Knockdown of Cyclin-Dependent Kinase Inhibitor 3 Inhibits Proliferation and Invasion in Human Gastric Cancer Cells.

Cyclin-dependent kinase inhibitor 3 (CDKN3) has been reported to promote tumorigenesis. Since it is unclear whether CDKN3 participates in the development of human gastric cancer, this study assessed the association between CDKN3 expression and cell biological function and demonstrated the clinical significance and prognosis of CDKN3 in human gastric cancer. In this study, we found that CDKN3 showed a high expression in 35 paired human gastric cancer tissues and was correlated with poor patient survival, AJCC clinical staging, and recurrence. Silencing of CDKN3 in human gastric cancer cells can significantly reduce proliferation, migration, invasion, and adhesion abilities. Also, silencing of CDKN3 in human gastric cancer cells can induce G0-G1 cell cycle arrest and apoptosis. Detection of cell cycle marker expression showed that CDKN3 knockdown promotes cell cycle arrest by decreasing the expression of CDK2, CDC25A, CCNB1, and CCNB2 in human gastric cancer cells. The results of this study will help elucidate the oncogene function of CDKN3 in human gastric cancer.

INTRODUCTION

Gastric cancer is one of the most common cancers and has been considered to be the second frequent cause of cancer-related deaths worldwide1, especially in China, with the incidence being 383,000 cases2. Despite recent advances in diagnostic and therapeutic approaches, the 5-year survival rate for patients suffering from gastric cancer in China is low, at 40%3, whereas the outlook for individuals with advanced gastric cancer is still disappointing. Poor prognosis is frequently explained by a lack of early diagnostic biomarkers and effective therapeutic treatments. Although significant advances have been achieved since the Human Genomic Project was finished, the molecular pathogenesis of gastric cancer still remains to be explored4. Therefore, it is of great clinical value to further understand the molecular mechanisms involved in gastric cancer and to find valuable diagnostic markers as well as novel therapeutic strategies.

Cyclin-dependent kinase inhibitor 3 (CDKN3) belongs to the protein phosphatase family and has a molecular function in regulating cell proliferation, cell cycle, and cell division through the regulation of cyclin-dependent protein kinase activity5,6. It is well known that CDKN3 has a dual function in cell cycling and that CDKN3 not only encodes a dual specificity phosphatase at the G1/S transition, which interacts with Cdk27,8, but also abolishes the induction of p21, a product of the p53 target gene, thus facilitating cell cycle progression9. CDKN3 may potentially function as either an oncogene or a tumor suppressor. CDKN3 was frequently overexpressed in hepatocellular carcinoma10 and in cervical11, breast12, and epithelial ovarian cancers13, and its expression was correlated with a poor clinical outcome. Paradoxically, overexpression of CDKN3 has been associated with the inhibition of cell proliferation in glioblastoma cell lines and has been proposed to be a tumor-suppressor gene in brain tumors14,15. Although CDKN3 was reported to be deleted or overexpressed in several kinds of cancers, the expression pattern and biological function of CDKN3 in human gastric cancer remain to be elucidated.

In this study, we found that CDKN3 acted as an oncogene in the tumorigenesis of human gastric cancer. Overexpression of CDKN3 was found in both gastric cancer tissues and cell lines and is associated with a poor survival time, clinical stage, and a high recurrence of patients with gastric cancer. Silencing CDKN3 inhibited cell proliferation, migration, and invasion; arrested G1/S transition; and increased the apoptotic rate in gastric cancer cell lines. Furthermore, we revealed that CDKN3 might reduce cell survival by regulating the expression of cell cycle-related protein, including CDK2, CDC25A, CCNB1, and CCNB2, in gastric cancer cell lines. Together, our experiments established an important role for CDKN3 in gastric cancer tumorigenesis and provide a potential new therapeutic target for the treatment of gastric cancer.

