CDC20通过稳定NLRP3的表达促进食管癌细胞增殖的研究

doi:10.19401/j.cnki.1007-3639.2024.05.004

中国癌症杂志 ›› 2024, Vol. 34 ›› Issue (5): 473-484.doi: 10.19401/j.cnki.1007-3639.2024.05.004

CDC20通过稳定NLRP3的表达促进食管癌细胞增殖的研究

关瑞瑞¹^,²(), 郝茜³, 张雅琦¹^,⁴, 孙庆港¹^,⁴, 陈怡恬¹^,⁴, 李秀敏²^,⁵, 周祥³(), 韩涛¹()

1.新乡医学院健康中原研究院，新乡市分子癌学重点实验室，河南新乡 453003
2.新乡医学院第三附属医院消化内科，河南新乡 453003
3.复旦大学附属肿瘤医院肿瘤研究所，复旦大学上海医学院肿瘤学系，上海 200032
4.新乡医学院基础医学院，河南新乡 453003
5.新乡医学院第一附属医院消化内科，河南新乡 453003

收稿日期:2023-12-27 修回日期:2024-03-06 出版日期:2024-05-30 发布日期:2024-06-07
通信作者: 韩涛（ORCID: 0000-0002-5943-8520），博士，教授；周祥（ORCID: 0000-0002-1172-7948），博士，研究员。
作者简介:关瑞瑞（ORCID: 0009-0000-0235-0723），硕士。
基金资助:
国家自然科学基金(81972330)

CDC20 facilitates the proliferation of esophageal carcinoma cell by stabilizing NLRP3 expression

GUAN Ruirui¹^,²(), HAO Qian³, ZHANG Yaqi¹^,⁴, SUN Qinggang¹^,⁴, CHEN Yitian¹^,⁴, LI Xiumin²^,⁵, ZHOU Xiang³(), HAN Tao¹()

1. Institutes of Health Central Plains, Xinxiang Key Laboratory for Molecular Oncology, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
2. Department of Gastroenterology, Third Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, Henan Province, China
3. Cancer Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
4. School of Basic Medical Sciences, Xinxiang Medical University, Xinxiang 453003, Henan Province, China
5. Department of Gastroenterology, First Affiliated Hospital of Xinxiang Medical University, Xinxiang 453003, Henan Province

Received:2023-12-27 Revised:2024-03-06 Published:2024-05-30 Online:2024-06-07
Contact: HAN Tao, ZHOU Xiang

摘要/Abstract

摘要：

背景与目的： 食管癌（esophageal carcinoma，ESCA）是死亡率较高的恶性肿瘤之一，其发生、发展的机制不明。CDC20被认为具有癌基因功能，其表达失调与肿瘤的发生、发展密切相关。CDC20在多种肿瘤中表达升高，敲低CDC20能够抑制肿瘤细胞增殖。NLRP3是炎症小体的主要成分之一，炎症小体失调与肿瘤的发生、发展也密切相关。本研究旨在探究CDC20是否通过NLRP3促进ESCA细胞增殖，同时分析其调控机制。方法： 通过癌症基因组图谱（The Cancer Genome Atlas，TCGA）数据库和GTEx公共数据库分析ESCA患者中CDC20和NLRP3基因的表达水平。收集新乡医学院第一附属医院收治的80例ESCA患者的临床病理学资料和组织，通过免疫组织化学染色检测NLRP3在ESCA患者中的蛋白表达水平。本研究通过新乡医学院第一附属医院伦理委员会的审批（编号：EC-021-137）。通过短发夹RNA（short hairpin RNA，shRNA）技术检测敲低CDC20和NLRP3基因后对食管鳞状细胞癌细胞系EC9706和KYSE150增殖能力的影响。通过免疫共沉淀（Co-immunoprecipitation，Co-IP）、蛋白酶体抑制剂和泛素化实验检测CDC20是否与NLRP3相互作用，以及阐明CDC20是否通过泛素化途径调控NLRP3表达。结果： TCGA数据库分析结果显示，ESCA组织中CDC20和NLRP3 mRNA表达水平明显高于癌旁组织。免疫组织化学结果也进一步显示，与癌旁组织相比，CDC20、NLRP3在ESCA组织中蛋白表达水平升高。敲低CDC20和NLRP3基因可抑制ESCA细胞增殖。Co-IP、蛋白酶体抑制剂和泛素化实验证实CDC20通过NLRP3的LRR区与NLRP3相互作用，且CDC20通过促进NLRP3泛素化稳定其表达。结论： CDC20和NLRP3在ESCA癌组织中表达上调，且CDC20通过泛素化NLRP3稳定其表达，从而促进ESCA细胞增殖，提示CDC20和NLRP3可能是ESCA潜在的诊断靶向标志物。

