CRISPR-Cas9-mediated CDC20 gene knockout inhibits cervical cancer cell proliferation, invasion and metastasis

doi:10.12122/j.issn.1673-4254.2025.06.09

Abstract

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

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.

Figures/Tables 12

Tab.1 Target sequence of sgRNA of CDC20

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

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

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.

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

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

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.

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.

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.

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.

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.

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.

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).

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