MATERIALS AND METHODS

Patient Tissue Samples

Ninety human gastric cancer tissues and their adjacent tissues, which were used for qRT-PCR and Western blot analysis, were collected from patients who underwent routine gastric resection at the Cancer Research Institute, The First Affiliated Hospital, China Medical University. None of the specimens had preoperative chemotherapy, radiation therapy, or any other treatment. The clinicopathologic factors are documented in Table 1. Study protocols were approved by the hospital ethics committee of the Cancer Research Institute, The First Affiliated Hospital, China Medical University, and written informed consent was obtained from patients based on the Declaration of Helsinki.

Table 1

Correlation of CDKN3 Expression and the Clinicopathological Characteristics of the Gastric Cancer Patients

Characteristics	CDKN3 Expression		p Value
	High [n (%)]	Low [n (%)]
Age (years)			0.791
≥65	24 (52.2)	22 (47.8)
<65	21 (47.7)	23 (52.3)
Gender			0.062
Male	25 (41.7)	35 (58.3)
Female	20 (66.7)	10 (33.3)
Pathological grading			0.962
II	20 (50.0)	20 (50.0)
III	21 (52.5)	19 (47.5)
IV	4 (40)	6 (60)
Distant metastasis			0.356
Yes	7 (70.0)	3 (30.0)
No	38 (47.5)	42 (52.5)
Tumor size (cm)			0.364
≥1.5	19 (44.2)	24 (55.8)
<1.5	26 (55.3)	21 (44.7)
AJCC clinical staging			0.012*
I	4 (40.0)	6 (60.0)
II	6 (23.1)	20 (76.9)
III	28 (63.6)	16 (36.4)
IV	7 (70.0)	3 (30.0)
Recurrence			0.021*
Yes	30 (69.8)	13 (30.2)
No	15 (31.9)	32 (68.1)

p < 0.05, chi-square test.

Immunohistochemistry

Immunohistochemistry (IHC) for the detection of CDKN3 was performed on sections of the patients’ tumor tissues. Rabbit anti-CDKN3 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used. The results of IHC staining were evaluated independently by two trained pathologists without knowledge of the clinical data. The CDKN3 immunostaining score was analyzed according to a semiquantitative scale. Cytoplasmic/nuclear immunostaining was considered positive staining.

Cell Cultures

Gastric cancer cell lines MKN-28, MKN-45, MKN-7, AGS, BGC-823, MGC-803, and SGC-7901 were obtained from the American Tissue Culture Collection (ATCC; Rockville, MD, USA). Cells were cultured and maintained in RPMI-1640, except MGC-803, which was maintained in DMEM, containing 10% (v/v) fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO2 at 37°C.

siRNA Transfection

siRNA sequences targeting the CDKN3 gene sequence and a negative control siRNA (NC) were designed on the basis of the principles for siRNA design and synthesized by Shanghai Genechem Co. Ltd. The constructs were then transfected into HEK293T cells with lentiviral packaging vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Viruses were collected 48 h after transfection and used to infect SGC-7901 cells.

Cell Viability Assay

Cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Gaithersburg, MD, USA) was used to evaluate the effect of CDKN3 on cell viability. Cells were seeded in 96-well plates at 5 × 103 cells/well in DMEM. Twenty-four hours later, CCK-8 solution was added to each well as the manufactory protocol suggests. Plates were incubated for 4 h at the same incubator conditions, after which the absorbance was read at 450 nm using VERSAmax tunable microplate reader (GE, Sunnyvale, CA, USA).

Cell Cycle Assay

The percentage of cells in different phases of the cell cycle was evaluated by determining the DNA content after propidium iodide (PI) staining (Biovision Inc., Mountain View, CA, USA). Cells were then fixed with 70% ethanol and made RNA free. PI (5 μmol/L) was added and incubated for 20 min in the dark before flow cytometry analysis (BD Biosciences, San Diego, CA, USA).

Cell Apoptosis Assay

Cell apoptosis was detected by annexin V/PI fluorescence-activated cell sorting (FACS) analysis as previously described16. Cells (3 × 105 cells/well) were harvested and washed in cold PBS. After fixation with 70% ethanol, cells were treated with RNase (5 mmol/L) and incubated for 10–15 min in the dark at 37°C. Subsequently, cells were stained with 195 μl of annexin V and 5 μl of PI. The fluorescence intensities were determined by FACS to analyze apoptotic cell percentage.

