KIF20A promotes cervical cancer progression by interacting with CLIP1

Abstract

Cervical cancer (CC) remains a major cause of cancer-related mortality among women worldwide. Although screening and vaccination have reduced its incidence, advanced-stage cases still pose a serious clinical challenge. This study aimed to identify novel biomarkers and therapeutic targets for CC through integrated bioinformatics and experimental approaches. Analysis of multiple datasets (GSE63514, GSE67522, and TCGA-GTEx) revealed 426 consistently upregulated genes in CC, among which several kinesin family members, including kinesin family member 20 A (KIF20A), were significantly overexpressed. KIF20A mRNA and protein levels were markedly elevated in CC patient tissues (n = 306 tumors vs. 22 normal controls). Functional assays demonstrated that KIF20A knockdown strongly inhibited cell proliferation, migration, and tumor growth in vitro and in vivo. Mechanistically, KIF20A interacts with CAP Gly domain containing linker protein 1 (CLIP1) and enhances its protein stability. Rescue experiments confirmed that CLIP1 silencing reversed the oncogenic effects of KIF20A overexpression. These results unveil a previously unrecognized KIF20A-CLIP1 regulatory axis in CC progression and suggest its potential as both a biomarker and a therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-42883-4.

Introduction

Cervical cancer (CC) remains a significant global health concern and a leading cause of cancer-related deaths among women worldwide. It is the fourth most commonly diagnosed cancer and the fourth leading cause of cancer death in women globally¹. In 2022, there were approximately 600,000 new cases of CC and about 340,000 deaths worldwide². The burden of disease is particularly high in developing countries, where incidence and mortality rates remain disproportionately elevated due to limited access to prevention and treatment services^3,4. China accounts for over 10% of global CC cases and deaths. In response to this burden, China implemented a nationwide screening program in 2009 aimed at identifying effective treatment strategies and reducing CC incidence⁵. Papanicolaou (Pap) smear screening and human papillomavirus (HPV) vaccination have significantly reduced CC mortality rates⁶. However, a substantial proportion of patients are diagnosed at advanced stages, where treatment options are limited⁷. This underscores the urgent need for novel biomarkers for early detection and targeted therapeutic strategies in CC.

The pathogenesis of CC is predominantly driven by persistent infection with high-risk human papillomavirus (HR-HPV) types, particularly HPV16 and 18, which account for approximately 70% of cases⁸. The viral oncoproteins E6 and E7 play a central role by degrading tumor suppressor p53 and retinoblastoma protein (pRb), respectively, leading to uncontrolled cell cycle progression and genomic instability⁹. Recent research has further elucidated that HPV E6/E7 can induce epigenetic modifications, such as histone acetylation (e.g., H3K27ac) via recruiters like CBP/p300, to activate super-enhancers driving oncogene expression or inhibit ferroptosis through acetylation of lncRNA-encoded peptides^10,11. Additionally, aberrant miRNA networks modulated by HPV (e.g., suppression of miR-34a/miR-375 and activation of miR-21/miR-93-5p) contribute to tumor progression by dysregulating key pathways like PI3K/AKT and TGF-β¹². The tumor microenvironment is also compromised through mechanisms such as M2 polarization of tumor-associated macrophages facilitating immune evasion¹³, while dysregulated Hippo-YAP pathway further promote proliferation, metastasis¹⁴. Despite these advances, the comprehensive pathogenesis of CC remains not fully elucidated. These mechanisms collectively underscore a complex interplay between viral factors, host genetic alterations, and immune modulation in cervical carcinogenesis, highlighting the critical need for further research to fully decipher the pathogenic mechanisms and identify novel therapeutic targets.

