CENPO as a potential biomarker for the prognosis and therapy of CSCC patients

CENPO as a potential biomarker for the prognosis and therapy of CSCC patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CENPO as a potential biomarker for the prognosis and therapy of CSCC patients Xiaoxin Li, Fei Li, Yan Tian, Ping Zhang, Hong Cai, Feng Xu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4446737/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Cutaneous squamous cell carcinoma (CSCC) is a common nonmelanoma skin cancer. There are limited targeted therapeutic options for treating CSCC. Methods This study explored the differential expression of CENPO in CSCC and its relationship with clinical prognosis via data from The Cancer Genome Atlas (TCGA) database. The CENPO gene knockdown lentivirus was constructed, and the biological function of CENPO was evaluated via CCK8 cell proliferation, scratch, invasion, and cell apoptosis experiments in vitro. Furthermore, CENPO was evaluated in vivo. Results The TCGA data and clinical immunohistochemical results confirmed that CENPO is significantly overexpressed in CSCC and that CENPO is upregulated with clinical grade. The CCK-8 results confirmed that cell proliferation decreased with CENPO knockdown. Scratch experiments confirmed that cell migration decreased with CENPO knockdown. The invasion experiments confirmed that the cell invasion ability decreased with CENPO knockdown. Flow cytometry experiments showed that cell apoptosis increased with CENPO knockdown. The in vivo assay results showed that the tumor growth rate significantly decreased with CENPO knockdown. Conclusions The proliferation, invasion, migration, and antiapoptotic ability of CSCC cells are enhanced by upregulating CENPO. The activity of CSCC cells was significantly inhibited by CENPO knockdown. CENPO could serve as a new biomarker for the diagnosis and treatment of CSCC. CSCC CENPO proliferation metastasis apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Cutaneous squamous cell carcinoma (CSCC) is the second most common nonmelanoma skin cancer [ 1 ]. The aggressive CSCC subtype is associated with increased metastatic and mortality rates[ 2 ]. The CSCC represents 20%~50% of skin cancers. Relevant data show that the incidence rate of CSCC is gradually increasing worldwide [ 3 ]. Currently, it is believed that actinic keratosis (AK) is the most common precancerous lesion derived from keratinocytes in humans[ 4 ]. Approximately 65% of CSCC is caused by precancerous lesions, such as AKs[ 5 ]. The risk factors that trigger CSCC include exposure to ultraviolet radiation, ionizing agents, chemical carcinogens, and human papillomavirus (HPV) infection. CSFCs usually occur in areas exposed to sunlight, such as the head and neck [ 6 ]. Related research has shown that patients receiving chemotherapy targeting BRAF often develop CSCC with RAS mutations. Organ transplant recipients who receive immunosuppressive regimens are 65 times more likely to develop CSCC than the general population. Human papillomavirus (HPV) infection is also associated with an increased risk of developing CSCC. Ninety-five percent of CSCCs can be surgically removed; however, 5% of patients (with a higher incidence of elderly squamous cell carcinoma) still experience metastasis and the formation of metastatic lymph nodes, which can only be treated with palliative care. The 3-year disease-free survival rate is 56%, and the 5-year survival rate is 25%-35%[ 7 ]. CSCC has become an important clinical issue at present. Therefore, identifying new therapeutic targets is important for treating CSCC. Malignant proliferation is an important characteristic of tumors. The human kinetochore protein CENPO is associated with cell mitosis. Depletion of CENPO results in monopolar spindles, raising the question of whether kinetochores contribute to centrosome separation[ 8 ]. Analysis of the TIMER 2.0 database revealed that CENPO is upregulated in various tumors. CENPO regulates the proliferation and apoptosis of CRC cells in a p53-dependent manner[ 9 ]. CENPO is associated with immune cell infiltration and is a potential diagnostic and prognostic marker for hepatocellular carcinoma[ 10 ]. CENPO expression regulates gastric cancer cell proliferation and is associated with poor patient prognosis[ 11 ]. CENPO may be a potential pancancer biomarker and oncogene, especially in LUAD. In addition, CENPO is associated with immune cell infiltration and may serve as a new molecular therapeutic target and effective prognostic marker for LUAD[ 12 ]. The CENPO-associated prognostic signature (CPS) was identified as an independent risk factor. The high-risk group for LUAD was identified based on CPS enrichment, which involved not only endocytosis, which transfers mitochondria to promote cell survival in response to chemotherapy but also cell cycle promotion, which leads to drug resistance. The removal of CENPO significantly suppressed metastasis and induced the arrest and apoptosis of LUAD cells. The involvement of CENPO in the immunosuppression of LUAD provides a prognostic signature for LUAD patients[ 13 ]. I identified the gene centromere protein o (CENPO), which is associated with immune cells and improves the prognosis of HCC patients. CENPO may be a potential biological therapeutic target for hepatocellular cancer treatment[ 14 ]. The main discovery was the determination of the promoting role of CENPO in LUAD, demonstrating that small molecule inhibitors targeting CENPO are a novel therapeutic strategy for LUAD[ 15 ]. Bioinformatics analysis revealed that CENPO is upregulated in skin squamous cell carcinoma. However, the role of CENPO in CSCC has not been reported. This work will further explore the role of CENPO in CSCC. Materials and methods Cell culture The A431 cell line was obtained from the Institute of Cell Biology of Shanghai, Chinese Academy of Sciences (Shanghai, China). The HSC-1 cell line was obtained from Beijing Zhongyuan Ltd. (Beijing, China). The cells were cultured in DMEM (Gibco, 10566016) supplemented with 10% FBS (Gibco, 10091148) and incubated at 37°C in a humidified 5% CO 2 atmosphere. Celigo cell counting assay A431 and HSC-1 cells were seeded into 96-well plates at 3×10 3 cells/100 µL/well. The cells were continuously monitored for 5 days by a Celigo Imaging Cytometer (Nexcelom). The number of GFP-positive cells in each well was accurately calculated. Woundhealing assay A431 and HSC-1 cells were seeded in a 24-well plate. When the cells reached 90% confluence, they formed a confluent monolayer. A wound was formed by dragging the tip of the pipette. Cell migration images were acquired at 0 and 36 h by microscopy. Migration area = 24 h-cell pixel area − 0 h-cell pixel area. Transwell assay The transwell upper chambers (8-µm pores, BD Biosciences) were coated with Matrigel. A431 and HSC-1 cells were seeded into the transwell upper chamber at a density of 1×104 cells/well in 100 µL of 5% FBS culture medium, and the lower compartment contained 400 µL of culture medium supplemented with 20% FBS. The cells were incubated at 37°C for 24 h. The upper chamber was washed with PBS, and the cells were removed. The migrating cells were fixed with methanol for 30 min and stained with 0.1% crystal violet for 20 min. Finally, the cells were observed with a microscope, images were captured, and the migrating cells were counted. Flow cytometry apoptosis assay A431 and HSC-1 cells were infected with lentivirus for 48 h. The cells were harvested and rinsed three times with PBS. Then, the cells were resuspended in 1× binding buffer and incubated with FITC-annexin V for 25 min and PI for 5 min in the dark at room temperature. The cells were detected by flow cytometry. The cell apoptosis rate was analyzed with FlowJo software. Q-PCR assay Total RNA was extracted with TRIzol solution (Invitrogen, 15596026). Reverse transcription of cDNA was performed with a M-MLV Reverse Transcriptase (Promega, M1705). qPCR was performed in triplicate with AceQ qPCR SYBR Green Mastermix (Vazyme, Q111-02). The following qPCR primers were used: CENPO: 5′- TGCTTTTGAGGGGAACCTATTG-3′ (Forward) and 5′- GGGGAATG AAGACTGGGACT-3′ (Reverse); GAPDH: 5’- TGACTTCAACAGCGACACCCA-3’ (Forward), 5’- CACCCTGTTGCTGTAGCCAAA-3’ (Reverse). Tumor formation assay in nude mice Female SCID mice (4–6 weeks old) (Cavens, China) were raised in a pathogen-free facility. A431 cells were harvested, washed, and resuspended in PBS. The cells (5×106 cells/100 µL) were injected into the left armpit of each mouse. There were 5 mice in each group. The tumor volume was measured with a Vernier caliper every three days. The mice were sacrificed and dissected, the tumors were photographed and weighed, and the tumor volumes were calculated using the following equation: length×width2×0.5. All animal experiments were carried out with the approval of the Animal Care and Use Committee of The Air Force Medical Center, PLA. IHC assay Tumor tissue was fixed, embedded, and sliced. The expression level of CENPO was detected by IHC with an anti-CENPO antibody (1:500, Proteintech, 20611-1-AP). Images were acquired with a microscope (Nikon, Tokyo, Japan). Statistical analysis The experimental data were collected and are expressed as the means ± standard deviations (SD), and the data were analyzed using GraphPad Prism 8.0 software. Significant differences between two groups were analyzed using Student’s t test. Each experiment in this study was repeated at least 3 times. *p value < 0.05, ** p value < 0.01 and *** p value < 0.001. Results The expression level of CENPO in CSCC/HNSC patients In this project, gene expression difference data were obtained with GEPIA2. Some CSCC occurs in the head and neck and is classified as HNSC[ 16 ]. A volcano plot of gene expression differences is shown in http://www.bioinformatics.com.cn . The expression of CENPO was upregulated in cancer tissues, and the adj. The p value was 4.49e-32, and the log2 (fold change) was 1.361 (Fig. 1 A). Furthermore, differences in the expression of these genes were analyzed in unpaired samples, and the results showed that the expression of CENPO was significantly upregulated in cancer tissues from unpaired samples (Fig. 1 B). Analysis of paired samples revealed that the expression of CENPO was significantly upregulated in cancer tissues compared with matched normal tissues (Fig. 1 C). The expression levels of CENPO in normal tissues and in stage I and stage II patients were detected by immunohistochemistry. The results showed that the expression levels of CENPO in stage I and stage II patients were greater than those in the control group (Fig. 1 D). It was further confirmed by UALCAN ( https://ualcan.path.uab.edu/analysis.html ) that the expression level increased with increasing stage compared with that in the control group, but there was no significant difference between the different stages (Fig. 1 E). The clinical relevance and differences in gene expression among cancers were analyzed Furthermore, the expression of CENPO was analyzed according to tumor grade. The results showed that the expression level of CENPO increased with increasing tumor grade (Fig. 2 A). The expression of CENPO was analyzed according to patient age. The results showed that the expression level of CENPO increased with patient age. However, there was no significant difference between the different patient age groups (Fig. 2 B). The expression of CENPO was analyzed according to nodal metastasis status. The results showed that the expression level of CENPO increased with increasing nodal metastasis status (Fig. 2 C). The expression of CENPO was analyzed according to HPV status. The results showed that the expression level of CENPO increased with increasing HPV status (Fig. 2 D). Pancancer expression differences were analyzed with TIMER 2.0, and the results showed that CENPO was significantly more highly expressed in BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, LIHC, LUAD, LUSC, PRAD, READ, STAD, THCA and UCEC tumor tissues (Fig. 2 E). The knockdown efficiency of the CENPO knockdown lentivirus was detected by Q-PCR To explore the biological functions of CENPO, the interference sequence CCTGGAAGATATGCAAA was designed based on the CENPO gene sequence and inserted into a lentiviral vector. The lentivirus was successfully packaged in 293T cells. A431 and HSC-1 cells were infected with a CENPO knockdown lentivirus. Both the knockdown and control lentiviruses had high infection efficiency (Fig. 3 A-B). RNA was extracted with TRIzol and reverse transcribed into cDNA. Then, the expression level of CENPO was detected by Q-PCR. The results showed that the CENPO knockdown lentivirus significantly reduced the expression level of CENPO in A431 and HSC-1 cells (Fig. 3 C-D). The CENPO knockdown lentivirus was successfully constructed in this study. This provides technical support for further exploration of the functions of CENPO. Knockdown of CENPO suppressed CSCC cell proliferation First, the effect of CENPO on the proliferation of CSCC cells was explored by Celigo. In A431 cells, the number of cells in the shCtrl group increased 6.139 ± 0.654-fold, and that in the shCENPO group increased 2.208 ± 0.093-fold (p = 0.0005) (Fig. 4 B). In the HSC-1 cells, the number of cells in the control group increased 11.444 ± 0.495-fold, the number of cells in the CENPO-sh group increased 1.598 ± 0.087-fold, and the p value was less than 0.0001 (Fig. 4 B). The results showed that knocking down the CENPO gene significantly reduced the proliferation ability of A431 and HSC-1 cells. Knockdown of CENPO suppressed CSCC cell migration and invasion Second, the effect of CENPO on the migration of CSCC cells was explored by cell scratch experiments. The migration area decreased from 0.4667 ± 0.055 to 0.277 ± 0.100 with CENPO knockdown in A431 cells, and the p value was 0.0451. The migration area decreased from 0.350 ± 0.017 to 0.110 ± 0.030 with CENPO knockdown in HSC-1 cells, and the p value was 0.0003. These results showed that knocking down the CENPO gene significantly reduced the migration ability of A431 and HSC-1 cells (Fig. 5 A). The effect of CENPO on the invasion of CSCC cells was explored by transwell assays. The number of migrating A431 cells decreased from 124 ± 2.646 to 19 ± 2.646 after CENPO knockdown, and the p value was less than 0.0001. The number of migrating HSC-1 cells decreased from 261 ± 4.583 to 33 ± 1.732 with CENPO knockdown, and the p value was less than 0.0001. These results showed that knocking down the CENPO gene significantly reduced the invasion ability of A431 and HSC-1 cells (Fig. 5 B). Knockdown of CENPO promoted CSCC cell apoptosis Then, the effect of CENPO on the apoptosis rate of CSCC cells was explored by flow cytometry. With CENPO knockdown, the percentage of apoptotic A431 cells increased from 3.480 ± 0.177 to 14.270 ± 0.710, and the p value was less than 0.0001. With CENPO knockdown, the percentage of apoptotic HSC-1 cells increased from 4.397 ± 0.430 to 14.383 ± 0.386, and the p value was less than 0.0001. These results showed that knocking down the CENPO gene significantly promoted CSCC cell apoptosis in A431 and HSC-1 cells (Fig. 6 ). Knockdown of CENPO inhibited subcutaneous tumor growth Finally, the effect of CENPO on tumor growth was explored in a subcutaneous transplantation tumor model. The tumor volume in the CENPO knockdown group decreased significantly (Fig. 7 A). The tumor growth curve showed that tumor growth was significantly inhibited in the CENPO knockdown group (Fig. 7 B). Compared with the tumor weight, the tumor quality was significantly lower in the CENPO knockdown group (Fig. 7 C). The protein expression level of Ki67 was detected by immunohistochemistry. The expression level of Ki67 decreased significantly with CENPO knockdown (Fig. 7 D). These results showed that subcutaneous tumor growth was significantly inhibited by CENPO knockdown. Discussion CSCC has become the most common subcutaneous tumor and is often ignored by people[ 17 ]. CSCC cells may metastasize when they are found and threaten