Wound Healing Assay

Cells were seeded in 35-mm tissue culture dishes at a density of 8 × 105 and further seeded until they reached 100% confluence. Then the confluent cultures were scratched using a pipette tip. After scratching, the well was gently washed twice with medium to remove the detached cells. Scratched cultures were photographed under a microscope at 0 and 18 h. Migration of cells was established by measuring the width of the scratched area at each time point in the scratched area at a magnification of 200×.

Cell Invasion Assay

For cell invasion, Transwell chambers (BD Biosciences, San Jose, CA, USA) were coated with Matrigel (BD Biosciences) and run as described in the manufacturer’s protocol. After transfection for 48 h, 1 × 105 cells in 500 μl of serum-free DMEM were seeded into the upper well of the chamber. The lower chamber was filled with 750 μl of DMEM containing 10% FBS. Cells were incubated at 37°C for 48 h. Afterward, the cells that were able to pass through the filter were fixed with 4% paraformaldehyde and stained by crystal violet solution for 30 min. Cells on the top surface of the insert were removed with a cotton swab and counted under a microscope in five fields (200×).

Cell Adhesion Assay

For adhesion assay, 1 × 105 cells/well were seeded on a fibronectin-coated 12-plate microplate and incubated for 1 h at 37°C in an atmosphere of 5% CO2. The cells were fixed with 4% paraformaldehyde (Gibco) for 15 min and stained with Giemsa (Gibco) for 30 min. Cells were photographed and counted under a microscope in 10 random fields with a magnification of 200×.

qRT-PCR

Total RNA was extracted using TRIzol reagent, and cDNA was synthesized using SuperScript II Reverse Transcriptase. qRT-PCR and data collection were performed with an ABI PRISM 7900HT sequence detection system. The following gene-specific primers were used in this study: CDKN3, 5′-AGCTGCACATCTATCATC-3′ (forward) and 5′-CACTGGTGGTTTCATTTC-3′ (reverse); GAPDH, 5′-CACCCACTCCTCCACCTTTG-3′ (forward) and 5′-CCACCACCCTGTTGCTGTAG-3′ (reverse). The relative expression level of CDKN3 was normalized to that of GAPDH using the comparative Δ CT (2−ΔΔCt) method.

Western Blot

Total protein was separated from tissues and cells using lysis buffer (Pierce, Rockford, IL, USA) and quantified by the Bradford method. Total protein (50 μg) was subjected to 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA, USA), which were blocked with blocking buffer (Beyotime, P.R. China) for 1 h at room temperature. Membranes were probed with specific antibodies. Blots were washed and probed with respective secondary peroxidase-conjugated antibodies, and the bands were visualized by chemoluminescence.

Statistical Analysis

Data were described as the mean ± SD. The statistical data were analyzed by SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The correlation between CDKN3 expression and gastric cancer patient clinicopathological features was analyzed using the chi-square tests. Overall survival in relation to CDKN3 expression was evaluated by the Kaplan–Meyer survival curve and the log-rank nonparametric test. The unpaired, two-tailed Student’s t-test was used to analyze the significance of difference between groups. Differences were considered significant with a value of p < 0.05.

RESULTS

CDKN3 Is Highly Expressed in Gastric Cancer Tissues

The mRNA levels of CDKN3 were detected in 35 out of 90 pairs of tumor and adjacent tissues. In most of these tissues, CDKN3 was more highly expressed in tumor tissues than in adjacent tissues at the transcriptional level (Fig. 1A and B). Western blot was further employed to detect the protein level of CDKN3 in tumor and adjacent tissues. In all 10 pairs, the CDKN3 protein showed a higher expression in tumor tissues than in adjacent tissues (Fig. 1C and D).

