敲除CDC20可明显抑制宫颈癌细胞的增殖及侵袭转移

doi:10.12122/j.issn.1673-4254.2025.06.09

摘要/Abstract

摘要：

目的探讨细胞分裂周期蛋白20（CDC20）在宫颈癌中的功能及其对宫颈癌C33A细胞增殖、迁移以及侵袭的影响和作用机制。方法 TCGA数据库分析CDC20在宫颈癌组织中的表达情况，免疫组化（IHC）检测宫颈癌组织与癌旁组织中的CDC20和β-Catenin蛋白表达水平。利用CRISPR/Cas9设计CDC20基因双靶点的sgRNA2＆7序列，构建重组质粒并测序验证。将宫颈癌细胞C33A按转染的质粒不同分为sgRNA-NC组和sgRNA-CDC20组，通过qRT-PCR和Western blotting检测CDC20的mRNA和蛋白表达水平，平板克隆检测其增殖能力，流式细胞术检测细胞周期和凋亡水平；Transwell小室法检测细胞迁移和侵袭能力。将2组C33A细胞分别接种于裸鼠（5只/组）双侧腋下左、右两侧，定期监测肿瘤体积并绘制其生长曲线，称取裸鼠体质量并脱臼处死。Western blotting分别检测细胞和瘤体组织中的CDC20和 β-Catenin蛋白的表达水平；免疫共沉淀（CoIP）和免疫荧光共定位验证CDC20与β-Catenin的相互作用。结果 CDC20和β-Catenin在宫颈癌组织中的表达高于癌旁组织（P<0.001）。体外细胞实验结果显示：与sgRNA-NC组细胞比较，sgRNA-CDC20敲除株的细胞增殖能力下降（P<0.001）且细胞凋亡显著升高（P<0.01）；迁移和侵袭能力受到明显抑制（P<0.05）。体内动物实验结果显示：与sgRNA-NC组比较，sgRNA-CDC20组的瘤体体积生长曲线明显减小（P<0.05），但裸鼠体质量无明显变化（P>0.05）；sgRNA-CDC20组中的细胞和组织中的CDC20和β-Catenin的蛋白水平明显降低（P<0.05），免疫共沉淀（CoIP）和免疫荧光验证CDC20和β-Catenin存在相关性。结论 CDC20在宫颈癌组织、细胞以及瘤体中的表达明显升高，敲除CDC20可明显抑制C33A细胞的增殖和侵袭转移能力，并促进凋亡，其机制可能与Wnt/β-Catenin信号通路密切相关。

关键词: CRISPR/Cas9, CDC20, 宫颈癌, Wnt/β-Catenin

Abstract:

Objective To study the effect of CDC20 knockdown on proliferation, migration and invasion of cervical cancer cells and its underlying mechanism. Methods CDC20 expression in cervical cancer tissues was analyzed using the TCGA database, and the protein expressions of CDC20 and β-Catenin in clinical specimens of cervical cancer and adjacent tissues were detected using immunohistochemistry. A dual target sgRNA2&7 sequence for CDC20 gene was designed for CDC20 gene knockdown in cervical cancer C33A cells using CRISPR/Cas9 technology, and CDC20 mRNA and protein expression levels in the transfected cells were detected using qRT-PCR and Western blotting. The changes in proliferation, cell cycle, apoptosis, migration and invasiveness of the transfected cells were evaluated using colony-forming assay, fluorescence activated cell sorting (FACS) and Transwell assay. In the animal experiment, naïve C33A cells and the cells with CDC20 knockdown were injected subcutaneously into the left and right axillae of nude mice (n=5) to observe tumor growth. The expressions of CDC20 and β-Catenin proteins in transfected cells and the xenograft were analyzed using Western blotting, and their interaction was confirmed by co-immunoprecipitation (CoIP) and immunofluorescence co-localization assays. Results Cervical cancer tissues expressed significantly higher CDC20 and β‑Catenin levels than the adjacent tissues. C33A cells with CDC20 knockdown showed reduced proliferation, increased apoptosis, and lowered migration and invasion abilities. CDC20 knockdown significantly suppressed the growth of C33A cell xenograft in nude mice, and the tumor-bearing mice did not exhibit obvious body mass changes. CDC20 and β-Catenin levels were both significantly lowered in C33A cells with CDC20 knockdown. Co-immunoprecipitation and co-localization assays confirmed the interaction between CDC20 and β‑Catenin. Conclusion CDC20 is highly expressed in cervical cancer tissues, and CDC20 knockdown can suppress proliferation, invasion, and metastasis while enhancing apoptosis of C33A cells, which is closely related with the regulation of the Wnt/β-Catenin signaling pathway.

