Identification of an oncogenic role of centromere protein M in non-small cell lung cancer via CDC20/MYBL2/Wnt signals

doi:10.21203/rs.3.rs-4818528/v1

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Research Article

Identification of an oncogenic role of centromere protein M in non-small cell lung cancer via CDC20/MYBL2/Wnt signals

https://doi.org/10.21203/rs.3.rs-4818528/v1

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Lung cancer is the most common carcinoma with high mortality. However, the underlying mechanism of pulmonary neoplasia and disease development remains poorly understood. Our study comprehensively analyzed the transcriptome profiles and clinical-pathological characteristics of 515 patients diagnosed with non-small cell lung cancer (NSCLC) retrieved from the TCGA database. We observed a significant upregulation of centromere protein M (CENPM) in tissues of NSCLC patients, which was positively correlated with adverse prognosis. Additionally, overexpression of CENPM significantly facilitated cell proliferation and enhanced tumorigenic potential of NSCLC cell lines (A549/NCI-H1299), resulting in accelerated tumor progression and shortened survival time in tumor-bearing mice. Mechanistically, CENPM activated the Wnt/β-catenin signaling pathway through cell division cycle 20 (CDC20)/ MYB proto-oncogene kike 2 (MYBL2) axis. Blockade of Wnt signaling or CDC20/MYBL2 axis suppressed the tumorigenic potential and proliferative characteristics-induced by CENPM. Our investigation highlighted an essential role of CENPM in promoting NSCLC development, and CENPM might represent a novel biomarker for predicting NSCLC progression in clinic.

CENPM

Wnt signaling

CDC20

MYBL2

Lung cancer is the leading cause of cancer-associated death worldwide (Rosell and Karachaliou, 2015). There are approximately 2.2 million new lung cancer diagnosed cases and 1.8 million deaths in 2020, representing nearly one-fifth (18.0%) cancer deaths (de Groot et al., 2018). NSCLC accounts for 85% of all lung carcinomas (Herbst et al., 2018). Though remarkable advance has been obtained in diagnosis and therapeutic approaches, it remains one of the poorest prognosis diseases. Recently, emerging researches have focused on discovering actionable molecular alterations and the molecularly driven treatment has been constantly evolving. Nonetheless, most NSCLC patients still suffered tumor recurrence or distant metastasis within 5 years of diagnosis. And the 5-year overall survival rate is 18.2% (Rotow and Bivona, 2017). Thus, innovative prognostic biomarkers and better understand of mechanism of tumor progression are essential for clinical diagnose and development of novel therapeutic drugs in NSCLC.

CENPM, also named as proliferation associated nuclear element 1 (PANE1), is a component of CENPA nucleosome‑associated complex (NAC) (Earnshaw and Rothfield, 1985). It plays important roles in kinetochore proteins assembly, chromosome segregation and mitotic progression. Aberrant function of CEAPM could induce chromosomal instability (CIN) (McKinley and Cheeseman, 2016). As is well known, one of the traditional phenotypes of cancer is increased CIN leading to aneuploidy and tumorigenesis (Pino and Chung, 2010). Moreover, the proliferative characteristics of cancer cells is tightly correlated with mitosis and chromosome separation (Wassmann and Benezra, 2001). Indeed, compelling investigations have indicated that abnormal expression of CENPA complex in human tumors has been observed in several tumor types. For example, the upregulation of CENPA stimulated tumor progression in colorectal cancer (Tomonaga et al., 2003), and increasing evidence suggested that CENPF contributed to a poor prognosis in patients with breast cancer and nasopharyngeal carcinoma (Sun et al., 2016). However, the underlying mechanisms of CENPA complex associated tumor progression remains unclear. Recently, CENPM has been excessively investigated in several cancer types and reported to serve as a novel prognosis maker in liver cancer, melanoma, breast cancer and bladder cancer (Xiao et al., 2019; Liu et al., 2020). But few research explored the correlation between CENPM and NSCLC progression. Although some studies suggested that CENPM gene may be involved in the evolution of lung adenocarcinoma (Zhou et al., 2021), the specific role of CENPM in NSCLC progression remains to be elucidated.