Figure 1

Expression of CDKN3 was upregulated in gastric cancer tissues. (A) CDKN3 mRNA expression was analyzed by qRT-PCR in gastric cancer and adjacent tissues. (B) Comparison of the expression levels of CDKN3 mRNA in gastric cancer and adjacent tissues. (C) CDKN3 protein expression was analyzed by Western blotting in gastric cancer and adjacent tissues. (D) Comparison of the expression levels of Cullin7 protein in gastric cancer and adjacent tissues. ***p < 0.001 compared with adjacent tissues.

Correlation of the Clinical Characteristics and CDKN3 Expression in the Gastric Cancer Cases

We then investigated the correlation between CDKN3 expression and clinicopathologic features of patients with gastric cancer. We followed up 90 gastric cancer patients for 5 years. Compared with normal tissue, CDKN3 showed a significantly high expression in tumor tissues (Fig. 2A). According to IHC staining results, all 70 gastric cancer tissue samples were divided into two groups. Group 1 was the high CDKN3 expression group, and group 2 was the low CDKN3 expression group. CDKN3 expression levels and clinicopathologic characteristics of gastric cancer patients are summarized in Table 1. High levels of CDKN3 expression were significantly associated with AJCC clinical staging and recurrence, but no significant correlation was noted with patient age, gender, pathological grading, distant metastasis, or tumor size. Overall survival was significantly reduced in patients with high CDKN3 expression compared with patients with low CDKN3 expression, suggesting that there was a negative correlation between CDKN3 and gastric cancer survival (Fig. 2B).

Figure 2

Negative correlations between CDKN3 and long survival in gastric cancer. (A) Expression of CDKN3 protein was measured by IHC in gastric cancer and normal gastric tissues. Representative images of CDKN3 expression in normal gastric tissues and gastric cancer tissues are shown. (B) Survival analysis of patients.

Depletion of CDKN3 Expression Suppresses SGC-7901 Cell Proliferation

CDKN3 showed a significantly high expression in SGC-7901 and AGE cells when compared to other cancer cells, including MKN-28, MKN-45, BGC-823, MGC-803, and MKN-7, in protein levels (Fig. 3A). Silencing of CDKN3 in cancer cells was established using SGC-7901 cell lines transfected with siRNA targeting CDKN3. The levels of CDKN3 in SGC-7901 cells were verified on mRNA and protein levels (Fig. 3B and C). The CCK-8 assay showed that downregulation of CDKN3 expression significantly reduced the viability of the SGC-7901 cells by 26.9 ± 1.06%, 32.8 ± 2.25%, and 33.3 ± 2.52% at 24, 48, and 72 h, respectively (Fig. 3D).

Figure 3

Expression level of CDKN3 was measured in gastric cancer cell lines. (A) Expression level of CDKN3 was measured by Western blotting in gastric cancer cell lines. The levels of CDKN3 were verified by qRT-CPR (B) and Western blotting (C) in SGC-7901 cells transfected with CDKN3 siRNA. (D) SGC-7901 cell proliferation was detected using the CCK-8 assay at 0, 24, 48, and 72 h. ***p < 0.001 compared with control.

Depletion of CDKN3 Expression Induces SGC-7901 Cell Cycle Arrest and Apoptosis

To explore the potential mechanism by which CDKN3 suppresses gastric cancer cell growth, we evaluated the cell cycle distribution in siRNA-CDKN3 transfected cells and siRNA-NC cells using flow cytometry. The results showed that knockdown of CDKN3 in SGC-7901 cells elicited an accumulation of cells in the G0–G1 phase (64.1 ± 1.10%) and a decrease in the S phase (14.7 ± 2.32%) compared with siRNA-NC-transfected cells (G0–G1, 48.9 ± 2.09%; S, 28.1 ± 1.72%) (Fig. 4A and B). Additionally, results from the annexin V/PI analysis showed that SGC-7901 cells transfected with siRNA-CDKN3 (23.5 ± 1.76%) underwent obvious apoptosis when compared to siRNA-NC-transfected cells (1.57 ± 0.25%) (Fig. 4C and D). Taken together, these data suggest that CDKN3 promotes cell proliferation and suppresses apoptosis of gastric cancer cells in vitro.