Key words: CRISPR/Cas 9, CDC20, cervical cancer, Wnt/β-Catenin

莫艳秀, 舒洋, 莫钰兰, 刘峻彤, 徐欧欧, 邓华菲, 王岐本. 敲除CDC20可明显抑制宫颈癌细胞的增殖及侵袭转移[J]. 南方医科大学学报, 2025, 45(6): 1200-1211.

Yanxiu MO, Yang SHU, Yulan MO, Juntong LIU, Ouou XU, Huafei DENG, Qiben WANG. CRISPR-Cas9-mediated CDC20 gene knockout inhibits cervical cancer cell proliferation, invasion and metastasis[J]. Journal of Southern Medical University, 2025, 45(6): 1200-1211.

图/表 12

表1 sgRNA靶序列扩增引物

Tab.1 Target sequence of sgRNA of CDC20

	Sequence name	Target sequences
	sgRNA2 target sequence	5' GTAACGGCAGGTCTTCCGGC 3'
	sgRNA7 target sequence	5' GTGGCCAGAACGTGAACCAC 3'

表2 CDC20引物序列

Tab.2 Primer sequences for qRT-PCR of CDC20

Sequence name		Sequences (5'→3')
CDC20	CDC20-F	ATGCGCCAGAGGGTTATCAG
CDC20	CDC20-R	AGGATGTCACCAGAGCTTGC
GAPDH	GAPDH-F	GCTGTAGCCAAATCGTTGT
GAPDH	GAPDH-R	CCAGGTGGTCTCCTCTGA

图1 CDC20在宫颈癌和癌旁组织中的差异表达以及患者生存预后的关系

Fig.1 Analysis of CDC20 expression in cervical cancer vs normal tissues and its impact on patient survival. A: Analysis of CDC20 levels in cancer (red) vs normal (green) tissues based on TCGA data. B: CDC20 expression level is correlated with survival of cervical cancer patients (red: high expression; green: low expression). ***P<0.001.

表3 宫颈癌患者的年龄

Tab.3 Age distribution of cervical cancer patients with different histopathological types

Patients' age (year)	Number	Percentage of the age distribution	Adenocarcinoma type	Squamous cell carcinoma type
20-30	3	1.38%	1	1
30-40	17	7.80%	2	3
40-50	66	30.28%	18	33
50-60	74	33.94%	23	31
60-70	45	20.64%	10	22
70-80	8	3.67%	3	1
80+	4	1.83%	2	2
Total	218	100%	59	93

表4 CDC20在不同临床病理特征的患者中表达情况分析（218例）

Tab.4 Analysis of CDC20 expression in patients with different clinicopathological characteristics (218 Cases)

CDC20 expression	Pathological type		Degree of differentiation		Clinical staging		Lymph node metastasis
CDC20 expression	Squamous cell carcinoma	Adenocarcinoma	Medium and low differentiation	High differentiation	I、II stage	III stage	No exist	Exist
Positive	93 (42.7%)	59 (27.1%)	159 (72.9%)	188 (86.2%)	46 (21.1%)	191 (87.6%)	12 (26.1%)	24 (52.2%)
Negative	125 (57.3%)	159 (72.1%)	59 (27.1%)	30 (13.8%)	172 (78.9%)	27 (12.4%)	9 (19.6%)	1 (2.2%)
P	>0.05		>0.05		<0.05		<0.05

图2 免疫组化检测(IHC)CDC20和β-Catenin在宫颈癌组织与癌旁组织中的表达情况

Fig.2 Immunohistochemical analysis of CDC20 and β-Catenin in cervical cancer and normal tissues (Original magnification: ×100). A, B: CDC20 expression in cervical cancer and adjacent tissues. C, D: β-Catenin expression in cervical cancer and adjacent tissues. E, F: Comparison of CDC20 and β-Catenin expression levels between cervical cancer and adjacent tissues (5 fields per section). Data are presented as Mean±SD (n=3). **P<0.01 vs Tumor.