the lives of patients at any time[ 18 ]. The CSCC is a skin problem that urgently needs to be solved[ 19 ]. Relevant studies have shown that CENPO is associated with immune cells and improves the prognosis of patients with HCC [ 20 ]. In this study, we found that CENPO was highly expressed in CSCC. Gao et al. reported that knockdown of CENPO contributed to GC cell growth inhibition and apoptosis induction[ 11 ]. To further study the gene function of CENPO, a CENPO knockdown lentivirus was constructed. The proliferation of A431 and HSC-1 cells decreased significantly with CENPO knockdown. Mitosis is an important process of cell proliferation[ 21 ]. A key step of mitosis is the congression of chromosomes to the spindle equator[ 22 ]. The CENP-O complex is constitutively localized at kinetochores throughout the cell cycle in vertebrates[ 23 ]. Therefore, CENPO is related to cell mitosis[ 24 ]. Knockdown of CENPO can inhibit cell mitosis and reduce cell proliferation. Similarly, CENP-U is a component of the CENP-O complex. CENP-U deficiency is associated with mitotic defects[ 25 ]. The survival rate of patients with cutaneous squamous cell carcinoma (cSCC) is reduced due to the presence of nodal metastases[ 26 ]. The data revealed that the migration and invasion ability of cells decreased significantly with CENPO knockdown. Therefore, CENPO plays an important regulatory role in the invasion and migration of tumor cells. The flow cytometry apoptosis assay results showed that the level of cell apoptosis increased significantly with CENPO knockdown. CENPO can block the G2/M phase transition[ 9 , 10 ] and inhibit cell mitosis. Relevant studies have confirmed that G2/M arrest can inhibit cell division and induce polyploidy and apoptosis [ 27 – 29 ]. Tetraploid and polyploid plants can activate the p53-dependent apoptosis pathway to drive cell apoptosis[ 30 , 31 ]. Finally, a mouse subcutaneous tumor model was constructed to evaluate tumor growth after CENPO knockdown. Compared with those of the control group, the tumor volume and weight of the knockdown group were significantly lower. Furthermore, the expression level of Ki67 was detected by IHC[ 32 ]. The results showed that the activity of the cells decreased after CENPO knockdown because tumor growth was slow. These results indicate that CENPO is related to the division, proliferation, invasion and migration of tumor cells. CENPO knockdown significantly inhibited the division, proliferation, invasion and migration of tumor cells. CENPO can be used as a new target for the treatment of CSCC. Conclusion CSCC promotes cell proliferation, metastasis and antiapoptotic ability by upregulating CENPO. CENPO knockdown can effectively inhibit the malignant progression of CSCC cells. Therefore, CENPO can be used as a new biomarker for the diagnosis and treatment of CSCC. Declarations Ethics Approval and Consent to Participate: The animal study was approved by the Experimental Animal Welfare Ethics Committee, Air Force Medical Center (approval NO. 2021-148-YJ01). Consent for publication: Not applicable Competing interests: The authors declare no conflicts of interest. Funding: This study was supported by the Capacity building project of the Dermatology Department, Air Force Medical Center, PLA (No. 2023PFKPY01). Author Contribution F.X. and X.L. developed the study concept and design; X.L. and F.L. acquired the data. F.X. and P.Z. analyzed and interpreted the data and performed the statistical analysis; H.C. and Y.T. provided technical and material support. X.L. and F.L. developed the methodology and wrote the manuscript; F.X. reviewed and revised the paper; and all the authors read and approved the final paper. Acknowledgement We acknowledge the TCGA, GEPIA2 and TIMER2.0 databases for free use. Availability of data and material: The authors confirm that the data supporting the findings of this study are available within the article. References Chang M, Azin M, Demehri S. Cutaneous Squamous Cell Carcinoma: The Frontier of Cancer Immunoprevention. Annu Rev Pathol. 2022;17:101–19. undefined u, undefined u, undefined u. Case Report: Dostarlimab for treatment of aggressive cutaneous squamous cell carcinoma. Front Med (Lausanne). 2024;11. Haasler L, von Montfort C, Kondadi A, Golombek M, Ebbert L, Wenzel C, et al. Involvement of necroptosis in the selective toxicity of the natural compound (±) gossypol on squamous skin cancer cells in vitro. Arch Toxicol. 2023;97:1997–2014. Ratushny V, Gober M, Hick R, Ridky T, Seykora J. From keratinocyte to cancer: the pathogenesis and modeling of cutaneous squamous cell carcinoma. J Clin Investig. 2012;122:464–72. Moseley I, Ahmed F, Lin E, Lim R, Hoang M, Baranwal N, et al. Host and primary tumor factors for the development of multiple cutaneous squamous cell carcinomas among a retrospective cohort in Rhode Island. Journal of the American Academy of Dermatology; 2023. Karampinis E, Aloizou A, Zafiriou E, Bargiota A, Skaperda Z, Kouretas D et al. Non-Melanoma Skin Cancer and Vitamin D: The Lost Sunlight Paradox and the Oxidative Stress Explanation. Antioxidants (Basel, Switzerland). 2023;12. Li Y, Hanna G, Laga A, Haddad R, Lorch J, Hammerman P. Genomic analysis of metastatic cutaneous squamous cell carcinoma. Clin cancer research: official J Am Association Cancer Res. 2015;21:1447–56. Toso A, Winter J, Garrod A, Amaro A, Meraldi P, McAinsh A. Kinetochore-generated pushing forces separate centrosomes during bipolar spindle assembly. J Cell Biol. 2009;184:365–72. Liu Z, Chen C, Yan M, Zeng X, Zhang Y, Lai D. CENPO regulated proliferation and apoptosis of colorectal cancer in a p53-dependent manner. Discover Oncol. 2022;13:8. He K, Xie M, Li J, He Y, Yin Y. CENPO is Associated with Immune Cell Infiltration and is a Potential Diagnostic and Prognostic Marker for Hepatocellular Carcinoma. Int J Gen Med. 2022;15:7493–510. Cao Y, Xiong J, Li Z, Zhang G, Tu Y, Wang L, et al. CENPO expression regulates gastric cancer cell proliferation and is associated with poor patient prognosis. Mol Med Rep. 2019;20:3661–70. Wang Y, Ye D, Li Y, Lv F, Shen W, Li H, et al. Prognostic and immune infiltrative biomarkers of CENPO in pancancer and its relationship with lung adenocarcinoma cell proliferation and metastasis. BMC Cancer. 2023;23:735. Shi T, Hu Z, Tian L, Yang Y. Advances in lung adenocarcinoma: A novel perspective on prognoses and immune responses of CENPO as an oncogenic superenhancer. Translational Oncol. 2023;34:101691. Xu Z. Gene mining of immune microenvironment in hepatocellular carcinoma. Medicine. 2022;101:e30453. Shi T, Hu Z, Tian L, Yang Y. Pancancer landscape of CENPO and its underlying mechanism in LUAD. Respir Res. 2023;24:113. undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Evolution of Head and Neck Cutaneous Squamous Cell Carcinoma Nodal Staging-An Australian Perspective. Cancers (Basel). 2022;14. undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Cutaneous Squamous Cell Carcinoma: Clinico-Dermoscopic and Histological Correlation: Approximately 72 Cases. Dermatol Pract Concept. 2024;14. undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Higher metastasis and death rates in cutaneous squamous cell carcinomas with lymphovascular invasion. J Am Acad Dermatol. 2021;86. undefined u, undefined u. Intralesional Treatments for Invasive Cutaneous Squamous Cell Carcinoma. Cancers (Basel). 2024;16. undefined u. Gene mining of immune microenvironment in hepatocellular carcinoma. Med (Baltim). 2022;101. undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in hepatocellular carcinoma. J Exp Clin Cancer Res. 2023;42. Bancroft J, Auckland P, Samora C, McAinsh A. Chromosome congression is promoted by CENP-Q- and CENP-E-dependent pathways. J Cell Sci. 2015;128:171–84. Okumura K, Kagawa N, Saito M, Yoshizawa Y, Munakata H, Isogai E, et al. CENP-R acts bilaterally as a tumor suppressor and as an oncogene in the two-stage skin carcinogenesis model. Cancer Sci. 2017;108:2142–8. Nguyen A, Fadel M, Cheeseman I. Differential requirements for the CENP-O complex reveal parallel PLK1 kinetochore recruitment pathways. Mol Biol Cell. 2021;32:712–21. Kagawa N, Hori T, Hoki Y, Hosoya O, Tsutsui K, Saga Y, et al. The CENP-O complex requirement varies among different cell types. Chromosome research: Int J Mol supramolecular evolutionary aspects chromosome biology. 