Figure 4

Silencing CDKN3 induces cell cycle arrest and apoptosis in SGC-7901 cells. (A, B) SGC-7901 cell cycle profiles were analyzed using flow cytometry. (C, D) SGC-7901 cells were stained with annexin V–fluorescein, and apoptotic rate was analyzed using flow cytometry. ***p < 0.001 compared with control.

Depletion of CDKN3 Expression Inhibits Migration, Invasion, and Adhesion of SGC-7901 Cells

Evidence has shown that a decrease in cell–cell and/or cell–matrix adhesion correlates with tumor invasion and metastasis. To investigate the migration-promoting function of CDKN3 in gastric cancer cells, the migration capacity of SGC-7901 cells was evaluated by a wound healing assay. Knockdown of CDKN3 in SGC-7901 cells significantly reduced the cell migration by 40.1 ± 2.08% compared with control cells (Fig. 5A and B). Also, the Transwell assay showed that the knockdown of CDKN3 in SGC-7901 cells also significantly reduced cell invasion (129.3 ± 14.05), compared with the control group (261.0 ± 15.13) (Fig. 5C and D). Similarly, silencing CDKN3 can also significantly decrease the number of adhesive SGC-7901 cells (19.3 ± 2.08) when compared with the control cells (77.0 ± 2.00) (Fig. 5E and F). Taken together, these findings demonstrate that CDKN3 promotes gastric cancer cell migration, invasion, and adhesion in vitro.

Figure 5

Silencing CDKN3 suppresses migration, invasion, and adhesion in SGC-7901 cells. (A, B) The migration of SGC-7901 cells was performed in in vitro scratch wound healing assay, and photographs were taken 0 and 18 h after the wound was made. (C, D) The invasion of SGC-7901 cells was performed by Transwell assay, and photographs were taken at 48 h after incubation in a Matrigel-precoated Transwell chamber (200×). (E, F) The adhesion of SGC-7901 cells was performed with fibronectin-coated microplate and stained with Giemsa, and photographs were taken at 1 h after incubation. ***p < 0.001 compared with control.

Cell Cycle-Associated Proteins Were Regulated by CDKN3

Five cell cycle-associated proteins (CDK2, CDC25A, CCNB1, and CCNB2) were predicted to be CDKN3 targets. To experimentally validate CDKN3 regulation of these genes, we decreased the expression of CDKN3 in SGC-7901 cells and detected the protein level of the target genes by Western blot. The protein levels of CDK2, CDC25A, CCNB1, and CCNB in SGC-7901 cells were significantly suppressed by CDKN3 siRNA, compared with the control group (Fig. 6A and B). These results demonstrate that CDKN3 promotes cell proliferation, migration, and invasion and inhibits cell apoptosis, possibly by regulating the expression of CDK2, CDC25A, CCNB1, and CCNB2 in gastric cancer cells.

Figure 6

Silencing CDKN3 suppresses cell cycle-associated protein expressions. (A, B) The protein levels of CDK2, CDC25A, CCNB1, and CCNB were detected by Western blot in SGC-7901 cells. Representative Western blots (A) and quantitative results are shown (B). ** p < 0.01, ***p < 0.001 compared with control.

DISCUSSION

Cell cycle regulation is the core event of cell proliferation regulation, which has a close relationship with cellular carcinogenesis. The main role of CDKN3 is cell cycle regulation, but it functions differently in different types of cancers, either inhibiting or stimulating cell proliferation. Overexpression of CDKN3 was observed in several kinds of cancers17,18. However, high levels of CDKN3 had a remarkable effect on the promotion of cancer cell proliferation and migration, as well as resistance to apoptosis and poor prognosis10,13,19. The possible clinical significance of CDKN3 has remained unclear in gastric cancer patients. Therefore, we examined the relationships between CDKN3 expression and the clinicopathologic characteristics of patients with gastric cancer.