Our study conducted an initial screen for the transcriptome expression of CENPM in 515 NSCLC patients. It was revealed that CENPM was upregulated in NSCLC tumor tissues compared to the normal group. On basis of this, we verified the clinical relevance of CENPM in clinical samples and indicated that CENPM expression was closely associated with distance metastasis and survival time in patients with NSCLC. Mechanically, overexpression of CENPM remarkably enhanced NSCLC cells proliferation and tumorigenic potential in vitro and in vivo through CDC20/MYBL2 mediated Wnt/β-catenin signaling activation. Hence, our study described the crucial role of CENPM in NSCLC development and provided a novel prognosis biomarker and a promising therapeutic strategy for NSCLC patients.

Cell lines and chemical reagents

Human NSCLC cell lines NCI-H1299 and A549 were purchased from the Global Bioresource Center (ATCC, USA). CENPM overexpression A549/NCI-H1299 cells were established by Tsingke (China) and determined by western blotting. Cancer cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Thermo Fisher, USA) containing 10% fetal calf serum (FBS) in a standard atmosphere with 5% CO₂, and at a temperature of 37°C. cAMP inhibitor PKI 14–22 amide, myristoylated (PKI), Wnt/β-catenin inhibitor PNU-74654 (PUN), hedgehog signaling inhibitor KAAD-Cyclopamine (KAAD) were purchased from Medchemexpress (USA).

Clinical specimens

Information of 59 normal tissues and 721 NSCLC tissues in the Cancer Genome Atlas Program (TCGA) databases were downloaded from Cbioportal database and http://ualcan.path.uab.edu/index.html. Paraffin sections of NSCLC tissues were obtained from Affiliated Central Hospital of Dalian University of Technology. 26 patients were divided into metastatic (M1) and non-metastatic (M0) groups according to their post-treatment diagnosis (five-years). Patients were informed with written consent and agreed to take part in our clinical experiments. All experiments were conducted according to the Declaration of Helsinki. Ethical review was granted by the Ethics Committee of the Affiliated Central Hospital of Dalian University of Technology.

Cell proliferation assay

Cell proliferation was determined by a cell counting kit (CCK8) purchased from Solarbio (China). 3000 pre-treated cells were seeded in 96-well plates. The absorbance value was measured at 0, 24, 48 and 72 hours after CCK8 reagent incubation according to the manufacturer’s instructions.

Colony formation assay

For tumorigenic potential assay, pre-treated cells were seeded in 6-well plates (500 cells/ well). Then cells were cultured with serum-free culture medium. After 10 days, cell colony formation was observed under the inverted optical microscope (Leica, Germany). Meanwhile, colonies were washed, and stained with crystal violet. The visible colonies were counted and analyzed.

Cell apoptosis assay

Cell apoptosis was evaluated using an Annexin V/PI staining kit (Solarbio, China) and flow cytometry analysis. Briefly, siRNA treated tumor cells were harvested and then stained by Annexin V/PI staining kit according to the manufacturer’s protocol. Then cell apoptosis was determined by flow cytometry (BD Pharmingen, CA).

Small interfering RNA (siRNA)

SiRNA for CDC20, CENPM and MYBL2 was purchased from Tsingke (China). The sequences of the CENPM siRNAs were as follows: 5′-GGACUCGAUGCUCAAAGAGTT-3′ and 5′-CUGAAGGUCCACUUGGCAATT-3′; The sequences of the CDC20 siRNAs were as follows: 5′-GGAGCUCAUCUCAGGCCA UUU-3′ and 5’- GGGAAUAUAUAUCCUCUGUUU-3’; The sequences of the MYBL2 siRNAs were as follows: 5’- GAGGGATAGCAAGTGCAAGGT-3’ and 5’-TTCCAGTCCTGCTGTCCAAA-3’; SiRNA interference was conducted using an Lipofectamine 3000 (Invitrogen, USA) protocol. The siRNA was added at the of concentration 100 nmol/l.

Western blotting

RIPA lysis buffer (Abcam, UK) was used for whole cell lysates extraction. Total proteins were separated by 10% SDS-PAGE, and samples were then transferred onto a nitrocellulose membrane (Thermo, USA). Subsequently, samples were incubated with the primary antibodies at 4°C overnight. After wash with TBST, samples were incubated with a horseradish peroxidase-conjugated secondary antibody for 2 hours at room temperature. Primary antibodies anti-β-catenin (ab32572), anti-CENPM (ab243820), anti-CDC20 (ab26483), anti-MYBL2 (ab191064) and anti-Wnt3a (ab219412) were purchased from Abcam (UK).