KIF20A, a member of the kinesin protein superfamily, is composed of 890 amino acids, including an N-terminal motor domain, a C-terminal tail domain, and a coiled-coil domain¹⁵. The N-terminal domain promotes microtubule polymerization in the presence of ATP, while the C-terminal domain regulates membrane fusion and vesicle transport¹⁶. KIF20A plays a pivotal role in spindle assembly and mitosis¹⁷. Abnormal KIF20A expression has been observed in various tumors, where its overexpression contributes to tumor progression^18–20. For example, KIF20A overexpression accelerates fibrosarcoma progression via activation of the PI3K-Akt signaling pathway²¹. Furthermore, KIF20A inhibits anti-tumor immunity in hepatocellular carcinoma (HCC) by suppressing c-Myc ubiquitination, thus reducing the efficacy of anti-PD-1 immunotherapy²⁰. Similarly, CAP-Gly domain-containing linker protein 1 (CLIP1) is a microtubule-binding protein involved in microtubule polymerization and cytoskeletal dynamics²². Aberrant CLIP1 expression has been implicated in the progression of various tumors^23,24. For example, in Lissencephaly 1 (LIS1)-driven salivary gland adenoid cystic carcinoma (SACC), CLIP1 interacts with LIS1, and its inhibition reverses tumor progression²⁵. Despite their distinct functions, the interaction between KIF20A and CLIP1 in CC progression remains underexplored. Despite established roles of kinesin and microtubule-associated proteins in cancer, the functional relationship between KIF20A and CLIP1—particularly in CC—remains largely unexplored. Although both molecules have been independently implicated in tumorigenesis, it is unclear whether they cooperate mechanistically in cervical carcinogenesis. Thus, the present study aims to test the hypothesis that KIF20A facilitates CC progression through a functional interaction with CLIP1. Elucidating this axis may provide new insights into CC biology and uncover potential therapeutic targets.

In this study, KIF20A was identified as a key biomarker in CC through analysis of public databases and clinical samples. Functionally, KIF20A knockdown using shRNA significantly inhibited CC cell proliferation and migration in vitro and in vivo. Mechanistically, KIF20A interacts with CLIP1 to stabilize its protein expression, thereby promoting tumor progression. Notably, CLIP1 knockdown effectively reversed the tumor-promoting effects of KIF20A, suggesting that the KIF20A-CLIP1 axis could serve as a promising therapeutic target for CC.

Materials and methods

Materials

The following antibodies were utilized in the experiments: anti-KIF20A (Proteintech, 15911-1-AP, 1:2000), anti-GAPDH (ZEN BIO, 200306-7E4, 1:10000), anti-CLIP1 (Proteintech, 23839-1-AP, 1:2000), anti-Rabbit IgG(H + L)-HRP secondary antibody (Abclonal, AS014, 1:10000), and anti-Mouse IgG(H + L)-HRP secondary antibody (Abclonal, AS003, 1:10000). The Immunohistochemical diaminobenzidine (DAB) staining kit was obtained from Solarbio Life Science (DA1010), and Lipofectamine 2000 (Invitrogen, 11668-027) was used for plasmid transfection. The plasmids used in this study are constructed by our lab.

Bioinformatics collection and analysis

RNA-seq and clinical data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) were downloaded from the UCSC Xena database (https://xenabrowser.net/datapages/). Statistical analysis was performed using R software v4.0.3. GEO datasets were downloaded from Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) and analyzed using GEO2R. Statistical significance was defined as p < 0.05.

Patient samples collection and immunohistochemistry

Cervical tumor and normal tissues were collected from the Second Hospital, Anhui Medical University, with ethical approval from the Institutional Ethics Committee. Tissue sections were deparaffinized and rehydrated using citrate buffer for antigen retrieval. The sections were then blocked with 2.5% goat serum (Solarbio, A8010), followed by incubation with the KIF20A antibody overnight at 4 °C. Afterward, the sections were incubated with a biotinylated secondary antibody at room temperature for 1 h. Signals were developed using the DAB substrate kit (VAZYME,36312ES50), and images were captured under a Leica microscope (Leica, LeicaDFC420C). KIF20A expression was graded based on staining intensity.

Cell culture and transfection

CC cell lines, Hela and SiHa, were purchased from American Type Culture Collection (ATCC CCL-2, HTB-35) and cultured in Dulbecco’s Modified Eagle medium (DMEM) (Gibco, 11965092) supplemented with 10% fetal bovine serum (FBS, Gibco, A5670701) at 37 °C in 5% Hela. For plasmid transfection, cells were transiently transfected with the indicated plasmids using Lipofectamine 2000 (Lipofectamine 2000, 11668019), following the manufacturer’s instructions.

Establishment of stable shRNA-mediated knockdown cell lines

HEK293T cells (ATCC, CRL-3216) were transfected with the PLKO.1 backbone plasmid, pMD2.G, and pSPAX2. After 48 and 72 h, the viral supernatant was collected. Cells were then infected with viral supernatants. After 24 h, the virus-containing medium is replaced with fresh culture medium. At 48–72 h post-transduction, puromycin (MCE, HY-B1743) is added to the medium at a predetermined concentration (2.5 µg/ml) that effectively kills non-transduced cells. The selection medium is refreshed every 2–3 days. After approximately 7–10 days, puromycin-resistant pools typically emerge. These clones are then expanded and validated for stable expression of the target gene via Western blot.