关键词: 食管癌, CDC20, NLRP3, 泛素化, 增殖

Abstract:

Background and purpose: Esophageal carcinoma (ESCA) is one of the malignant tumors with high mortality rate, and the underlying mechanism of its development is largely unknown. CDC20 plays an important role in tumorigenesis, and its dysregulated expression is closely related to tumor occurrence and development. The expression of CDC20 is increased in a variety of tumors, and knocking down CDC20 can inhibit tumor cell proliferation. NLRP3 is the main component of the inflammasome, and inflammasome is also closely related to tumor occurrence and development. Here, our study aimed to investigate whether CDC20 promotes the proliferation of ESCA cells through NLRP3 and its regulatory mechanism. Methods: The expression levels of CDC20 and NLRP3 genes in ESCA patients were analyzed using The Cancer Genome Atlas (TCGA) detabase and GTEx public database. We collected clinical and pathological data and tissues from 80 ESCA patients at the First Affiliated Hospital of Xinxiang Medical College, and detected the protein expression of NLRP3 in ESCA patients through immunohistochemistry staining. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xinxiang Medical College (Number: EC-021-137). We studied the effects of knocking down CDC20 and NLRP3 gene on the proliferation ability of esophageal squamous cell carcinoma cells EC9706 and KYSE150 using short hairpin RNA (shRNA) technology. Co-immunoprecipitation (Co-IP), proteasome inhibitors and ubiquitination experiments were used to detect whether CDC20 interacts with NLRP3, and to elucidate whether CDC20 regulates NLRP3 expression through the ubiquitination pathway. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xinxiang Medical College (Number: EC-021-137). Results: The TCGA database analysis showed that the expression levels of CDC20 and NLRP3 mRNA were significantly higher in the cancer tissues of ESCA patients than in the adjacent tissues. The immunohistochemistry results further showed that compared with adjacent tissues, the protein expression levels of CDC20 and NLRP3 were increased in ESCA tissues. Knocking down CDC20 and NLRP3 genes inhibited the proliferation of ESCA cells. Co-IP, proteasome inhibitors and ubiquitination experiments confirmed that CDC20 interacted with NLRP3 through its leucine-rich repeat (LRR), and CDC20 stabilized its expression by promoting NLRP3 ubiquitination. Conclusion: CDC20 and NLRP3 are upregulated in ESCA tissues, and CDC20 stabilizes their expression through ubiquitination of NLRP3, promoting ESCA cell proliferation. This suggests that CDC20 and NLRP3 may be potential diagnostic targets for ESCA.

Key words: Esophageal carcinoma, CDC20, NLRP3, Ubiquitination, Proliferation

中图分类号:

R735.1

关瑞瑞, 郝茜, 张雅琦, 孙庆港, 陈怡恬, 李秀敏, 周祥, 韩涛. CDC20通过稳定NLRP3的表达促进食管癌细胞增殖的研究[J]. 中国癌症杂志, 2024, 34(5): 473-484.

GUAN Ruirui, HAO Qian, ZHANG Yaqi, SUN Qinggang, CHEN Yitian, LI Xiumin, ZHOU Xiang, HAN Tao. CDC20 facilitates the proliferation of esophageal carcinoma cell by stabilizing NLRP3 expression[J]. China Oncology, 2024, 34(5): 473-484.

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Pathologic feature	Total n	CDC20 expression n		χ² value	P value	NLRP3 expression n		χ² value	P value
Pathologic feature	Total n	High	Low	χ² value	P value	High	Low	χ² value	P value
Organizational classification				5.788	0.016			4.290	0.038
G1	54	24	30			36	18
G2-G3	26	19	7			11	15
Depth of infiltration				8.459	0.004			4.921	0.027
T1-T2	15	3	12			5	10
T3-T4	65	40	25			42	23
Lymph node metastasis				5.729	0.020			5.071	0.024
No	57	26	31			29	28
Yes	23	17	6			18	5
TNM staging
Ⅰ	27	9	18	3.884	0.049	13	14	3.884	0.049
Ⅱ	21	13	8			16	5
Ⅲ	25	19	6	5.153	0.020	17	8	6.412	0.011
Ⅳ	7	2	5			1	6

CDC20通过稳定NLRP3的表达促进食管癌细胞增殖的研究

CDC20 facilitates the proliferation of esophageal carcinoma cell by stabilizing NLRP3 expression

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