2014;22:293–303. undefined u, undefined u, undefined u, undefined u. Tumor Recurrence, Depth of Invasion, and Temple Location as Independent Prognostic Parameters of Lymph Node Metastases of Head and Neck Cutaneous Squamous Cell Carcinomas. Int J Clin Pract. 2024;2024. undefined u, undefined u, undefined u, undefined u. Methyl protodioscin induces G2/M arrest and apoptosis in K562 cells with the hyperpolarization of mitochondria. Cancer Lett. 2005;224. undefined u, undefined u, undefined u, undefined u, undefined u. Cyclin A1 is a p53-induced gene that mediates apoptosis, G2/M arrest, and mitotic catastrophe in renal, ovarian, and lung carcinoma cells. Cell Mol Life Sci. 2006;63. undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Curcumin-Piperlongumine Hybrid Molecule Increases Cell Cycle Arrest and Apoptosis in Lung Cancer through JNK/c-Jun Signaling Pathway. J Agric Food Chem. 2024. undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis. EMBO Rep. 2020;21. undefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Inhibition of cancer progression by a novel trans-stilbene derivative through disruption of microtubule dynamics, driving G2/M arrest, and p53-dependent apoptosis. Cell Death Dis. 2018;9. Raverot G, Ilie M, Lasolle H, Amodru V, Trouillas J, Castinetti F, et al. Aggressive pituitary tumors and pituitary carcinomas. Nat reviews Endocrinol. 2021;17:671–84. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4446737","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308538549,"identity":"17cd6608-aa1e-4879-acf6-9398801bde65","order_by":0,"name":"Xiaoxin Li","email":"","orcid":"","institution":"Air Force Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxin","middleName":"","lastName":"Li","suffix":""},{"id":308538551,"identity":"22414554-d31e-4a4f-9d52-7ba90aa16ac7","order_by":1,"name":"Fei Li","email":"","orcid":"","institution":"Air Force Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Li","suffix":""},{"id":308538552,"identity":"67922cc3-8280-4255-b3f5-7368981fa529","order_by":2,"name":"Yan Tian","email":"","orcid":"","institution":"Air Force Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Tian","suffix":""},{"id":308538555,"identity":"038dd16b-8ccd-48f1-a933-c88c41a55dba","order_by":3,"name":"Ping Zhang","email":"","orcid":"","institution":"Air Force Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Ping","middleName":"","lastName":"Zhang","suffix":""},{"id":308538556,"identity":"70649430-8d8b-4e2f-8fa9-898abb6c3945","order_by":4,"name":"Hong Cai","email":"","orcid":"","institution":"Air Force Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Cai","suffix":""},{"id":308538557,"identity":"8221f708-a7ff-4654-ac59-16688d2755f2","order_by":5,"name":"Feng Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYFAD9uaDDz4Y2NiRoIXnWLLhjIK0ZBK0SPioCfN8OMTYQEih/Izcg48r2+wS+2fwsDHbGBxgZmA/fHQDPi0GN/KSDc+2JSfOuN177HGOwR0+Bp60tBt4tUjkmEk2tjEnNtw5l26cY/CMmUGCxwyvFvkZOeY/G9vqE+ffyDGTtjA4zNhASAsDUCVjY9vhxA0gLQzEaDE48y5ZsuHcceONZ4CB3GOQlsxGyC/y7bkHPzaUVcvOOw6Myh9/bOz42Q8fw+8wBh4GBkY2BscGGJ8Nv3KoFoY/DPaEFY6CUTAKRsGIBQAE6VFRxMFXFgAAAABJRU5ErkJggg==","orcid":"","institution":"China Academy of Chinese Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Feng","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-05-20 05:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4446737/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4446737/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58077022,"identity":"87a676cc-2762-47df-9f26-984efe0db0c7","added_by":"auto","created_at":"2024-06-10 22:23:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4291057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferences in the expression of CENPO were analyzed.\u003c/strong\u003e (A) Volcanic map of gene expression differences. (B) Expression difference analysis in unpaired samples. (C) Expression difference analysis in paired samples. (D) Differences in gene expression were detected by immunohistochemistry. (E) The expression differences during the staging period were analyzed with UALCAN. ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/ca2c7fa1496a59721a8fb6a2.png"},{"id":58077020,"identity":"7442cb22-fe77-4446-9075-acab44ce701d","added_by":"auto","created_at":"2024-06-10 22:23:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2219184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe clinical relevance and differences in gene expression among cancers were analyzed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The expression of CENPO based on tumor grade. (B) The expression of CENPO based on patient age. (C) The expression of CENPO based on nodal metastasis status. (D) The expression of CENPO based on HPV status. (E) The differences in pancancer expression were analyzed with TIMER 2.0.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/3c7f68089d19ecc8828815a1.png"},{"id":58077021,"identity":"a9bfda59-6294-4ecc-af60-5049a61f9cbf","added_by":"auto","created_at":"2024-06-10 22:23:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6350432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe infection efficiency and knockdown efficiency of the CENPO knockdown lentivirus were analyzed. \u003c/strong\u003e(A) The fluorescence intensity of the cells was observed with a fluorescence microscope. (B) The knockdown efficiency of CENPO was determined by Q-PCR. ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/f14f559c42922908d5258643.png"},{"id":58077245,"identity":"63fad38b-42fe-42a2-935c-113194099b0d","added_by":"auto","created_at":"2024-06-10 22:31:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1345349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of CENPO knockdown on cell proliferation were analyzed by Celigo.\u003c/strong\u003e (A) The proliferation of A431 cells was analyzed. (B) Cell proliferation was analyzed in HSC-1 cells. ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/5082b690c6e8c7e2253755e4.png"},{"id":58077246,"identity":"fa3a2411-ae54-4d5a-9817-cdfbec94bb1a","added_by":"auto","created_at":"2024-06-10 22:31:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9602371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of CENPO knockdown on cell migration and invasion ability were analyzed by cell scratch assays and transwell assays.\u003c/strong\u003e (A) The migration ability of A431 and HSC-1 cells was analyzed. (B) The invasion ability of A431 and HSC-1 cells was analyzed. ***p\u0026lt;0.001, *p\u0026lt;0.05\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/3a65140e0e02d9a17211dd81.png"},{"id":58077023,"identity":"e926b4be-8ec6-4080-afde-9f44e1b7eaf4","added_by":"auto","created_at":"2024-06-10 22:23:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1134937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of CENPO knockdown on cell apoptosis were analyzed by flow cytometry. \u003c/strong\u003e(A) The percentage of A431 cells undergoing apoptosis was analyzed. (B) The cell apoptosis rate was analyzed in HSC-1 cells. ***p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/7cfc0fa23d553ce3fd46a68a.png"},{"id":58077026,"identity":"33877bcd-f856-4559-8970-a4f36ebe79f7","added_by":"auto","created_at":"2024-06-10 22:23:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3399189,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of CENPO knockdown on tumor growth were analyzed in subcutaneous transplantation tumor models.\u003c/strong\u003e (A) Tumor image. (B) Tumor growth curve. (C) Tumor weight. (D) Ki67 expression levels were determined by IHC.