Immunostaining

Sections from NSCLC patients or mice were blocked with 5% bovine serum albumin for 30 minutes, then incubated with following primary antibodies anti-CENPM (ab243820, Abcam, UK) anti-Wnt3a (ab219412, Abcam, UK), anti-β-catenin (ab32572, Abcam, UK) and anti-Ki67 (ab15580, Abcam, UK) overnight at 4℃. Subsequently, sections were incubated with secondary antibody for 2 hours and developed at room temperature, according to the manufacturer’s instructions. Protein expression intensity was calculated by Image-Pro Plus 5.0 (Media cybernetics, USA).

Animal protocols

NOD-SCID mice (female, 6 ~ 8 weeks) were obtained from Vitalriver (USA) and raised in a specific pathogen free (SPF) room. For tumor growth assay in vivo, Immune deficient mice were subcutaneously injected with 1×10⁶ A549 cells (6 mice per group). Tumor tissues were collected for further protein expression assay. Tumor volume was calculated by the formula: tumor volume = length × width ²/2. Animal experiments were approved and monitored by the Animal Ethics Committee of Affiliated Central Hospital of Dalian University of Technology. The animal experiments were performed in accordance with the Public Health Service Policy and the WHO guidelines for the humane use and care of animals.

Statistical analysis

All data in our study are presented as the mean values ± SD. Each experiment was conducted for at least three independent times. Variation analysis was performed using one-way ANOVA or t test. Multiple comparisons among the groups were analyzed by Sidak correction method. The survival rates were determined by Kaplan–Meier survival analysis. Statistical analysis was conducted by SPSS 23.0 software (IBM, USA) or GraphPad Prism version 6.0 (GraphPad Software, USA). P-values < 0.05 were considered as a statistically significant difference.

Upregulated CEMPM was found in NSCLC tissues and correlated with poor prognosis

To assess the potential clinically relevance between CENPM and NSCLC development, we evaluated the transcriptome expression of CENPM in 515 NSCLC patients in comparison with 59 normal tissues using TCGA databases. Those results indicated that CENPM was markedly upregulated in tumor tissues compared to normal group, and similar CENPM level was found in NSCLC patients with different clinical stages (Fig. 1a). Given the strong expression of CENPM expression in NSCLC tissues, we sought to assess whether enriched CENPM influenced overall survival of NSCLC patients. Here, 211 NSCLC patients were divided into high-CENPM (n = 103) and low-CENPM (n = 108) groups. Accordingly, patients with lower CENPM expression level displayed a significantly extended survival time in comparison with the high expression group (Fig. 1b), indicating that CENPM might correlated with the poor prognosis in NSCLC. Subsequently, to evaluate the role of CENPM in regulating NSCLC metastasis, 26 NSCLC tissues from clinical patients were divided into metastatic (M1) and non-metastatic (M0) groups by their post-treatment diagnosis (five-years). Immunohistochemistry was performed to evaluated CENPM expression in 26 tumor tissues. Consistently, increased expression level of CENPM was observed in metastatic patients (Fig. 1c). Those data provided evidence that CENPM facilitated tumorigenesis and contributed to the poor prognosis in NSCLC patients.

CENPM promoted cell proliferation and tumorigenic potential in NSCLC

Given that enriched CENPM contributed to the poor prognosis in NSCLC patients, we further investigated the molecular mechanism of CENPM-induced NSCLC progression. Here, NSCLC cell lines NCI-H1299 and A549 were treated with CENPM siRNA and cultured for 3 days (Fig. 1d). However, an increase of cell apoptosis was found in CENPM silenced A549/NCI-H1299 cells (Fig. 1e), indicating the essential role of CENPM in regulating the cell survival of NSCLC. To better explore the influence of CENPM on NSCLC progression, we further established CENPM overexpression A549/NCI-H1299 cells (Fig. 1f) and assessed their cell proliferation and colony formation capability. Notably, a prominent increase of cell proliferation was observed in CENPM overexpression cells when compared to vector group (Fig. 1g). Meanwhile, CENPM overexpression also strengthened colony formation capability of A549/NCI-H1299 cells (Fig. 1h). These results indicated that excess CENPM expression facilitated NSCLC cell proliferation, and tumorigenic potential.