The following oligonucleotides (5’ to 3’) were used for KIF20A and CLIP1 knockdown:

KIF20A RNAi-1#:

5’-CCGGGCAGTGCAAAGCAGAGCTAAACTCGAGTTTAGCTCTGCTTTGCACTGCTTTTTG-3’;

KIF20A RNAi-2#:

5’-CCGGCCGATGACGATGTCGTAGTTTCTCGAGAAACTACGACATCGTCATCGGTTTTTG-3’;

CLIP1 RNAi-1#:

5’-CCGGCCCGTCAACAAATCTAGGAAACTCGAGTTTCCTAGATTTGTTGACGGGTTTTTG-3’.

Establishment of KIF20A‑overexpressing Cell Lines

Hela and SiHa cells were transfected with either an empty vector or a Flag-KIF20A expression plasmid using Lipofectamine 2000. In parallel, the Flag-KIF20A plasmid was also transfected into CLIP1-knockdown cell lines. At 48 h post-transfection, cells were harvested for Western blot analysis to examine KIF20A and CLIP1 protein expression. The remaining cells were used for subsequent functional assays.

Immunoblotting

Cells were lysed with Radio Immunoprecipitation Assay buffer (RIPA) (Solarbio, R0010) containing PMSF (Solarbio, P0100) for 30 m on ice. After centrifugation at 12,000 g for 15 min, the supernatants were boiled with loading buffer for 5 min. Equal amounts of protein were separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk for 1 h. Following blocking, primary antibodies were applied and incubated with the membranes overnight at 4 °C. Excess primary antibodies were washed with phosphate buffered saline with tween-20 (PBST), and signals were detected using horseradish peroxidase-conjugated anti-mouse/rabbit secondary antibodies.

CCK-8 and wound healing assay

The cell viability was assessed using the Cell Counting Kit-8 (VAZYME, A311-01) according to the manufacturer’s instructions. Briefly, cells were seeded in 96-well plates at a density of 2 × 10³cells per well and cultured under appropriate conditions. After treatments,10µL of Cell Counting Kit-8 (CCK-8) reagent was added to each well followed by incubation at 37 °C for 2 h. The absorbance was measured at 450 nm using a microplate reader. The cell viability was calculated as a percentage relative to the control group. Cell migration ability was evaluated using a wound healing assay. Cells were seeded in 6-well plates and cultured until they reached 90–95% confluence. A sterile 200 µL pipette tip was used to create a straight scratch wound across the cell monolayer. The detached cells were removed by washing twice with phosphate-buffered saline (PBS). Fresh serum-free medium was then added. Images of the same wound fields were captured at 0 h and 24 h using an inverted microscope (Nikon, Japan). The migration distance was quantified using ImageJ software. The wound closure rate was calculated as follows:

Wound closure (%) = [(Area at 0 h - Area at 24 h)/Area at 0 h]×100%.

Immunoprotein co-precipitation (Co-IP)

HEK293T cells were transfected with plasmids encoding Flag-KIF20A and HA-CLIP1. After 24 h, the cells were washed with PBS and lysed using RIPA lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF). The lysate was centrifuged at 14,000 × g for 10 m at 4 °C to collect the supernatant. Immunoprecipitation was performed by incubating the supernatant with the appropriate antibodies overnight at 4 °C. Subsequently, the mixture was incubated with protein A/G–agarose beads for 2 h at 4 °C. The beads were then washed five times with lysis buffer, followed by boiling in loading buffer for 5 min. Co-IP with an antibody against the endogenous protein was performed. Briefly, SiHa cells were washed with ice-cold PBS and lysed using RIPA buffer. The cell lysate supernatant was incubated with 1 µg of the corresponding antibody at 4 °C for 3 h. Subsequently, protein A/G–agarose beads were added to the mixture, followed by further incubation at 4 °C for 2 h. The beads were then washed five times with cell lysis buffer. Finally, the immunoprecipitated complexes were eluted by boiling in loading buffer for 5 min. The samples were finally analyzed by SDS–PAGE.