图3 C33A-CRISPR-CDC20 sgRNA 基因组PCR及测序结果

Fig.3 Results of C33A-CRISPR-CDC20 sgRNA genome PCR and sequencing. A: Agarose gel of PCR products. B: Sequencing results of the C33A-CRISPR-CDC20 sgRNA cells and sgRNA-NC cells.

图4 CDC20 基因敲除效果验证

Fig.4 Validation of CDC20 gene knockdown (Mean±SD, n=3). A: Fluorescent transfection using sgRNA-CDC20 (×100). B: CDC20 knockout confirmed by qRT-PCR. C: sgRNA-CDC20 knockdown shown by Western blotting. D: Quantification of CDC20 expression levels. *P<0.05 vs sgRNA-NC group.

图5 CDC20敲除对细胞克隆、周期与凋亡的影响

Fig.5 Impact of CDC20 knockdown on clone formation (A, B), cell cycle (C, D) and apoptosis (E, F) in cervical cancer C33A cells (Mean±SD, n=3). **P<0.01, ***P<0.001 vs sgRNA-NC group.

图6 敲除CDC20抑制宫颈癌细胞的迁移和侵袭能力

Fig.6 CDC20 knockdown inhibits migration and invasion of cervical cancer C33A cells (Mean±SD, n=3). A, B: Impact of CDC20 knockdown on migration and invasion of C33A cells (×100). C, D: Quantitative analysis of cell migration and invasion data. *P<0.05 vs sgRNA-NC group.

图7 CDC20敲除抑制裸鼠移植瘤生长

Fig.7 CDC20 knockdown suppresses xenograft tumor growth in nude mice (Mean±SD, n=3). A: Sizes and HE staining of the dissected tumors (×100). B: Body mass changes of the nude mice in the two groups. C: Tumor growth curves in the two groups. D: Observation of the tumor-bearing nude mice in the two groups. E: In vivo imaging of nude mice in the two groups. **P<0.001 vs sgRNA-NC group.

图8 敲除CDC20通过β-Catenin信号通路抑制宫颈癌细胞迁移和侵袭

Fig.8 CDC20 knockdown suppresses cervical cancer cell migration and invasion via the β-Catenin pathway. A, B: Western blotting for detecting β-Catenin levels in C33A cells (Mean±SD, n=3; ***P<0.001 vs sgRNA-NC group). C, D: Western blotting for detecting CDC20 and β‑Catenin expressions in tumor tissues from nude mic (Mean±SD, n=5; *P<0.05 vs sgRNA-NC group). E: Co-IP confirms CDC20 and β-Catenin interaction in mouse tissues. F: Immunofluorescence co-localization assay showing CDC20 and β-Catenin co-expression in C33A cells (×100).