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/db079269222907d2de604fec.png"},{"id":105729316,"identity":"829ec59b-2b60-468f-9d42-5da37ba05cd8","added_by":"auto","created_at":"2026-03-30 11:14:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36766711,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4446737/v1/27e11562-dc33-448f-a229-81ba222ba1f6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CENPO as a potential biomarker for the prognosis and therapy of CSCC patients","fulltext":[{"header":"Background","content":"\u003cp\u003eCutaneous squamous cell carcinoma (CSCC) is the second most common nonmelanoma skin cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The aggressive CSCC subtype is associated with increased metastatic and mortality rates[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The CSCC represents 20%~50% of skin cancers. Relevant data show that the incidence rate of CSCC is gradually increasing worldwide [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Currently, it is believed that actinic keratosis (AK) is the most common precancerous lesion derived from keratinocytes in humans[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Approximately 65% of CSCC is caused by precancerous lesions, such as AKs[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The risk factors that trigger CSCC include exposure to ultraviolet radiation, ionizing agents, chemical carcinogens, and human papillomavirus (HPV) infection. CSFCs usually occur in areas exposed to sunlight, such as the head and neck [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Related research has shown that patients receiving chemotherapy targeting BRAF often develop CSCC with RAS mutations. Organ transplant recipients who receive immunosuppressive regimens are 65 times more likely to develop CSCC than the general population. Human papillomavirus (HPV) infection is also associated with an increased risk of developing CSCC. Ninety-five percent of CSCCs can be surgically removed; however, 5% of patients (with a higher incidence of elderly squamous cell carcinoma) still experience metastasis and the formation of metastatic lymph nodes, which can only be treated with palliative care. The 3-year disease-free survival rate is 56%, and the 5-year survival rate is 25%-35%[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. CSCC has become an important clinical issue at present. Therefore, identifying new therapeutic targets is important for treating CSCC.\u003c/p\u003e \u003cp\u003eMalignant proliferation is an important characteristic of tumors. The human kinetochore protein CENPO is associated with cell mitosis. Depletion of CENPO results in monopolar spindles, raising the question of whether kinetochores contribute to centrosome separation[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Analysis of the TIMER 2.0 database revealed that CENPO is upregulated in various tumors. CENPO regulates the proliferation and apoptosis of CRC cells in a p53-dependent manner[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. CENPO is associated with immune cell infiltration and is a potential diagnostic and prognostic marker for hepatocellular carcinoma[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CENPO expression regulates gastric cancer cell proliferation and is associated with poor patient prognosis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CENPO may be a potential pancancer biomarker and oncogene, especially in LUAD. In addition, CENPO is associated with immune cell infiltration and may serve as a new molecular therapeutic target and effective prognostic marker for LUAD[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The CENPO-associated prognostic signature (CPS) was identified as an independent risk factor. The high-risk group for LUAD was identified based on CPS enrichment, which involved not only endocytosis, which transfers mitochondria to promote cell survival in response to chemotherapy but also cell cycle promotion, which leads to drug resistance. The removal of CENPO significantly suppressed metastasis and induced the arrest and apoptosis of LUAD cells. The involvement of CENPO in the immunosuppression of LUAD provides a prognostic signature for LUAD patients[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. I identified the gene centromere protein o (CENPO), which is associated with immune cells and improves the prognosis of HCC patients. CENPO may be a potential biological therapeutic target for hepatocellular cancer treatment[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The main discovery was the determination of the promoting role of CENPO in LUAD, demonstrating that small molecule inhibitors targeting CENPO are a novel therapeutic strategy for LUAD[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBioinformatics analysis revealed that CENPO is upregulated in skin squamous cell carcinoma. However, the role of CENPO in CSCC has not been reported. This work will further explore the role of CENPO in CSCC.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e A431 cell line was obtained from the Institute of Cell Biology of Shanghai, Chinese Academy of Sciences (Shanghai, China). The HSC-1 cell line was obtained from Beijing Zhongyuan Ltd. (Beijing, China). The cells were cultured in DMEM (Gibco, 10566016) supplemented with 10% FBS (Gibco, 10091148) and incubated at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCeligo cell counting assay\u003c/h2\u003e \u003cp\u003eA431 and HSC-1 cells were seeded into 96-well plates at 3\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/100 \u0026micro;L/well. The cells were continuously monitored for 5 days by a Celigo Imaging Cytometer (Nexcelom). The number of GFP-positive cells in each well was accurately calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWoundhealing assay\u003c/h2\u003e \u003cp\u003eA431 and HSC-1 cells were seeded in a 24-well plate. When the cells reached 90% confluence, they formed a confluent monolayer. A wound was formed by dragging the tip of the pipette. Cell migration images were acquired at 0 and 36 h by microscopy. Migration area\u0026thinsp;=\u0026thinsp;24 h-cell pixel area \u0026minus;\u0026thinsp;0 h-cell pixel area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTranswell assay\u003c/h2\u003e \u003cp\u003eThe transwell upper chambers (8-\u0026micro;m pores, BD Biosciences) were coated with Matrigel. A431 and HSC-1 cells were seeded into the transwell upper chamber at a density of 1\u0026times;104 cells/well in 100 \u0026micro;L of 5% FBS culture medium, and the lower compartment contained 400 \u0026micro;L of culture medium supplemented with 20% FBS. The cells were incubated at 37\u0026deg;C for 24 h. The upper chamber was washed with PBS, and the cells were removed. The migrating cells were fixed with methanol for 30 min and stained with 0.1% crystal violet for 20 min. Finally, the cells were observed with a microscope, images were captured, and the migrating cells were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry apoptosis assay\u003c/h2\u003e \u003cp\u003eA431 and HSC-1 cells were infected with lentivirus for 48 h. The cells were harvested and rinsed three times with PBS. Then, the cells were resuspended in 1\u0026times; binding buffer and incubated with FITC-annexin V for 25 min and PI for 5 min in the dark at room temperature. The cells were detected by flow cytometry. The cell apoptosis rate was analyzed with FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQ-PCR assay\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted with TRIzol solution (Invitrogen, 15596026). Reverse transcription of cDNA was performed with a M-MLV Reverse Transcriptase (Promega, M1705). qPCR was performed in triplicate with AceQ qPCR SYBR Green Mastermix (Vazyme, Q111-02). The following qPCR primers were used:\u003c/p\u003e \u003cp\u003eCENPO: 5\u0026prime;- TGCTTTTGAGGGGAACCTATTG-3\u0026prime; (Forward) and 5\u0026prime;- GGGGAATG AAGACTGGGACT-3\u0026prime; (Reverse); GAPDH: 5\u0026rsquo;- TGACTTCAACAGCGACACCCA-3\u0026rsquo; (Forward), 5\u0026rsquo;- CACCCTGTTGCTGTAGCCAAA-3\u0026rsquo; (Reverse).