Generation of tumor xenograft models

Six-week-old female nude mice were purchased from Charles River and housed in a specific pathogen-free (SPF) facility. The mice were randomly divided into two groups (n = 5 per group). SiHa cells were washed with ice-cold PBS, digested with trypsin. Each mouse was subcutaneously inoculated with 5 × 10⁶ SiHa cells in the inguinal mammary gland. Tumor size was measured every five days using a vernier caliper, and tumor volume was calculated using the formula: volume = (length × width²)/2. Tumor growth curves were plotted based on the volume measurements. Following the final behavioral test, mice were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg) and then euthanized by cervical dislocation to ensure death. After the completion of the experiment, euthanasia was performed on all mice. The method involved a two-step process: first, the animals were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (100 mg/kg) to achieve a surgical plane of anesthesia. Subsequently, cervical dislocation was performed as a secondary physical method to confirm death.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 5.0. Data were analyzed using a Student’s t-test, and results are presented as mean ± SD from two independent experiments, each performed in triplicate. A p-value of < 0.05 was considered statistically significant.

All human procedures in this study were reviewed and approved by the Ethics Committee of Anhui Medical University (Approval No. LLSC20241076) in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants prior to inclusion in the study, as approved by the Institutional Review Board of Anhui Medical University Second Affiliated Hospital. The animal experiments were conducted under the approval of the Animal Ethics Committee of Anhui Medical University (Approval No. LLSC20240765) and complied with the ARRIVE guidelines. All procedures were performed in accordance with the institutional guidelines for the care and use of laboratory animals.

Results

Screening of differentially expressed genes (DEGs)

To explore the differential expression of genes between patients with CC and paired normal tissues, this study utilized two GEO datasets (GSE63514 and GSE67522) along with the TCGA and GTEx datasets to identify reliable DEGs. The selection criteria were set to a fold change > 1.5 and a p-value < 0.05, with DEGs visualized through volcano plots and heatmaps. Analysis of the GSE63514 dataset, which includes 28 patients with CC and 24 normal tissues, revealed 2,603 upregulated genes and 1,461 downregulated genes in CC samples (Fig. 1A and B). Similarly, in the GSE67522 dataset, which consists of 20 patients with CC and 22 normal tissues, 1,140 genes were upregulated, and 1,228 genes were downregulated in the cancer samples (Fig. 1C and D). Further, an analysis of 306 patients with CC and 22 normal tissues from the TCGA and GTEx datasets showed 4,216 upregulated genes and 4,583 downregulated genes in CC (Fig. 1E and F).

Fig. 1.

Identification of differentially expressed genes (DEGs) between cervical cancer and normal samples across three cohorts. (A, B) Volcano plots and heatmap of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets. Analysis of the GSE63514 dataset (28 CC vs. 24 normal tissues) identified 2,603 upregulated (red) and 1,461 downregulated (blue) genes in CC, with thresholds of |log2 fold change| > 1.5 and p-value < 0.05. Panel A shows the volcano plot, and Panel B shows the corresponding heatmap of top DEGs. (C, D) Volcano plots and heatmap of the GSE63514 dataset. Analysis of the GSE67522 dataset (20 CC vs. 22 normal tissues) identified 1,140 upregulated and 1,228 downregulated genes in CC. Panel C shows the volcano plot, and Panel D shows the heatmap. (E, F) Volcano plots and heatmap of the GSE67522 dataset. Combined analysis of TCGA-CC (306 samples) and GTEx-normal (22 samples) identified Differentially expressed genes (DEGs) which include 4,216 upregulated and 4,583 downregulated genes in CC. Panel E shows the volcano plot, and Panel F shows the heatmap. Each volcano plot displays genes with significant upregulation (red), downregulation (blue), or non-significant change (gray). Heatmaps illustrate expression patterns of selected DEGs across samples (rows: genes, columns: samples; red indicates high expression, blue indicates low expression).

Identification of hub up-regulation and down-regulation genes

To identify the co-upregulated and co-downregulated genes across the three datasets, a Venn diagram analysis was performed (Fig. 2A and B). A total of 426 genes were consistently upregulated in GSE63514, GSE67522, and TCGA-GTEx datasets. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis^26–28 revealed significant enrichment in biological processes such as cell cycle regulation, DNA metabolic processes, and retinoblastoma gene-related cancer pathways (Fig. 2C and E). These findings suggest that the shared upregulated genes are involved in common pathways and biological functions across datasets. Similarly, 150 genes were consistently downregulated, and they are involved in processes such as negative regulation of cell proliferation, extracellular matrix (ECM) remodeling, and mesenchymal cell differentiation (Fig. 2D and F). Among the upregulated genes, seven kinesin family members—KIFC1, KIF2A, KIF2C, KIF11, KIF14, KIF15, KIF18A, KIF20A, and KIF23 (Table 1)—were found to be overexpressed in patients with CC based on bioinformatics analyses. These results suggest that the kinesin family may play a pivotal role in CC progression and could serve as potential prognostic markers for the disease.