参考文献 34

1	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49. doi：10.3322/caac.21660
2	Lin M, Ye MM, Zhou JH, et al. Recent advances on the molecular mechanism of cervical carcinogenesis based on systems biology technologies[J]. Comput Struct Biotechnol J, 2019, 17: 241-50. doi：10.1016/j.csbj.2019.02.001
3	Bruni L, Serrano B, Roura E, et al. Cervical cancer screening programmes and age-specific coverage estimates for 202 countries and territories worldwide: a review and synthetic analysis[J]. Lancet Glob Health, 2022, 10(8): e1115-27. doi：10.1016/s2214-109x(22)00241-8
4	Wang S, Chen B, Zhu Z, et al. CDC20 overexpression leads to poor prognosis in solid tumors: a system review and meta-analysis[J]. Medicine: Baltimore, 2018, 97(52): e13832. doi：10.1097/md.0000000000013832
5	Gayyed MF, El-Maqsoud NMRA, Tawfiek ER, et al. A comprehensive analysis of CDC20 overexpression in common malignant tumors from multiple organs: its correlation with tumor grade and stage[J]. Tumor Biol, 2016, 37(1): 749-62. doi：10.1007/s13277-015-3808-1
6	Song C, Lowe VJ, Lee S. Inhibition of Cdc20 suppresses the metastasis in triple negative breast cancer (TNBC)[J]. Breast Cancer, 2021, 28(5): 1073-86. doi：10.1007/s12282-021-01242-z
7	Xi X, Cao T, Qian Y, et al. CDC20 is a novel biomarker for improved clinical predictions in epithelial ovarian cancer[J]. Am J Cancer Res, 2022, 12(7): 3303-17.
8	Liu Y, Zou SH, Gao X. Bioinformatics analysis and experimental validation reveal that CDC20 overexpression promotes bladder cancer progression and potential underlying mechanisms[J]. Genes Genom, 2024, 46(4): 437-49. doi：10.1007/s13258-024-01505-x
9	Zhang Q, Huang H, Liu A, et al. Cell division cycle 20 (CDC20) drives prostate cancer progression via stabilization of β-catenin in cancer stem-like cells[J]. EBioMedicine, 2019, 42: 397-407. doi：10.1016/j.ebiom.2019.03.032
10	Chu Z, Zhang X, Li Q, et al. CDC20 contributes to the development of human cutaneous squamous cell carcinoma through the Wnt/β-catenin signaling pathway[J]. Int J Oncol, 2019, 54(5): 1534-44.
11	Pang F, Shi D, Yuan L. Screening and identification of key genes for cervical cancer, Ovarian Cancer and endometrial cancer by combinational bioinformatic analysis[J]. Curr Bioinform, 2023, 18(8): 647-57. doi：10.2174/1574893618666230428095114
12	Lin Y, Kong LL, Zhao YT, et al. The oncogenic role of EIF4A3/CDC20 axis in the endometrial cancer[J]. J Mol Med, 2024, 102(11): 1395-410. doi：10.1007/s00109-024-02486-w
13	Yu SJ, Zhao RR, Zhang BC, et al. Research progress and application of the CRISPR/Cas9 gene-editing technology based on hepato-cellular carcinoma[J]. Asian J Pharm Sci, 2023, 18(4): 100828. doi：10.1016/j.ajps.2023.100828
14	Chunjing L, Yujie Y, Wei Z, et al. Effect of silencing CDC20 on proliferation and cell cycle of endometrial cancer cells by inhibiting Wnt/β‑catenin signaling pathway[J]. Journal of Jilin University (Medicine Edition), 2024, 50(5): 1305-12.
15	Wang BQ, Li XP, Liu L, et al. β‑Catenin: oncogenic role and therapeutic target in cervical cancer[J]. Biol Res, 2020, 53(1): 33. doi：10.1186/s40659-020-00301-7
16	许晓燕, 冯群, 夏嫱. Wnt/β-catenin信号通路在宫颈癌发生发展中的研究进展[J]. 医学信息, 2021, 34(5): 1-5. doi：10.3969/j.issn.1006-1959.2021.05.001