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTumor formation assay in nude mice\u003c/h2\u003e \u003cp\u003eFemale SCID mice (4\u0026ndash;6 weeks old) (Cavens, China) were raised in a pathogen-free facility. A431 cells were harvested, washed, and resuspended in PBS. The cells (5\u0026times;106 cells/100 \u0026micro;L) were injected into the left armpit of each mouse. There were 5 mice in each group. The tumor volume was measured with a Vernier caliper every three days.\u003c/p\u003e \u003cp\u003eThe mice were sacrificed and dissected, the tumors were photographed and weighed, and the tumor volumes were calculated using the following equation: length\u0026times;width2\u0026times;0.5. All animal experiments were carried out with the approval of the Animal Care and Use Committee of The Air Force Medical Center, PLA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIHC assay\u003c/h2\u003e \u003cp\u003eTumor tissue was fixed, embedded, and sliced. The expression level of CENPO was detected by IHC with an anti-CENPO antibody (1:500, Proteintech, 20611-1-AP). Images were acquired with a microscope (Nikon, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were collected and are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations (SD), and the data were analyzed using GraphPad Prism 8.0 software. Significant differences between two groups were analyzed using Student\u0026rsquo;s t test. Each experiment in this study was repeated at least 3 times. *p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and *** p value\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe expression level of CENPO in CSCC/HNSC patients\u003c/h2\u003e \u003cp\u003eIn this project, gene expression difference data were obtained with GEPIA2. Some CSCC occurs in the head and neck and is classified as HNSC[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A volcano plot of gene expression differences is shown in \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.com.cn\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.com.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The expression of CENPO was upregulated in cancer tissues, and the adj. The p value was 4.49e-32, and the log2 (fold change) was 1.361 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, differences in the expression of these genes were analyzed in unpaired samples, and the results showed that the expression of CENPO was significantly upregulated in cancer tissues from unpaired samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Analysis of paired samples revealed that the expression of CENPO was significantly upregulated in cancer tissues compared with matched normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The expression levels of CENPO in normal tissues and in stage I and stage II patients were detected by immunohistochemistry. The results showed that the expression levels of CENPO in stage I and stage II patients were greater than those in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). It was further confirmed by UALCAN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ualcan.path.uab.edu/analysis.html\u003c/span\u003e\u003cspan address=\"https://ualcan.path.uab.edu/analysis.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) that the expression level increased with increasing stage compared with that in the control group, but there was no significant difference between the different stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe clinical relevance and differences in gene expression among cancers were analyzed\u003c/h2\u003e \u003cp\u003eFurthermore, the expression of CENPO was analyzed according to tumor grade. The results showed that the expression level of CENPO increased with increasing tumor grade (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The expression of CENPO was analyzed according to patient age. The results showed that the expression level of CENPO increased with patient age. However, there was no significant difference between the different patient age groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The expression of CENPO was analyzed according to nodal metastasis status. The results showed that the expression level of CENPO increased with increasing nodal metastasis status (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). The expression of CENPO was analyzed according to HPV status. The results showed that the expression level of CENPO increased with increasing HPV status (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Pancancer expression differences were analyzed with TIMER 2.0, and the results showed that CENPO was significantly more highly expressed in BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, LIHC, LUAD, LUSC, PRAD, READ, STAD, THCA and UCEC tumor tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe knockdown efficiency of the CENPO knockdown lentivirus was detected by Q-PCR\u003c/h2\u003e \u003cp\u003eTo explore the biological functions of CENPO, the interference sequence CCTGGAAGATATGCAAA was designed based on the CENPO gene sequence and inserted into a lentiviral vector. The lentivirus was successfully packaged in 293T cells. A431 and HSC-1 cells were infected with a CENPO knockdown lentivirus. Both the knockdown and control lentiviruses had high infection efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). RNA was extracted with TRIzol and reverse transcribed into cDNA. Then, the expression level of CENPO was detected by Q-PCR. The results showed that the CENPO knockdown lentivirus significantly reduced the expression level of CENPO in A431 and HSC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). The CENPO knockdown lentivirus was successfully constructed in this study. This provides technical support for further exploration of the functions of CENPO.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of CENPO suppressed CSCC cell proliferation\u003c/h2\u003e \u003cp\u003eFirst, the effect of CENPO on the proliferation of CSCC cells was explored by Celigo. In A431 cells, the number of cells in the shCtrl group increased 6.139\u0026thinsp;\u0026plusmn;\u0026thinsp;0.654-fold, and that in the shCENPO group increased 2.208\u0026thinsp;\u0026plusmn;\u0026thinsp;0.093-fold (p\u0026thinsp;=\u0026thinsp;0.0005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In the HSC-1 cells, the number of cells in the control group increased 11.444\u0026thinsp;\u0026plusmn;\u0026thinsp;0.495-fold, the number of cells in the CENPO-sh group increased 1.598\u0026thinsp;\u0026plusmn;\u0026thinsp;0.087-fold, and the p value was less than 0.0001 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The results showed that knocking down the CENPO gene significantly reduced the proliferation ability of A431 and HSC-1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of CENPO suppressed CSCC cell migration and invasion\u003c/h2\u003e \u003cp\u003eSecond, the effect of CENPO on the migration of CSCC cells was explored by cell scratch experiments. The migration area decreased from 0.4667\u0026thinsp;\u0026plusmn;\u0026thinsp;0.055 to 0.277\u0026thinsp;\u0026plusmn;\u0026thinsp;0.100 with CENPO knockdown in A431 cells, and the p value was 0.0451. The migration area decreased from 0.350\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017 to 0.110\u0026thinsp;\u0026plusmn;\u0026thinsp;0.030 with CENPO knockdown in HSC-1 cells, and the p value was 0.0003. These results showed that knocking down the CENPO gene significantly reduced the migration ability of A431 and HSC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effect of CENPO on the invasion of CSCC cells was explored by transwell assays. The number of migrating A431 cells decreased from 124\u0026thinsp;\u0026plusmn;\u0026thinsp;2.646 to 19\u0026thinsp;\u0026plusmn;\u0026thinsp;2.646 after CENPO knockdown, and the p value was less than 0.0001. The number of migrating HSC-1 cells decreased from 261\u0026thinsp;\u0026plusmn;\u0026thinsp;4.583 to 33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.732 with CENPO knockdown, and the p value was less than 0.0001. These results showed that knocking down the CENPO