to explore the effect of CDCA8 knockdown on the proliferation, invasion, and migration of GC cells. Flow cytometry was conducted to analyze the effect of CDCA8 knockdown on cell cycle progression and apoptosis. The relationship between CDCA8, epithelial-mesenchymal transition (EMT), and Akt pathway activity was determined by western blot analysis. 
Results: Proliferation, invasion, and migration of GC cells were significantly inhibited by CDCA8 knockdown. Knockdown of CDCA8 induced cell cycle arrest in G1 phase and apoptosis, and inhibited EMT and Akt pathway activity. 
Conclusion: Knockdown of CDCA8 inhibits GC growth and metastasis in vitro by reducing Akt pathway activity. Thus, this molecule presents a potential strategy for the management of GC. 
 
 
 



INTRODUCTION
Gastric cancer (GC) is one of the most aggressive common malignancies, with approximately 1,000,000 new cases and 781,000 deaths according to global cancer statistics in 2018 [1]. GC has the second highest morbidity and mortality in China, with 679,100 new cases and 49,800 deaths in 2015 [2]. GC progresses rapidly to advanced stages, and the high recurrence rate and high invasiveness of advanced GC are the main causative factors of patient deaths. The high invasiveness of GC is also the main obstacle to improving patients' survival rate after surgical resection [3].
The mechanisms that drive cancer occurrence and metastasis include those that promote proliferation, migration, invasion, and angiogenesis, and lead cells to evade aging and death [4,5]. Cell division cycle associated 8 (CDCA8) is a component of vertebrate chromosomal passenger complex (CPC), and the overexpression of CPC is associated with the occurrence of cancer [6][7][8].
CDCA8 targets CPC components to the centrosome, corrects kinetochore attachment errors, and stabilizes the bipolar axis of human cells, and thus has important regulatory functions during mitosis [6,9,10]. Overexpression of CDCA8 is associated with malignant progression of cancer and poor patient prognosis in a variety of human cancers, including pancreatic ductal adenocarcinoma [11] and lung cancer [12]. Therefore, CDCA8 is considered a potential new therapeutic target and prognostic marker for cancer.
It has been reported that abnormal expressions of chromosomal guest proteins that regulate mitosis is associated with poor prognosis of GC [13], but the role of CDCA8 in GC is not clear. Our study investigates whether the aberrant expression of CDCA8 affects the malignant progression of GC and, if it does, its potential mechanism of action. Our overall goal is to provide new effective therapeutic targets for advanced GC.

EXPERIMENTAL Cell culture and transfection
The GC cell lines SGC-7901 (CL-0206) and AGS (CL-0022) were obtained from Procell Life Sciences (Wuhan, China), and cultured in RPMI 1640 medium (Thermo Fisher Scientific, MA, USA) containing 10 % fetal bovine serum (FBS) (Gibco) at 37 °C in an incubator containing 5 % CO2 humidified air. The pLKO.1 vector and CDCA8 shRNA were acquired from Addgene (Watertown, MA, USA). The CDCA8 shRNA was cloned into the pLKO.1 vector and lentiviral transfection was used to construct SGC-7901 and AGS cell lines with stable knockdown of CDCA8 expression.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from GC cells using TRIzol reagent (Life Technologies, Thermo Fisher Scientific) according to the manufacturer's instructions. One μg of total RNA was reverse transcribed into cDNA using reverse transcriptase and Oligo dT primers (Takara, Japan). The cDNA was then amplified via PCR using specific primers. The CDCA8 primers used for PCR were 5`-GTTCCAAGGAGAAGCCC ACA-3` (forward) and 5`-CCGGTCCACATTC TCTGGAA-3` (reverse). The primers used to amplify β-actin as internal control were 5`-CATGTACGTTGCTATCCAGGC-3` (forward) and 5`-CTCCTTAATGTCACG CACGAT-3` (reverse). Cycle threshold (Cq) values were obtained in each sample and relative expression levels were calculated using the 2 -ΔΔCt method [14].

Western blot
GC cells were lysed with lytic buffer (Merck Millipore, USA), and extracted proteins were quantified using the bicinchoninic acid assay. Ten -20μg of extracted protein was separated using 12 % SDS-PAGE and transferred to a PVDF membrane (Merck Millipore). After blocking non-specific sites with 5 % skim milk at room temperature for 2 h, the PVDF membrane was incubated overnight at 4 °C with the appropriate primary antibody dilution as shown in Table 1. Then, the membrane was incubated with a 1:2000 dilution of goat anti-rabbit IgG H&L (HRP) (ab205718, Abcam) at 20 °C for 2 h. The immunoreactive proteins were visualized using chemiluminescence (Santa Clara, USA).