Fig. 2.

Hub gene selection and signaling pathway enrichment. (A, B) Venn diagram of the up- and downregulated hub genes across three cohorts. Venn diagrams showing the overlap of upregulated (A) and downregulated (B) genes among the three analyzed datasets (GSE63514, GSE67522, TCGA/GTEx). A core set of 426 genes was consistently upregulated, and 150 genes were consistently downregulated across all datasets. (C, D) Gene Ontology (GO) enrichment analyses of upregulated and downregulated DEGs in Fig. 2(A, B). (E, F) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of upregulated and downregulated DEGs in Fig. 2(A, B). The Count indicates the number of input genes associated with a given term/pathway. The enrichment P value was calculated via hypergeometric test and adjusted for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) correction. Data were obtained from the KEGG database (Kanehisa et al., 2025).

Table 1.

Description of the KIFs members.

Gene	Description	Gene ID	Aliases
KIFC1	Kinesin Family Member C1	3833	HSET, KNSL2
KIF2A	Kinesin Family Member 2 A	3796	CDCBM3, HK2,
KIF2C	Kinesin Family Member 2 C	11,004	CT139, NSL6, MCAK
KIF11	Kinesin Family Member 11	3832	EG5, HKSP, KNSL1, MCLMR
KIF14	Kinesin Family Member 14	9928	MCPH20, MKS12
KIF15	Kinesin Family Member 15	56,992	KLP2, KLP2, KNSL7
KIF18A	Kinesin Family Member 18 A	81,930	MS-KIF18A, PPP1R99
KIF20A	Kinesin Family Member 20 A	10,112	MKLP2, RAB6KIFL, RCM6
KIF23	Kinesin Family Member 23	9493	CDAN3A, CHO1, KNSL5

The overexpression of KIF20A in cervical cancer

Building on current research and analysis, the role and pathogenicity of KIF20A in CC progression were investigated. The results revealed a significant upregulation of KIF20A mRNA expression in patients with CC (n = 306) compared to normal controls (n = 22) (Fig. 3A). Elevated KIF20A expression was also observed in both cervical squamous cell carcinoma (CSCC) and cervical adenosquamous carcinoma (CASC), the two major subtypes of CC (Fig. 3B and C). To assess the clinical significance of KIF20A expression, immunohistochemical (IHC) staining was performed on specimens from a cohort of 10 patients with CC and paired adjacent non-tumor tissues. High KIF20A expression was consistently detected in tumor tissues compared to adjacent non-tumor tissues (Fig. 3D). To confirm these findings at the protein level, protein lysates were extracted from patient samples and subjected to western blot analysis. In accordance with the mRNA and IHC results, KIF20A protein levels were significantly higher in CC samples than in normal controls (Fig. 3E). In conclusion, KIF20A was significantly upregulated in CC at both the mRNA and protein levels, suggesting its potential involvement in cervical tumorigenesis and progression.

Fig. 3.

Aberrant upregulation of KIF20A in cervical cancer. (A-C) mRNA expression profile of KIF20A in cervical cancer (CC), cervical squamous cell carcinoma (CSCC), and cervical adenosquamous carcinoma (CASC) based on the TCGA-GTEx database. KIF20A mRNA expression levels are significantly higher in CC tumor tissues (n = 306) compared to normal control tissues (n = 22) based on data from the TCGA and GTEx databases (A). Elevated KIF20A mRNA expression is confirmed in the two major CC histological subtypes: CSCC (B) and CASC (C). (D) IHC staining of KIF20A in cervical cancer (CC) and control patient samples. Representative image (left panel) and quantitative analysis (right panel) showing higher KIF20A protein expression in tumor tissues compared to paired adjacent non-tumor tissues from 10 CC patients. (E) Western blot analysis confirms that KIF20A protein levels are significantly elevated in CC samples (T) versus paired normal tissue samples (N). In conclusion, KIF20A is consistently upregulated in CC at both the mRNA and protein levels. ***p < 0.001, **p < 0.01, *p < 0.05.