gene significantly reduced the invasion ability of A431 and HSC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of CENPO promoted CSCC cell apoptosis\u003c/h2\u003e \u003cp\u003eThen, the effect of CENPO on the apoptosis rate of CSCC cells was explored by flow cytometry. With CENPO knockdown, the percentage of apoptotic A431 cells increased from 3.480\u0026thinsp;\u0026plusmn;\u0026thinsp;0.177 to 14.270\u0026thinsp;\u0026plusmn;\u0026thinsp;0.710, and the p value was less than 0.0001. With CENPO knockdown, the percentage of apoptotic HSC-1 cells increased from 4.397\u0026thinsp;\u0026plusmn;\u0026thinsp;0.430 to 14.383\u0026thinsp;\u0026plusmn;\u0026thinsp;0.386, and the p value was less than 0.0001. These results showed that knocking down the CENPO gene significantly promoted CSCC cell apoptosis in A431 and HSC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eKnockdown of CENPO inhibited subcutaneous tumor growth\u003c/h2\u003e \u003cp\u003eFinally, the effect of CENPO on tumor growth was explored in a subcutaneous transplantation tumor model. The tumor volume in the CENPO knockdown group decreased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The tumor growth curve showed that tumor growth was significantly inhibited in the CENPO knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Compared with the tumor weight, the tumor quality was significantly lower in the CENPO knockdown group (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). The protein expression level of Ki67 was detected by immunohistochemistry. The expression level of Ki67 decreased significantly with CENPO knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These results showed that subcutaneous tumor growth was significantly inhibited by CENPO knockdown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eCSCC has become the most common subcutaneous tumor and is often ignored by people[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. CSCC cells may metastasize when they are found and threaten the lives of patients at any time[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The CSCC is a skin problem that urgently needs to be solved[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Relevant studies have shown that CENPO is associated with immune cells and improves the prognosis of patients with HCC [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we found that CENPO was highly expressed in CSCC. Gao et al. reported that knockdown of CENPO contributed to GC cell growth inhibition and apoptosis induction[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To further study the gene function of CENPO, a CENPO knockdown lentivirus was constructed. The proliferation of A431 and HSC-1 cells decreased significantly with CENPO knockdown. Mitosis is an important process of cell proliferation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. A key step of mitosis is the congression of chromosomes to the spindle equator[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The CENP-O complex is constitutively localized at kinetochores throughout the cell cycle in vertebrates[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, CENPO is related to cell mitosis[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Knockdown of CENPO can inhibit cell mitosis and reduce cell proliferation. Similarly, CENP-U is a component of the CENP-O complex. CENP-U deficiency is associated with mitotic defects[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The survival rate of patients with cutaneous squamous cell carcinoma (cSCC) is reduced due to the presence of nodal metastases[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The data revealed that the migration and invasion ability of cells decreased significantly with CENPO knockdown. Therefore, CENPO plays an important regulatory role in the invasion and migration of tumor cells. The flow cytometry apoptosis assay results showed that the level of cell apoptosis increased significantly with CENPO knockdown. CENPO can block the G2/M phase transition[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and inhibit cell mitosis. Relevant studies have confirmed that G2/M arrest can inhibit cell division and induce polyploidy and apoptosis [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Tetraploid and polyploid plants can activate the p53-dependent apoptosis pathway to drive cell apoptosis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Finally, a mouse subcutaneous tumor model was constructed to evaluate tumor growth after CENPO knockdown. Compared with those of the control group, the tumor volume and weight of the knockdown group were significantly lower. Furthermore, the expression level of Ki67 was detected by IHC[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The results showed that the activity of the cells decreased after CENPO knockdown because tumor growth was slow.\u003c/p\u003e \u003cp\u003eThese results indicate that CENPO is related to the division, proliferation, invasion and migration of tumor cells. CENPO knockdown significantly inhibited the division, proliferation, invasion and migration of tumor cells. CENPO can be used as a new target for the treatment of CSCC.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCSCC promotes cell proliferation, metastasis and antiapoptotic ability by upregulating CENPO. CENPO knockdown can effectively inhibit the malignant progression of CSCC cells. Therefore, CENPO can be used as a new biomarker for the diagnosis and treatment of CSCC.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics Approval and Consent to Participate:\u003c/h2\u003e \u003cp\u003eThe animal study was approved by the Experimental Animal Welfare Ethics Committee, Air Force Medical Center (approval NO. 2021-148-YJ01).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication:\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported by the Capacity building project of the Dermatology Department, Air Force Medical Center, PLA (No. 2023PFKPY01).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eF.X. and X.L. developed the study concept and design; X.L. and F.L. acquired the data. F.X. and P.Z. analyzed and interpreted the data and performed the statistical analysis; H.C. and Y.T. provided technical and material support. X.L. and F.L. developed the methodology and wrote the manuscript; F.X. reviewed and revised the paper; and all the authors read and approved the final paper.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the TCGA, GEPIA2 and TIMER2.0 databases for free use.\u003c/p\u003e\u003ch2\u003eAvailability of data and material:\u003c/h2\u003e \u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChang M, Azin M, Demehri S. Cutaneous Squamous Cell Carcinoma: The Frontier of Cancer Immunoprevention. Annu Rev Pathol. 2022;17:101\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u. Case Report: Dostarlimab for treatment of aggressive cutaneous squamous cell carcinoma. Front Med (Lausanne). 2024;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaasler L, von Montfort C, Kondadi A, Golombek M, Ebbert L, Wenzel C, et al. Involvement of necroptosis in the selective toxicity of the natural compound (\u0026plusmn;) gossypol on squamous skin cancer cells in vitro. Arch Toxicol. 2023;97:1997\u0026ndash;2014.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRatushny V, Gober M, Hick R, Ridky T, Seykora J. From keratinocyte to cancer: the pathogenesis and modeling of cutaneous squamous cell carcinoma. J Clin Investig. 2012;122:464\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoseley I, Ahmed F, Lin E, Lim R, Hoang M, Baranwal N, et al. Host and primary tumor factors for the development of multiple cutaneous squamous cell carcinomas among a retrospective cohort in Rhode Island. Journal of the American Academy of Dermatology; 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarampinis E, Aloizou A, Zafiriou E, Bargiota A, Skaperda Z, Kouretas D et al. Non-Melanoma Skin Cancer and Vitamin D: The Lost Sunlight Paradox and the Oxidative Stress Explanation. Antioxidants (Basel, Switzerland). 