CENPM promoted NSCLC development through Wnt/β-catenin signaling

To elucidate molecular pathways driven by CENPM for promoting NSCLC development, we next divided 211 NSCLC patients into high CENPM (n = 126, relative CENPM expression > 100) and low CENPM (n = 85, relative CENPM expression < 100) groups by utilizing TCGA database. Differential expressed genes and CENPM involved main pathways were identified by enrichment analysis (Fig. 2a). Notably, cAMP, Wnt and Hedgehog pro-survival signaling pathways have been reported to play essential roles in tumor progression. Thus, vector and CENPM overexpression A549/NCI-H1299 cells were treated with cAMP inhibitor PKI, Wnt/β-catenin inhibitor PNU and Hedgehog signaling inhibitor KAAD respectively. We found blockade of Wnt/β-catenin signaling inhibited the proliferation (Fig. 2b) and tumorigenic capability (Fig. 2c) in CENPM overexpressed A549 and NCI-H1299 cells, while limited influence were found in PKI or KAAD treated groups. This promoted us to speculate that CENPM mediated NSCLC progression in a Wnt/β-catenin dependent manner. To validate our hypothesis, we conducted western blotting assay to assess the expression of Wnt-3a and β-catenin in CENPM overexpression A549/NCI-H1299 cells. Consistently, elevated expression of Wnt-3a and β-catenin was found in CENPM overexpression A549/NCI-H1299 when compared to vector group (Fig. 2d). Moreover, immunostaining results further indicated that Wnt-3a was upregulated in metastatic patients by analyzing 26 tumor tissues derived from patients (Fig. 2e). Collectively, our data suggested that CENPM facilitated NSCLC development through Wnt/β-catenin signals.

CENPM upregulated CDC20 and MYBL2 to mediate Wnt/β-catenin signaling activation

Given the role of Wnt/β-catenin signals in CENPM-induced tumor progression, we next wondered how CENPM mediated Wnt signaling activation. Here, we further analyzed the upregulated genes in CENPM-high patients, compared to the CENPM-low patients (n = 211). Upregulated genes in CENPM-high patients were listed (Fig. 3a). Intriguingly, significant upregulation of CDC20 and MYBL2 were observed in our CENPM high group. And previous study has reported that MYBL2/CDC20 axis was able to stimulated gastric cancer cell proliferation through Wnt/β-catenin signals (Deng et al., 2021). Thus, we detected the expression of CDC20 and MYBL2 in CENPM overexpression A549/NCI-H1299 cells. Increased expression of CDC20 and MYBL2 were found (Fig. 3b), indicating that CENPM upregulated CDC20 and MYBL2 in NSCLC cells. Subsequently, to explore whether CENPM mediated Wnt/β-catenin signaling activation through CDC20/MYBL2 axis. CDC20 and MYBL2 were silenced using siRNA technology in CENPM overexpression A549/NCI-H1299 cells respectively (Fig. 3c and d). Meanwhile, western blotting assay was conducted to evaluate Wnt-3a/β-catenin expression in CDC20 or MYBL2 silenced tumor cells. Consistent to our hypothesis, silence of CDC20 or MYBL2 suppressed upregulation of Wnt-3a and β-catenin in CENPM overexpression A549/NCI-H1299 cells (Fig. 3e and f). Moreover, CDC20 or MYBL2 silence suppressed cell proliferation (Fig. 3g and h) and tumorigenic potential (Fig. 3i and j) induced by CENPM. Those results suggested that CENPM upregulated CDC20 and MYBL2 to regulate Wnt/β-catenin signaling activation.

CENPM promoted NSCLC progression in vivo

Next, to evaluate the pro-tumor effects of CENPM in vivo, we sought to evaluate the tumor growth of CENOM overexpressed cells in vivo. 1×10⁶ vector or CENPM overexpressed A549 cells were subcutaneously implanted into immunodeficient mice respectively. As excepted, CENPM overexpressed A549 formed rapidly growing tumor in comparison with vector group (Fig. 4a). As excepted, CENPM overexpression A549 bearing mice also exhibited shortened survival time (Fig. 4b). Consistent to our results in vitro, the protein level of CDC20, MYBL2, Wnt-3a and β-catenin were upregulated in CENPM overexpressed tumor tissues in comparison with vector group (Fig. 4c and d). We also assessed the expression of Ki67, the cell proliferation marker, which was remarkably upregulated in CENPM overexpression tumor tissues (Fig. 4e). Together, those results suggested that CENPM regulated CDC20/MYBL2/Wnt signaling to accelerate NSCLC progression.

