CDKN3高表达促进胃癌细胞的迁移和侵袭：基于调控p53/NF-κB信号通路和抑制胃癌细胞凋亡

doi:10.12122/j.issn.1673-4254.2025.04.21

摘要/Abstract

摘要：

目的探究周期蛋白依赖性激酶抑制因子3（CDKN3）在胃癌中的表达情况，并分析其影响胃癌患者预后的潜在作用机制。方法纳入114例胃癌患者资料，分析CDKN3在胃癌组织中表达水平及其对胃癌患者术后5年生存率的影响；GO及KEGG富集分析预测CDKN3的生物学功能及可能的作用机制；通过慢病毒转染技术干预CDKN3的表达。采用Transwell、CCK-8、TUNEL染色、流式细胞术及Western blotting验证生物学功能。结果 CDKN3在胃癌组织的表达水平显著高于癌旁组织（P<0.01），且CDKN3的表达水平与CEA、CA19-9、T分期及N分期相关（P<0.05）。单因素联合多因素分析显示，CDKN3高表达是影响胃癌患者术后5年生存率的独立风险因子（P<0.05）且CDKN3高表达对胃癌患者远期预后具有预判价值（P<0.01）。生物信息学富集分析提示CDKN3的功能可能与凋亡有关。Transwell实验结果显示，下调CDKN3可抑制胃癌细胞迁移、侵袭，上调则反之（P<0.01）。TUNEL染色结果显示，癌旁组织中细胞凋亡水平高于胃癌组织（P<0.01）。CCK-8和流式结果显示，降低CDKN3表达可抑制胃癌细胞的增殖，升高胃癌细胞凋亡率（P<0.05）。Western blotting结果显示过表达CDKN3后，p53、p21和促凋亡蛋白Bax表达降低，p-p65和抗凋亡蛋白Bcl-2表达升高（P<0.05）。结论 CDKN3在胃癌组织中高表达并影响患者预后，其可能通过p53/NF-κB信号通路调控胃癌细胞凋亡并抑制其增殖、侵袭与迁移有关。

关键词: 胃癌, CDKN3, 凋亡, 增殖, 预后, p53/NF-κB

Abstract:

Objective To investigate the expression of CDKN3 in gastric cancer and its impact on prognosis of gastric cancer patients. Methods We analyzed CDKN3 expression in clinical specimens from 114 gastric cancer patients and assessed its association with 5-year postoperative survival of the patients. GO and KEGG enrichment analyses were used to predict the biological function and possible mechanism of CDKN3. The effects of lentivirus-mediated CDKN3 knockdown on biological behaviors of gastric cancer cells were evaluated using Transwell assay, CCK-8 assay, TUNEL staining, flow cytometry, and Western blotting. Results CDKN3 expression was significantly higher in gastric cancer tissues than in the adjacent tissues with significant correlations with CEA level, CA19-9 level, and T and N staging (P<0.05). High CDKN3 expression was an independent risk factor affecting 5-year postoperative survival of the patients and predictive for long-term prognosis (P<0.01). Enrichment analyses suggested a probable association of CDKN3 with apoptosis. In MGC-803 cells, CDKN3 knockdown significantly lowered migration and invasion capacities of the cells, while CDKN3 overexpression produced the opposite effects. TUNEL staining revealed a significantly lower level of cell apoptosis in gastric cancer tissues than in adjacent tissues (P<0.01). CDKN3 knockdown obviously inhibited proliferation and increased apoptosis of MGC-803 cells. CDKN3 overexpression down-regulated the expressions of p53, p21 and Bax and up-regulated the expressions of p-p65 and Bcl-2. Conclusion CDKN3 is highly expressed in gastric cancer tissues and affects patient prognosis. CDKN3 overexpression promotes proliferation, invasion and migration and suppressed apoptosis of gastric cancer cells possibly through the p53/NF-κB signaling pathway.

Key words: gastric cancer, CDKN3, apoptosis, proliferation, prognosis, p53/NF-κB

张毅, 沈昱, 万志强, 陶嵩, 柳亚魁, 王栓虎. CDKN3高表达促进胃癌细胞的迁移和侵袭：基于调控p53/NF-κB信号通路和抑制胃癌细胞凋亡[J]. 南方医科大学学报, 2025, 45(4): 853-861.

Yi ZHANG, Yu SHEN, Zhiqiang WAN, Song TAO, Yakui LIU, Shuanhu WANG. High expression of CDKN3 promotes migration and invasion of gastric cancer cells by regulating the p53/NF-κB signaling pathway and inhibiting cell apoptosis[J]. Journal of Southern Medical University, 2025, 45(4): 853-861.

图/表 11

图1 CDKN3在胃癌及癌旁组织中的表达情况

Fig.1 Expression of CDKN3 in gastric cancer (GC) and adjacent tissues. A: Immunohistochemistry of CDKN3 in gastric cancer (GC) and adjacent tissues. B: Relative IOD values of CDKN3 expression. **P<0.01.