2023;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Hanna G, Laga A, Haddad R, Lorch J, Hammerman P. Genomic analysis of metastatic cutaneous squamous cell carcinoma. Clin cancer research: official J Am Association Cancer Res. 2015;21:1447\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToso A, Winter J, Garrod A, Amaro A, Meraldi P, McAinsh A. Kinetochore-generated pushing forces separate centrosomes during bipolar spindle assembly. J Cell Biol. 2009;184:365\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu Z, Chen C, Yan M, Zeng X, Zhang Y, Lai D. CENPO regulated proliferation and apoptosis of colorectal cancer in a p53-dependent manner. Discover Oncol. 2022;13:8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe K, Xie M, Li J, He Y, Yin Y. CENPO is Associated with Immune Cell Infiltration and is a Potential Diagnostic and Prognostic Marker for Hepatocellular Carcinoma. Int J Gen Med. 2022;15:7493\u0026ndash;510.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Y, Xiong J, Li Z, Zhang G, Tu Y, Wang L, et al. CENPO expression regulates gastric cancer cell proliferation and is associated with poor patient prognosis. Mol Med Rep. 2019;20:3661\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Ye D, Li Y, Lv F, Shen W, Li H, et al. Prognostic and immune infiltrative biomarkers of CENPO in pancancer and its relationship with lung adenocarcinoma cell proliferation and metastasis. BMC Cancer. 2023;23:735.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi T, Hu Z, Tian L, Yang Y. Advances in lung adenocarcinoma: A novel perspective on prognoses and immune responses of CENPO as an oncogenic superenhancer. Translational Oncol. 2023;34:101691.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Z. Gene mining of immune microenvironment in hepatocellular carcinoma. Medicine. 2022;101:e30453.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi T, Hu Z, Tian L, Yang Y. Pancancer landscape of CENPO and its underlying mechanism in LUAD. Respir Res. 2023;24:113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Evolution of Head and Neck Cutaneous Squamous Cell Carcinoma Nodal Staging-An Australian Perspective. Cancers (Basel). 2022;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Cutaneous Squamous Cell Carcinoma: Clinico-Dermoscopic and Histological Correlation: Approximately 72 Cases. Dermatol Pract Concept. 2024;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Higher metastasis and death rates in cutaneous squamous cell carcinomas with lymphovascular invasion. J Am Acad Dermatol. 2021;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u. Intralesional Treatments for Invasive Cutaneous Squamous Cell Carcinoma. Cancers (Basel). 2024;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u. Gene mining of immune microenvironment in hepatocellular carcinoma. Med (Baltim). 2022;101.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in hepatocellular carcinoma. J Exp Clin Cancer Res. 2023;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBancroft J, Auckland P, Samora C, McAinsh A. Chromosome congression is promoted by CENP-Q- and CENP-E-dependent pathways. J Cell Sci. 2015;128:171\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkumura K, Kagawa N, Saito M, Yoshizawa Y, Munakata H, Isogai E, et al. CENP-R acts bilaterally as a tumor suppressor and as an oncogene in the two-stage skin carcinogenesis model. Cancer Sci. 2017;108:2142\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen A, Fadel M, Cheeseman I. Differential requirements for the CENP-O complex reveal parallel PLK1 kinetochore recruitment pathways. Mol Biol Cell. 2021;32:712\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKagawa N, Hori T, Hoki Y, Hosoya O, Tsutsui K, Saga Y, et al. The CENP-O complex requirement varies among different cell types. Chromosome research: Int J Mol supramolecular evolutionary aspects chromosome biology. 2014;22:293\u0026ndash;303.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u. Tumor Recurrence, Depth of Invasion, and Temple Location as Independent Prognostic Parameters of Lymph Node Metastases of Head and Neck Cutaneous Squamous Cell Carcinomas. Int J Clin Pract. 2024;2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u. Methyl protodioscin induces G2/M arrest and apoptosis in K562 cells with the hyperpolarization of mitochondria. Cancer Lett. 2005;224.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u. Cyclin A1 is a p53-induced gene that mediates apoptosis, G2/M arrest, and mitotic catastrophe in renal, ovarian, and lung carcinoma cells. Cell Mol Life Sci. 2006;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Curcumin-Piperlongumine Hybrid Molecule Increases Cell Cycle Arrest and Apoptosis in Lung Cancer through JNK/c-Jun Signaling Pathway. J Agric Food Chem. 2024.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. PIDDosome-induced p53-dependent ploidy restriction facilitates hepatocarcinogenesis. EMBO Rep. 2020;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eundefined u, undefined u, undefined u, undefined u, undefined u, undefined u, et al. Inhibition of cancer progression by a novel trans-stilbene derivative through disruption of microtubule dynamics, driving G2/M arrest, and p53-dependent apoptosis. Cell Death Dis. 2018;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaverot G, Ilie M, Lasolle H, Amodru V, Trouillas J, Castinetti F, et al. Aggressive pituitary tumors and pituitary carcinomas. Nat reviews Endocrinol. 2021;17:671\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"CSCC, CENPO, proliferation, metastasis, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-4446737/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4446737/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCutaneous squamous cell carcinoma (CSCC) is a common nonmelanoma skin cancer. There are limited targeted therapeutic options for treating CSCC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis study explored the differential expression of CENPO in CSCC and its relationship with clinical prognosis via data from The Cancer Genome Atlas (TCGA) database. The CENPO gene knockdown lentivirus was constructed, and the biological function of CENPO was evaluated via CCK8 cell proliferation, scratch, invasion, and cell apoptosis experiments in vitro. Furthermore, CENPO was evaluated in vivo.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe TCGA data and clinical immunohistochemical results confirmed that CENPO is significantly overexpressed in CSCC and that CENPO is upregulated with clinical grade. The CCK-8 results confirmed that cell proliferation decreased with CENPO knockdown. Scratch experiments confirmed that cell migration decreased with CENPO knockdown. The invasion experiments confirmed that the cell invasion ability decreased with CENPO knockdown. Flow cytometry experiments showed that cell apoptosis increased with CENPO knockdown. The in vivo assay results showed that the tumor growth rate significantly decreased with CENPO knockdown.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe proliferation, invasion, migration, and antiapoptotic ability of CSCC cells are enhanced by upregulating CENPO. The activity of CSCC cells was significantly inhibited by CENPO knockdown. CENPO could serve as a new biomarker for the diagnosis and treatment of CSCC.\u003c/p\u003e","manuscriptTitle":"CENPO as a potential biomarker for the prognosis and therapy of CSCC patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-10 22:23:29","doi":"10.21203/rs.3.rs-4446737/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4f1eeb2f-c6bf-44ab-b227-ae05a34858e4","owner":[],"postedDate":"June 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T06:42:36+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-10 22:23:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4446737","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4446737","identity":"rs-4446737","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}