CENPM is not only the microtubules-related kinetochores protein to module chromosome separation, but also participates in the regulation of cell cycle (Earnshaw, 2015). It has reported that enriched CENPM expression was able to facilitate uneven chromosome separation, contributing an unequal number of chromosomes allocation. These aneuploid cells are prone to chromosomal aberrations and can carry more visible aberrations than normal cells (Dobie et al., 1999). Consistently, it has been illustrated that CENPM is upregulated in proliferating cells, for example, activated lymphoid cells and cancer cells (Earnshaw, 2015). Previous studies have defined that the expression of CENPM was tightly associated with the prognosis of patients with hepatocellular carcinoma progression and could be a complement to AFP in the diagnosis of liver cancer (Z. H. Wu and Yang, 2020). Moreover, recent investigations addressed that CENPM might contribute to tumor metastasis and recurrence in melanoma (Chen et al., 2019), bladder cancer (Kim et al., 2018) and pancreatic cancer (Zheng et al., 2020). However, limited researches investigated the influence of CENPM on the oncogenesis of NSCLC. Our investigation systematically analyzed the transcriptome profiles and clinicopathological characteristics of 515 NSCLC patients from TCGA dataset, and described that upregulated CENPM was found in tumor tissues and was closely associated with the distant metastasis and poor survival in NSCLC patients. These suggested that CENPM might promoted NSCLC progression and might correlate with tumor cells proliferation and invasiveness. Our investigation further indicated that the depletion of CENPM inhibited cell proliferation and tumorigenesis potential in vitro. Additionally, we established CENPM overexpressed xenograft mouse model to demonstrate that CENPM played an essential role in tumor growth and disease progression in vivo. Taken together, these evidences implied that CENPM may act as a novel biomarker in clinical early diagnosis or prognosis of NSCLC and provided a potential chemotherapeutics target for NSCLC treatment.

Given that CENPM played a central role in NSCLC development, we further investigated the molecular mechanism of CENPM associated cells proliferation and tumorigenesis. Previous studies reported that CENPM regulated cell proliferation/invasion through the mTOR/p70S6K axis in pancreatic cancer (Zheng et al., 2020). Meanwhile, it has been documented that CENPM was able to suppress cell apoptosis and facilitate cell cycle progression through P53 signaling in hepatocarcinogenesis (Xiao et al., 2019). Noteworthy, a study investigated the protein interaction network of CENPM and demonstrated that CENPM mediated tumor development by modulating cell cycle-associated proteins, such as KIF4A, HMMR, CKS2, and CDC25C, which are considered as important drivers of carcinogenesis (Xiao et al., 2019). In agreement with prior reports, this study determined a significant upregulation of CDC20 and MYBL2 in CENPM overexpressed NSCLC cell lines as well as tumor tissues (Yu, 2002). CDC20 and MYBL2 are both recognized as central regulators of cell proliferation or differentiation involved in tumorigenesis. As is well known, CDC20 is a key component in spindle assembly checkpoint, and correlated with cell cycle, proliferation and apoptosis. The overexpression of CDC20 enhances cells proliferation and migratory capacity and serves as a vital role in tumorigenesis and progression in several types of cancer (Deng et al., 2021). Meanwhile, previous evidences suggested that up-regulated MYBL2, along with CDC20, could be used as a biomarker for disease severity in several cancers, including glioma, gastric cancer, lung cancer and breast cancer (Wang et al., 2015; Musa et al., 2017). Notably, recent researches revealed that MYBL2 and FOXM1 regulated an crucial “driver-network” consist of 26 genes, including CDC20, which is correlated with modulation of cell cycle or proliferation in lung cancer (Ahmed, 2019). However, to date, the documented researches were limited to be gene expression network analysis, and the specific underlying molecular mechanism still remains largely unclear. Our experiments, for the first time, demonstrated that the CENPM mediated CDC20 and MYBL2 up-regulation could further activate downstream Wnt/β-catenin signals in NSCLC.