Cell proliferation assay
Cell Counting Kit 8 assay (CCK-8, Dojindo Laboratories, Kumamoto, Japan) was performed to evaluate cell viability. SGC-7901 or AGS cells (1 × 10 3 ) were spread in 96-well plates, and incubated for 24 h. CCK-8 reagent was added to the wells and incubation was continued for 1 h, and the absorbance was then measured at 450 nm using an automatic enzyme labeling instrument (Synergy4, VT, USA).

Colony formation assay
The cells were digested with trypsin, inoculated into a 6-well plate at a density of 1 × 10 3 cells / well, and incubated at 37 °C for 14 days. Cells were fixed with 20 % methanol solution and later stained with 0.1 % crystal violet. The colonies were observed under an inverted microscope, then counted and analyzed.

Cell cycle analysis
SGC-7901 or AGS cells were placed in 70% cold ethanol overnight. Ethanol was removed by centrifugation and the cells were incubated gradually with RNase A (10 μg/ml) and propidium iodide (PI) solution (50 μg/ml) at 37 °C. Finally, the DNA content of the cells was analyzed via flow cytometry.

Apoptosis assay
Apoptosis was analyzed using the FITC Annexin V Apoptosis Detection Kit 1 (BD Pharmingen, CA, USA). Cells in logarithmic phase were inoculated in a 6-well plate at a density of 1 × 10 5 cells per well and incubated for 24 h. The cells were collected, and washed and stained under dark conditions with Annexin V FITC and PI for 15 min. Cytomics FC 500 (Beckman Coulter, CA, USA) was used for flow cytometric analysis.

Transwell assay
Invasion and migration assays were performed in transwell chambers pre-coated with or without Matrigel, for invasion and migration, respectively. Briefly, 5 × 10 4 SGC-7901 or AGS cells were inoculated into the upper chamber which was covered with 200 μl serum-free medium. The lower chamber contained 600 μL medium and 10 % FBS added as a chemical attractant. After incubation for 24 h, the cells that had migrated or invaded the lower chamber were fixed with formaldehyde and stained with 0.5 % crystal violet for 5 min. The cells were photographed and counted under an inverted microscope.

Statistical analysis
The results were statistically analyzed with SPSS 22.0 (IBM Corp., Armonk, NY, USA) software. All values were expressed as mean ± SEM. The experiments were repeated 3 times independently. Differences between paired samples were analyzed using the t-test and were statistically significant when p < 0.05.

Knockdown of CDCA8 inhibits the growth of GC cells in vitro
CDCA8 is often reported as an oncogene, which suggests that CDCA8 may promote the malignant growth of GC. In order to test this conjecture, a recombinant vector was constructed from shRNA against CDCA8 and transfected into SGC-7901 and AGS cells. CDCA8 shRNA significantly decreased the expression of CDCA8 mRNA and protein in GC cell lines, and pLKO-1# CDCA8 knockdown had the greatest effect (Figure 1 A and B). Therefore, this shRNA was selected for experimental exploration and named pLKO CDCA8. The CCK-8 assay showed that knockdown of CDCA8 expression significantly decreased cell viability (Figure 1 C). The colony formation test demonstrated that the number of colonies formed by each cell line decreased significantly after knocking down CDCA8 (Figure 1 D). These results show that knockdown of CDCA8 inhibits the growth of GC cells in vitro.

CDCA8 regulates the mitotic process of cells and promotes the growth of GC cells in vitro.
Therefore, knockdown of CDCA8 may induce cell cycle arrest. This speculation was tested using flow cytometry, which showed that CDCA8 knockdown induced cell cycle arrest in G1 phase (Figure 2 A). Furthermore, the protein levels of CyclinD1, p21 and p27 were detected via western blot (Figure 2 B). The data showed that after CDCA8 knockdown, the expression of CyclinD1 was significantly decreased, while the expression of cyclin-dependent kinase inhibitors (p21 and p27) was significantly increased. These results confirmed that CDCA8 knockdown induced cell cycle arrest.