Loss of the function of KIF20A inhibits cervical cancer cell proliferation and migration

To investigate the oncogenic function of KIF20A in CC, KIF20A expression was silenced using short hairpin RNA (shRNA) interference in SiHa and Hela cells. Knockdown efficiency was confirmed, showing a significant reduction in KIF20A expression in shRNA-treated groups compared to the control groups (Fig. 4A). Functionally, KIF20A knockdown significantly inhibited the proliferation of Hela and SiHa cells, as demonstrated by colony formation and cell viability assays (CCK-8) (Fig. 4B, C and E). These results suggest that KIF20A is critical for promoting CC cell proliferation in vitro. Moreover, cell migration was significantly reduced in KIF20A-silenced cells, as demonstrated by the cell scratch assays (Fig. 4D). To assess the impact of KIF20A on tumor growth in vivo, subcutaneous xenograft experiments were conducted using a mouse model. Tumors derived from KIF20A-silenced cells exhibited significantly reduced size and weight compared to those in the control group (Fig. 4F and H). In summary, these findings underscore the oncogenic role of KIF20A in CC progression, showing its essential functions in promoting proliferation, migration in vitro, and facilitating tumor growth in vivo.

Fig. 4.

Inhibition of CC progression by KIF20A silencing. (A) KIF20A protein expression in scramble and shKIF20A cell lines. Efficient knockdown of KIF20A expression in Hela and SiHa cell lines was confirmed after transduction with specific short hairpin RNA (shKIF20A), compared to control (Scramble) cells. (B-C) CCK-8 viability assays demonstrate that KIF20A knockdown markedly inhibits the growth rate of cervical cancer cells over time. (D) Wound healing (scratch) assays show that the migratory capacity of Hela and SiHa cells is significantly impaired upon KIF20A silencing. (E) Colony formation assays show that silencing KIF20A significantly reduces the colony-forming ability of both Hela and SiHa cells. (F-H) KIF20A knockdown suppresses tumor growth in vivo. Representative images of tumors resected from nude mice subcutaneously injected with shKIF20A or Scramble SiHa cells (F). Tumor growth curves show that tumors derived from shKIF20A cells grew significantly slower than control tumors (G). Tumor weights confirm that KIF20A-silenced tumors are significantly smaller and lighter than control tumors (H). ***p < 0.001, **p < 0.01, *p < 0.05.

KIF20A interacts with CLIP1

To explore the molecular mechanism underlying KIF20A’s regulation of CC progression, the STRING online database (https://cn.string-db.org/) was used to identify potential proteins interacting with KIF20A. The analysis suggested that CLIP1 is a potential interacting partner of KIF20A (Fig. 5A). To validate this interaction, HEK293T cells were co-transfected with Flag-tagged KIF20A and HA-tagged CLIP1 plasmids. Co-immunoprecipitation of cell lysates showed that HA-CLIP1 was present in Flag-KIF20A immunoprecipitates and vice versa (Fig. 5B and C), confirming the interaction between KIF20A and CLIP1. Further, the interaction of endogenous KIF20A with CLIP1 was assessed in SiHa cells (Fig. 5D and E). To investigate the regulatory function of KIF20A in stabilizing CLIP1, knockdown of KIF20A led to the degradation of CLIP1 in both SiHa and Hela cells (Fig. 5F). In contrast, overexpression of KIF20A resulted in the stabilization of CLIP1 (Fig. 5G), suggesting that KIF20A regulates CLIP1 protein stability.

Fig. 5.

Interaction between KIF20A and CLIP1. (A) Prediction of potential KIF20A-interacting proteins using the STRING database, identifying CLIP1 as a high-confidence candidate. (B-C) Validation of the KIF20A-CLIP1 interaction by co-immunoprecipitation (Co-IP) in HEK293T cells. Cells were co-transfected with Flag-tagged KIF20A and HA-tagged CLIP1 plasmids. Immunoprecipitation with an anti-Flag antibody co-precipitated HA-CLIP1 (B), and reciprocally, immunoprecipitation with an anti-HA antibody co-precipitated Flag-KIF20A (C). (D-E) Endogenous interaction between KIF20A and CLIP1 in SiHa cervical cancer cells, confirmed by reciprocal Co-IP assays using antibodies against the native proteins. (F) KIF20A knockdown promotes CLIP1 degradation. Silencing KIF20A in SiHa and Hela cells using shRNA reduced the protein level of CLIP1, as shown by western blot. (G) KIF20A overexpression stabilizes CLIP1. Ectopic expression of KIF20A in SiHa and Hela cells increased the protein level of CLIP1.