17	莫艳秀, 徐欧欧, 李梁政, 等.基于TCGA数据库探讨CDC20在宫颈癌组织中的表达及其对癌细胞自噬的影响[J].现代肿瘤医学, 2025,33(3): 375-87. doi：10.3969/j.issn.1672-4992.2025.03.004
18	莫艳秀, 陈淑娟, 范云鹏, 等. 顺铂作用于人恶性睾丸生殖细胞瘤NT2细胞后对肿瘤干细胞标志物的影响[J]. 生命科学研究, 2018, 22(5): 390-6. doi：10.16605/j.cnki.1007-7847.2018.05.008
19	莫艳秀, 姚飞虹, 刘峻彤, 等. SP600125对人宫颈癌HeLa细胞增殖和侵袭的影响[J]. 肿瘤防治研究, 2022, 49(4): 304-13. doi：10.3971/j.issn.1000-8578.2022.21.0807
20	Mo YX, Fan YP, Fu W, et al. Acute immune stress improves cell resistance to chemical poison damage in SP600125-induced pol-yploidy of fish cells in vitro [J]. Fish Shellfish Immunol, 2019, 84: 656-63. doi：10.1016/j.fsi.2018.10.063
21	Burmeister CA, Khan SF, Schäfer G, et al. Cervical cancer therapies: Current challenges and future perspectives[J]. Tumour Virus Res, 2022, 13: 200238. doi：10.1016/j.tvr.2022.200238
22	Mondal G, Sengupta S, Panda CK, et al. Overexpression of Cdc20 leads to impairment of the spindle assembly checkpoint and aneuplo-idization in oral cancer[J]. Carcinogenesis, 2007, 28(1): 81-92. doi：10.1093/carcin/bgl100
23	Ni K, Hong L. Current progress and perspectives of CDC20 in female reproductive cancers[J]. Curr Mol Med, 2023, 23(3): 193-9. doi：10.2174/1573405618666220321130102
24	Tang J, Lu M, Cui Q, et al. Overexpression of ASPM CDC20 and TTK confer a poorer prognosis in breast cancer identified by gene co-expression network analysis[J]. Front Oncol, 2019, 9: 310. doi：10.3389/fonc.2019.00310
25	严龙君, 夏萌, 李明, 等. CDC20、TOP2A、NEK2的表达特征以及对食管鳞状细胞癌细胞增殖和迁移的影响[J]. 临床和实验医学杂志, 2022, 21(21): 2273-7. doi：10.3969/j.issn.1671-4695.2022.21.009
26	石峰, 孙文功, 刘增振, 等. CDC20基因的表达对前列腺癌患者临床病理特征及预后的影响[J]. 解放军医学杂志, 2021, 46(11): 1092-7.
27	Gu Q, Li F, Ge S, et al. CDC20 knockdown and acidic microenvironment collaboratively promote tumorigenesis through inhibiting autophagy and apoptosis[J]. Molecular Therapy-Oncolytics, 2020, 17: 94-106. doi：10.1016/j.omto.2020.03.015
28	Shang G, Ma X, Lv G. Cell division cycle 20 promotes cell proliferation and invasion and inhibits apoptosis in osteosarcoma cells[J]. Cell Cycle, 2018, 17(1): 43-52. doi：10.1080/15384101.2017.1387700
29	Cheng S, Castillo V, Sliva D. CDC20 associated with cancer metastasis and novel mushroom-derived CDC20 inhibitors with antimetastatic activity[J]. Int J Oncol, 2019, 54(6): 2250-6.
30	Gao Y, Zhang B, Wang Y, et al. Cdc20 inhibitor apcin inhibits the growth and invasion of osteosarcoma cells[J]. Oncology reports, 2018, 40(2): 841-8.
31	Wang B, Wang M, Li X, et al. Variations in the Wnt/β-catenin pathway key genes as predictors of cervical cancer susceptibility[J]. Pharmgenomics Pers Med, 2020, 13: 157-65. doi：10.2147/pgpm.s248548
32	Song P, Gao ZR, Bao YG, et al. Wnt/β-catenin signaling pathway in carcinogenesis and cancer therapy[J]. J Hematol Oncol, 2024, 17(1): 46. doi：10.1186/s13045-024-01563-4
33	Deng Q, Wu L, Li Y, et al. MYBL2 in synergy with CDC20 promotes the proliferation and inhibits apoptosis of gastric cancer cells[J]. Advances in Clinical and Experimental Medicine, 2021, 30(9): 957-66. doi：10.17219/acem/135938
34	Li K, Mao Y, Lu L, et al. Silencing of CDC20 suppresses metastatic castration-resistant prostate cancer growth and enhances chemo-sensitivity to docetaxel[J]. International Journal of Oncology, 2016, 49(4): 1679-85. doi：info:doi/10.3892/ijo.2016.3671