CDCA8 knockdown induces apoptosis in GC cells
Apoptosis is one of the main pathways that leads to cell growth inhibition. The ratios of apoptotic to non-apoptotic SGC-7901 and AGS cells were determined by flow cytometry. The results showed that knockdown of CDCA8 significantly increased the ratio of apoptotic GC cells (Figure  3 A). Similarly, the expression of apoptosisrelated proteins was analyzed by western blot. After knocking down CDCA8, the expression of the anti-apoptotic protein Bcl-2 was inhibited, while the expression of the pro-apoptotic protein Bax was significantly increased (Figure 3 B). Combined with the results of flow cytometry and western blot analysis, these results confirm that knockdown of CDCA8 significantly induces apoptosis of GC cells.

CDCA8 knockdown inhibit invasion and migration of GC cells
To measure the influence of CDCA8 knockdown on cell metastasis, its influence on SGC-7901 and AGS cell invasion and migration was assessed using the transwell assay. As shown in Figure 4 A, knockdown of CDCA8 inhibited invasion and migration of GC cells. In addition, the effect of CDCA8 knockdown on the expression of epithelial-mesenchymal transition (EMT)-related proteins was analyzed using western blotting (Figure 4 B). The results showed that the expression of E-cadherin protein in SGC-7901 and AGS cells increased, while the expression of N-cadherin decreased after CDCA8 knockdown.

CDCA8 knockdown inhibit the activity of Akt signal pathway
Knocking down CDCA8 down-regulated p-PI3K and p-Akt ( Figure 5). Therefore, CDCA8 knockdown inhibits the growth and invasiveness of GC cells, at least in part, by inactivating the Akt pathway.

DISCUSSION
In recent years, research on GC targets and prognostic markers has accelerated. Although no study has reported a role for CDCA8 in the development of GC, its regulation of the mitotic processes [9] and the pro-carcinogenic role it exhibits in common cancers such as lung [12] and pancreatic cancer [11] are noteworthy. In terms of cell growth, this study revealed, through CCK-8 and colony formation assays, that knockdown of CDCA8 inhibited GC cell viability and proliferation ability. This result is consistent with previous reports that CDCA8 knockdown inhibited the proliferation of skin melanoma cell lines [17], and bladder [18] and breast cancer [19]. Can et al [20] found that the CDCA8 promoter has high activity in several cancer cell lines, as well as in undifferentiated human embryonic stem cells (hESCs) and early mouse embryos, which share the common feature of retaining high proliferative activity. This suggests that the activation of CDCA8 transcription contributes to cell growth, and explains the inhibition of GC cell proliferation by CDCA8 knockdown found in this study. Furthermore, the results of flow cytometry demonstrated that knockdown of CDCA8 induced cell cycle G1 arrest and induced apoptosis. As early as 2003, a study on the cell cycle reported that CDCA8 regulates mitosis in cells [10]. In addition, Hayama et al. [12] confirmed that knocking down CDCA8 in lung and ovarian cancer cells can induce cell cycle arrest, which is consistent with the findings of the present work.
This study also revealed that CDCA8 knockdown inhibited the migration and invasiveness of GC cells. CDCA8 may be a key gene involved in the metastasis of clear cell renal cell carcinoma [21] and may promote metastasis of prostate cancer [22] and melanoma [17]. In addition, this study found that knockdown of CDCA8 upregulated E-cadherin and downregulated Ncadherin expression. During EMT, E-cadherin, which is responsible for normal epithelial cell adhesion, is replaced by N-cadherin, which provides better linkage flexibility and enhances cell motility [23]. This suggests that knockdown of CDCA8 inhibits EMT. This is the first report that abnormal expression of CDCA8 affects EMT, not only in the field of GC research, but also in the field of cancer research.
This study further investigated the potential mechanism of CDCA8 knockdown in inhibiting GC progression and found that CDCA8 knockdown led to inactivation of the Akt signaling pathway. Activation of the PI3K/Akt signaling pathway has previously been shown to promote GC occurrence and invasion [24]. Also, inactivation of the PI3K/Akt signaling pathway led to GC cell autophagy and apoptosis [25]. These reports on the association of PI3K/Akt signaling pathway activity with GC support the conclusion of this study that CDCA8 knockdown inhibits GC growth and metastasis by inducing inactivation of the Akt pathway.

CONCLUSION
This study is the first to investigate the effect of aberrant expression of CDCA8 on GC progression and revealed that CDCA8 knockdown inhibits GC cell growth and metastasis in vitro by inducing cell cycle arrest and Akt pathway inactivation. It fills a critical gap in our understanding of the involvement of CDCA8 in GC and is crucial to assess the value of CDCA8 as a therapeutic target and prognostic indicator in GC. Further research will involve animal experiments to illuminate the effect of CDCA8 on gastric cancer growth and metastasis in vivo.