KIF20A promotes cervical cancer progression via CLIP1

To investigate the functional role of CLIP1 in KIF20A-mediated CC progression, CLIP1 was first knocked down in KIF20A-overexpressing cells (Fig. 6A). Colony formation and CCK-8 assays demonstrated that KIF20A overexpression significantly accelerated CC progression. However, silencing CLIP1 in these cells notably reversed the tumorigenic effects induced by KIF20A (Fig. 6B and D). Furthermore, cell scratch assays showed that CLIP1 knockdown significantly suppressed the migratory ability of CC cells (Fig. 6C). Collectively, these results indicate that KIF20A promotes CC progression by stabilizing and functionally regulating CLIP1, both in vitro and in vivo. Finally, CLIP1 expression was found to be elevated in patients with CC (Fig. 6E), suggesting that CLIP1 may serve as a biomarker for CC progression.

Fig. 6.

Impact of CLIP1 on KIF20A-induced cervical cancer progression. (A) Western blot confirming the experimental setup: CLIP1 was knocked down (shCLIP1) in SiHa and Hela cells that stably overexpress Flag-KIF20A (KIF20A-OE). (B) CCK-8 viability assay confirms that the pro-growth effect of KIF20A overexpression is abolished when CLIP1 is silenced. CCK-8 assay of Vector, KIF20A-OE, and KIF20A-OE/shCLIP1 cells in SiHa and Hela cells. (C) CLIP1 knockdown inhibits KIF20A-enhanced cell migration. Wound healing (scratch) assays demonstrate that the increased migratory capacity driven by KIF20A overexpression is suppressed by CLIP1 knockdown. (D) Colony formation assay shows that KIF20A overexpression (KIF20A-OE) enhances colony formation, which is significantly attenuated by concomitant CLIP1 knockdown (KIF20A-OE/shCLIP1). (E) Western blot detection of CLIP1 in cervical cancer (T) and normal patient samples (N). ***p < 0.001, **p < 0.01, *p < 0.05.

Discussion

The disruption of microtubule dynamics is a critical factor in cancer development, with motor proteins playing an essential role in this process²⁹. Kinesins, a superfamily of motor proteins, utilize ATP to drive their functions within the microtubule network³⁰. In vertebrates, 45 kinesin family members (KIFs) are classified into 15 families based on the position of the motor domain within the KIF structure³¹. Dysregulation of KIF functions has been implicated in various diseases, including several types of cancer. Previous studies have shown that KIF family members, such as KIF3A and KIF13A, promote cancer cell migration and invasion via the MT1-MMP vesicle transport-dependent pathway³². In our study, bioinformatics analysis and clinical sample testing revealed that KIF20A is overexpressed in patients with CC, suggesting its potential as a molecular marker for CC screening. KIF20A plays a critical role in embryonic development and maintaining intracellular homeostasis, with its dysregulation being crucial in the initiation and progression of cancer^33–35. Previous research has also demonstrated that KIF20A is upregulated in HCC³⁶, contributing to cancer cell proliferation, migration, and invasion³⁷, suggesting that KIF20A regulates these processes in cancer cells through these mechanisms.

Our findings are consistent with and extend previous reports implicating KIF20A in other cancer types. For instance, a recent study demonstrated that KIF20A promotes immune evasion in hepatocellular carcinoma by stabilizing c-Myc and reducing PD-L1 ubiquitination, thereby compromising anti-tumor immunity²⁰. Similarly, Zhu et al. reported that KIF20A facilitates the development of fibrosarcoma through activation of the PI3K/AKT pathway²¹. Our work now establishes a pivotal role for KIF20A in CC and reveals CLIP1—a microtubule-associated protein—as a novel functional binding partner. This mechanism has not been previously reported in HPV-related malignancies and suggests that cytoskeletal regulatory networks may play broader roles in gynecological cancers than previously recognized.

The cervix, a vital organ in the female reproductive system, consists of mucus-secreting columnar epithelium in the endocervical canal and stratified squamous epithelium on the exo-cervix³⁸. HPV, particularly types 16 and 18, infects the cervical mucosal tissue and is a major cause of CC³⁹. Although the HPV vaccine is an effective preventive measure, vaccination rates remain low in developing countries due to economic and healthcare challenges. Early detection of CC, especially in its initial stages, is crucial for timely diagnosis, particularly for low-income populations⁴⁰. Therefore, discovering new biomarkers for CC could improve early detection, and our findings of KIF20A upregulation suggest that targeting KIF20A may offer a promising therapeutic strategy for CC treatment.