The current study revealed that CDKN3 was upregulated in gastric cancer tissues and cell lines. A high level of CDKN3 expression was found to significantly correlate with clinical stage, recurrence, and the prognosis of gastric cancer, and it may play a significant role in tumor carcinogenesis and gastric cancer progression. This was the first time we evaluated the relationship among CDKN3, clinicopathological features, and prognosis in gastric cancer. Consistent with our findings that expression of CDKN3 was significantly associated with FIGO stage, recurrence, and residual tumor size, CDKN3 status is a significant prognostic factor for epithelial ovarian cancer and lung adenocarcinoma patients13,18. The mechanism of a high expression of CDKN3 may be the hypomethylation of its promoter region20 and remains to be further studied.

To better understand the biological function of CDKN3, we investigated whether depletion of CDKN3 reduces the malignant phenotypes (such as cell proliferation, apoptotic resistance, and invasion) in gastric cancer cell lines. In the case of hepatocellular carcinoma, it was found that overexpression of CDKN3 could dramatically promote the proliferation of HepG2 and MHCC-LM3 cells through the induction of G1/S transition10, suggesting a positive role for CDNK3 in cell proliferation and cell cycle. In line with the previous study, depletion of CDKN3 showed a significant decrease in cell proliferation, inhibition of G1/S transition, and induction of apoptosis. However, this was in contrast with our findings that overexpression of CDKN3 was sufficient to prevent K562 leukemic cells from entering the S phase of the cell cycle and promote apoptosis21, suggesting that CDKN3 may negatively regulate proliferation of leukemic cells.

Invasion and migration are biological characteristics of malignant tumors and pose the most problems for clinical treatment22,23. The role of CDKN3 in cell invasion and migration is rarely reported and prompted us to investigate whether CDKN3 has a relationship with cell invasion and migration. The functional study demonstrated that there were no changes in cell invasion after CDKN3 knockdown or overexpression in epithelial ovarian cancer13 and hepatocellular carcinoma cells10. However, we found a positive correlation between CDKN3 and gastric cancer invasion and migration, suggesting that cells with positive CDKN3 expression may promote gastric cancer cell invasion and migration. In addition, CDKN3 may have a different influence on cancer cells in various cancer types. To our knowledge, this is the first report of CDKN3 on tumor invasion and migration other than its proliferation functions.

Another issue involves the mechanism on how CDKN3 promotes gastric cancer progression. To further investigate the molecular mechanism of CDKN3, Western blot analysis was used to identify possible partners of CDKN3 in gastric cancer cells. We identified that depletion of CDKN3 significantly reduced the protein expression of CDK2, CDC25A, CCNB1, and CCNB2 in gastric cancer cells. CDK2 is a key protein running through the G1/S and G2/M phase restriction point in the cell cycle24. Overexpression of CDK2 is closely related to tumor progression and poor prognosis25. CDC25A enhances the activity of cyclin E/A-CDK2 and thereby facilitates S phase entry and progression26. In addition, upregulation of CDC25A promotes gastric cancer cell proliferation27. Besides, CCNB1 could accumulate in the cytoplasm through the S and G2 phases and translocate to the nucleus during prophase28. CCNB2 also binds to transforming growth factor-β RII, and thus CCNB2 may play a key role in transforming growth factor-β-mediated cell cycle control29. CCNB1 depletion or stable gene silencing of CCNB1 inhibits proliferation and induce apoptosis in human tumor cells30,31. Elevated cytoplasmic CCNB2 protein levels were strongly associated with short-term disease-specific survival of breast cancer patients32. All of these reports show a common point, that downregulation of CDK2, CDC25A, CCNB1, and CCNB2 inhibits cancer cell proliferation, indicating that the inhibition of gastric cancer cell viability by CDKN3 downregulation may occur via CDK2, CDC25A, CCNB1, and CCNB2.

In summary, our results have shown that CDKN3 is frequently upregulated in gastric cancer tissues and cell lines and is related to advanced clinical stage, recurrence, and poor clinical outcome in gastric cancer. The functional data strongly suggest that CDKN3 behaves as an oncogene in gastric cancer, and downregulation of CDKN3 could inhibit gastric cancer cell proliferation, migration, and invasion, and induce cell cycle arrest and apoptosis. Our findings suggest that targeting CDKN3 could be a novel therapeutic strategy for the prevention and treatment of human gastric cancer.