表1 CDKN3表达量与胃癌患者临床病理参数间的关系

Tab.1 Relationship between the expression level of CDKN3 in GC tissues and clinicopathological parameters of the patients

Clinicopatholological parameters	n	CDKN3		χ²	P
Clinicopatholological parameters	n	Low expression (n=57)	High expression (n=57)	χ²	P
Gender
Male	81	43 (53.09%)	38 (46.91%)	1.066	0.302
Female	33	14 (42.42%)	19 (57.58%)	1.066	0.302
Age (year)
<60	46	22 (47.83%)	24 (52.17%)	0.146	0.703
≥60	68	35 (51.47%)	33 (48.53%)	0.146	0.703
CEA (μg/L)
<5	69	46 (66.67%)	23 (33.33%)	19.422	<0.001
≥5	45	11 (24.44%)	34 (75.56%)	19.422	<0.001
CA19-9 (kU/L)
<37	86	53 (61.63%)	33 (38.37%)	18.937	<0.001
≥37	28	4 (14.29%)	24 (85.71%)	18.937	<0.001
Tumor size (cm)
<5	67	38 (56.72%)	29 (43.28%)	2.932	0.087
≥5	47	19 (40.43%)	28 (59.57%)	2.932	0.087
Histological type
Adenocarcinoma	104	53 (50.96%)	51 (49.04%)	0.438	0.508
Other	10	4 (40.00%)	6 (60.00%)	0.438	0.508
Grade of differentiation
Well	10	4 (40.00%)	6 (60.00%)	4.001	0.135
Moderate	38	24 (63.16%)	14 (36.84%)
Poor	66	29 (43.94%)	37 (56.06%)
T Stage
T₁-T₂	37	24 (64.86%)	13 (35.14%)	4.842	0.028
T₃-T₄	77	33 (42.86%)	44 (57.14%)	4.842	0.028
N Stage
N₀-N₁	71	41 (57.75%)	30 (42.25%)	4.518	0.034
N₂-N₃	43	16 (37.21%)	27 (62.79%)	4.518	0.034

图2 胃癌组织中CDKN3表达水平与CA19-9及CEA的相关性分析

Fig.2 Correlation analysis between the expression level of CDKN3 in GC and the levels of CA19-9 and CEA in peripheral blood. A: Correlation analysis between CDKN3 and CA19-9. B: Correlation analysis between CDKN3 and CEA.

图3 胃癌组织中CDKN3表达量与患者远期预后之间的关系

Fig.3 Correlation of the expression level of CDKN3 in gastric cancer tissues with long-term prognosis of the patients.

表2 影响胃癌根治术后5年生存率的单因素及多因素分析

Tab.2 Univariate and multivariate analyses of the factors affecting the 5-year survival rate after radical gastrectomy for gastric cancer patients

Factors	Univariate analysis		Multivariate analysis
Factors	Log-rank χ²	P	HR	95% CI	P
Gender (female vs male)	0.108	0.742	-	-	-
Age (≥60 years vs <60 years)	0.162	0.687	-	-	-
CDKN3 expression (high vs low)	34.036	<0.001	2.819	1.396-5.692	0.004
CEA(≥5 μg/L vs <5 μg/L)	20.961	<0.001	1.954	1.084-3.522	0.026
CA19-9 (≥37 kU/L vs <37 kU/L)	22.695	<0.001	1.847	1.048-3.257	0.034
Tumor size (≥5 cm vs <5 cm)	6.139	0.013	0.978	0.552-1.730	0.938
Histological type (other vs adenocarcinoma)	0.309	0.578	-	-	-
Grade of differentiation (well vs moderate vs poor)	5.623	0.060	-	-	-
T Stage (T₃-T₄vs T₁-T₂)	13.586	<0.001	2.438	1.160-5.120	0.019
N Stage (N₂-N₃vs N₀-N₁)	20.178	<0.001	2.099	1.177-3.744	0.012
HR: Hazard ratio; CI: Confidence interval.

图4 对胃癌患者预后的评估价值

Fig.4 Assessment of the prognostic value of CDKN3 for gastric cancer patients.

图5 CDKN3促进胃癌细胞的迁移与侵袭

Fig.5 CDKN3 overexpression promotes gastric cancer cell migration and invasion. A,B: The expression of CDKN3 protein after lentivirus transfection was detected by Western blotting. C:Transwell experiment for analyzing the impact of CDKN3 on the migration and invasion abilities of MGC803 cells. D: Quantitative analysis of the number of migrated cells. E: Quantitative analysis of the number of invaded cells. *P<0.05, **P<0.01 vs control.

图6 CDKN3的KEGG和GO富集分析

Fig.6 KEGG and GO enrichment analysis of CDKN3. A: KEGG analysis of CDKN3 in gastric cancer. B: GO analysis of CDKN3 in gastric cancer.

图7 TUNEL染色检测胃癌及癌旁组织中细胞凋亡情况

Fig.7 TUNEL staining was used to detect cell apoptosis in gastric cancer and adjacent tissues. A,B: TUNEL staining and scoring of apoptosis in gastric cancer tissues sections and adjacent tissues sections. **P<0.01vs gastric cancer. GC: Gastric cancer.

图8 CDKN3对胃癌细胞凋亡和增殖的影响

Fig.8 Effect of CDKN3 on apoptosis and proliferation of MGC803 cells. A,B: Cell apoptosis rate detected by flow cytometry after lentivirus transfection. C: Expression of Bcl-2 and Bax in MGC803 cells. D: Quantitative analysis of Bcl-2/Bax ratio. E: Effect of CDKN3 on proliferation of MGC-803 cells. *P<0.05, **P<0.01 vs control.

图9 CDKN3对胃癌细胞p53/NF-κB信号通路的影响

Fig.9 Effect of CDKN3 on p53/NF-κB signaling in MGC803 cells. A: Expression of p53, p21, p65 and p-p65 in MGC803 cells. B, C: Quantitative analysis of p53, p21, p65 and p-p65 ratio. *P<0.05, **P<0.01 vs control.

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