Cytoplasmic linker protein 1 (CLIP1), a microtubule end-binding protein⁴¹, binds to dissociated α/β tubulin monomers in the cytoplasm, facilitating their transport to the plus end of microtubules, thereby contributing to microtubule elongation⁴². CLIP1 primarily promotes microtubule growth and cell migration⁴³. Additionally, CLIP1 is involved in tumor progression^44,45 and acts as an effector of various regulatory factors in cancer development^46,47. In the present study, KIF20A interacted with CLIP1 and enhanced its protein stability, a process associated with CC progression. Moreover, silencing CLIP1 significantly mitigated the CC progression induced by KIF20A, suggesting that CLIP1 functions as a downstream effector of KIF20A in CC. This finding aligns with recent work, which identified CLIP1 as a key mediator of microtubule-mediated invasion in cancer^23,48, further supporting its broader role in cancer metastasis.

In summary, our study identifies KIF20A as a significantly upregulated gene in CC through integrated bioinformatics analysis and experimental validation. We demonstrate that KIF20A promotes CC cell proliferation, migration, and tumor growth in vivo. Importantly, we reveal a novel molecular mechanism in which KIF20A interacts with and stabilizes CLIP1, thereby facilitating CC progression. Rescue experiments further confirm that CLIP1 knockdown effectively reverses the oncogenic effects induced by KIF20A, highlighting the functional importance of the KIF20A-CLIP1 axis in cervical carcinogenesis. The novelty of our work lies in the identification of KIF20A as a potential diagnostic biomarker and therapeutic target, as well as the discovery of its previously unrecognized partnership with CLIP1—a mechanism not yet reported in CC. These findings provide new insights into microtubule-related oncogenic signaling and suggest that targeting the KIF20A-CLIP1 interaction could represent a promising strategy for CC treatment. However, this study has several limitations. First, the clinical sample size was relatively small; further validation in larger multi-center cohorts is needed to strengthen the clinical relevance of KIF20A and CLIP1. Second, the precise molecular details of how KIF20A stabilizes CLIP1—such as post-translational modifications or involvement of ubiquitin-proteasome pathways—remain unclear and warrant further investigation. Despite these limitations, our findings establish a foundation for understanding the functional role of kinesin proteins in CC and open new avenues for biomarker development and targeted therapies.

Ethics declarations

The ethical approval for this study was granted by the Ethics Committee of Anhui Medical University (Approval No. LLSC20241076). All procedures were performed in accordance with the ethical standards of the Declaration of Helsinki. Informed consent was obtained from all participants prior to inclusion in the study, as approved by the Institutional Review Board of Anhui Medical University Second Affiliated Hospital. The animal experiments were conducted under the approval of the Animal Ethics Committee of Anhui Medical University (Approval No. LLSC20240765) and complied with the ARRIVE guidelines. All procedures were performed in accordance with the institutional guidelines for the care and use of laboratory animals.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

X.F.M. and Z.L.X. contributed equally to this work. They designed and performed the in vitro experiments, including the establishment of stable cell lines, functional characterization of gene knockdown/overexpression through CCK-8, EdU, colony formation, wound healing, and Transwell invasion assays. Y.C. conducted all bioinformatics and statistical analyses, prepared the figures and graphs, and interpreted the data. E.L.W. conceived the study, secured funding, wrote the original manuscript, and coordinated the entire project. W.Y.W. was responsible for the collection, annotation, and ethical compliance of all human clinical tissue specimens used in the study. F.J. and Y.Y.J. performed key experimental work, including immunohistochemistry (IHC) staining, scoring of IHC results, and assisted in animal studies. All authors participated in the discussion of the results, critically reviewed the manuscript, and approved the final version.

Funding

The Natural Science Foundation of Anhui medical University [grant no. 2022xkj037].

The Natural Science Foundation of Anhui medical University [grant no .2023xkj016].

Data availability

The RNA expression datasets generated and analyzed during the current study are available in publicly accessible repositories. The data are available from the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) under the following accession numbers: GSE63514: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE63514GSE67522: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE67522TCGA-Cervical Cancer (TCGA-CC) dataset: [https://portal.gdc.cancer.gov](https:/portal.gdc.cancer.gov).

Declarations

Competing interests

The authors declare no competing interests.