[1]	朱胤福, 李怡燃, 王奕, 黄颖而, 龚昆翔, 郝文波, 孙玲玲. 桂枝茯苓丸活性成分常春藤皂苷元通过抑制JAK2/STAT3通路抑制宫颈癌细胞的生长[J]. 南方医科大学学报, 2025, 45(7): 1423-1433.
[2]	岳雅清, 牟召霞, 王希波, 刘艳. Aurora-A过表达通过激活NF-κBp65/ARPC4信号轴促进宫颈癌细胞的侵袭和转移[J]. 南方医科大学学报, 2025, 45(4): 837-843.
[3]	张文静, 张诺, 杨子, 张小凤, 孙奥飞, 王炼, 宋雪, 耿志军, 李静, 胡建国. BZW1 高表达促进胃癌细胞的侵袭和转移：基于调控Wnt//β-catenin通路和促进上皮间质转化[J]. 南方医科大学学报, 2024, 44(2): 354-362.
[4]	刘欢, 饶阳, 孙传铸, 王洋涛, 齐顺, 李想, 田萌, 禹汛, 穆允凤. 宫颈癌放疗后癌因性失眠患者的大脑功能网络与个体-经颅磁刺激干预的相关性：基于图论分析[J]. 南方医科大学学报, 2023, 43(9): 1629-1635.
[5]	郭雨军, 李婷, 杨鑫, 祁振宇, 陈利, 黄思娟. 放疗计划定量评估在宫颈癌外照射精准放疗流程管理中的应用[J]. 南方医科大学学报, 2023, 43(6): 1035-1040.
[6]	毛建英, 杨文静, 郭和, 董瑞丽, 任丽芳, 李树斌. 体外重建宫颈癌的三维培养模型：基于人宫颈癌组织的去细胞支架[J]. 南方医科大学学报, 2023, 43(2): 157-165.
[7]	肖姝喆, 成燕玲, 朱云, 唐芮, 古建标, 兰林, 何志华, 刘丹琼, 耿岚岚, 程旸, 龚四堂. WNT2b高表达的成纤维细胞破坏肠道黏膜屏障[J]. 南方医科大学学报, 2023, 43(2): 206-212.
[8]	梁健, 余凤姿, 朱金汉, 宋婷. 准直器叶片到位精度对宫颈癌容积调强计划验证的影响[J]. 南方医科大学学报, 2022, 42(7): 1089-1094.
[9]	赵行宇, 杨昆, 宋梓桐, 何涵, 张巍. 胡桃醌通过促进c-Myc泛素化降解抑制宫颈癌细胞的生长和促进凋亡[J]. 南方医科大学学报, 2022, 42(7): 1026-1031.
[10]	霍叶琳, 王月, 安娜, 杜雪. TIM-3 基因在上皮性卵巢癌中高表达并促进癌细胞的增殖和迁移[J]. 南方医科大学学报, 2022, 42(2): 190-200.
[11]	林小丽, 何金梅, 卢佳蕴, 黄承颖, 刘楠. 纳米炭示踪前哨淋巴结在宫颈癌诊治中的应用价值[J]. 南方医科大学学报, 2022, 42(12): 1896-1901.
[12]	孙子久, 胡静, 胡凯, 唐敏, 孙恃雷, 方玉婷, 余伙梅, 张彦. LncRNA SNHG3对宫颈癌细胞SiHa增殖、迁移及侵袭的影响[J]. 南方医科大学学报, 2021, 41(6): 931-936.
[13]	朱梦云, 崔双慧, 郝泽宇, 王文锐, 杨清玲, 陈昌杰, 王剑锋, 周琦. 姜黄素通过 Wnt/β-catenin 信号通路诱导人晶状体上皮细胞的凋亡和细胞周期阻滞[J]. 南方医科大学学报, 2021, 41(5): 722-728.
[14]	张涛, 李维丽, 邱晓拂, 刘百川, 李高远, 冯才鑫, 廖俊发, 林康健. TEAD1基因敲除对糖尿病性ED大鼠阴茎海绵体平滑肌细胞表型转化的影响[J]. 南方医科大学学报, 2021, 41(4): 567-573.
[15]	周丽婷, 叶颖, 原海波, 吴超逸, 吴淑燕. gsdmd基因敲除巨噬细胞RAW 264.7的构建：基于CRISPR/Cas9 系统[J]. 南方医科大学学报, 2021, 41(1): 116-122.