The Wnt/β-catenin signals exert integral roles in many biological processes ranging from embryogenesis to growth-associated diseases (MacDonald et al., 2009). Aberrant activation of this pathway is a key driver of tumor replase and growth in various cancers (Fodde and Brabletz, 2007).The pro-tumor role of Wnt/β-catenin siganls has been widely investigated in recent years. For example, Teng et al demonstrated that activated Wnt/β-catenin pathway up-regulated the stem-like properties in lung cancer, including increased colony formation, tumorigenesis and drug resistant abilities (Teng et al., 2010). In addition, accumulated evidences indicated that the upregulation of Wnt/β-catenin axis promoted the EMT transition, migration and invasiveness of cancer cells via different dependent manner. Nevertheless, the mutations of β-catenin are one of the contributors of aberrant activation of Wnt proteins in some tumor types, for example, gastric cancer and colon cancer, whereas that are rarely detected in NSCLC (Y. Wu et al., 2012). Therefore, identifying regulators to Wnt signaling could provide innovative insights in molecular mechanism investigation in NSCLC. Our study that CENPM up-regulated CDC20 and MYBL2 remarkably increase Wnt-3a and β-catenin provided a novel mechanism to supports the pro-tumor role of Wnt/β-catenin in lung cancer. It is noteworthy that conductin, which was also named as axin2/axil, is a negative regulator of Wnt/β-catenin signals, and localizes and function at centrosomes and the mitotic apparatus, while CDC20 could promoted the degradation of axin2/axil (Hadjihannas et al., 2012). Therefore, it is reasonable that CDC20 activates Wnt/β-catenin aixs via conductin degradation. Our investigation sheds light on the important of CDC20 and MYBL2 mediated Wnt/β-catenin activation of NSCLC progression, which provides a future research direction. Meanwhile, discovering and validating CDC20 or MYBL2 targeted small molecule inhibitors might serve as a potential therapeutic strategy for NSCLC.

Given the limitations of previous studies, this study identified the important clinical relevance of CENPM in NSCLC by analyzing transcript profiles and clinical characteristics from TCGA database as well as clinical pathological sections. Initially, we determined the essential role of CENPM in NSCLC. Then, we established CENPM overexpressed cell lines and CDC20 knockdown as well as MYBL2 knockdown cell lines to further investigated the underlying molecular mechanism. On basis of these, our evidences illustrated that overexpressed CENPM leads to inhibited apoptosis, increased cell proliferation and tumorigenesis in NSCLC via CDC20/MYBL2 mediated Wnt/β-catenin signaling activation. Notably, our CENPM overexpressed NSCLC mice model further verified this pro-tumor mechanism in vivo. Our investigation highlighted that CENPM and associated activated pathway might serve as potential prognosis markers and therapeutical targets. However, our study remains several limitations. The population of patients was small and the measured pathological sections were limited, which made the clinically meaningful power limited, however, our preliminary results still benefit current ongoing investigations. Additionally, the synergistic interaction as well as the molecular mechanism between CDC20 and MYBL2 remain to be further explored. Meanwhile, there were still more details to be investigated in signaling pathway analysis. For example, other proteins in the Wnt pathway, such as TCF4, were not analyzed in our investigation. Taken together, our research indicated that CENPM can promote NSCLC progression via CDC20/MYBL2/Wnt signaling, and provided a reliable biomarker for the individualized treatment for NSCLC patients.

14-22 amide, myristoylated (PKI);

PNU-74654 (PUN);

KAAD-Cyclopamine (KAAD);

Non-small cell lung cancer (NSCLC);

The Cancer Genome Atlas Program (TCGA);

Centromere Protein M (CENPM);

Cell Division Cycle 20 (CDC20);

MYB Proto-Oncogene Like 2 (MYBL2);

chromosomal instability (CIN);

Kyoto Encyclopedia of Genes and Genomes (KEGG);

proliferation associated nuclear element 1 (PANE1);

nucleosome‑associated complex (NAC);

Funding: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing Interests: The authors have no relevant financial or non-financial interests to disclose.

Author Contributions: Q.K., L.W., and J.L. were collectively responsible for the comprehensive research design. J.L. and L.W. performed the experiments and wrote the manuscript. H.W. and X.C. assisted with the bioinformatics analysis and data visualization. Q.K. reviewed the article and made revisions. All authors contributed to the article and approved the final submitted version.

Data Availability: The article contains all the data in the study.

Ethics approval: We confirmed that all experiments involving human subjects were conducted by The Code of Ethics of the World Medical Association (Declaration of Helsinki). Ethical review was granted by the Ethics Committee of the Affiliated Central Hospital of Dalian University of Technology. All animal experiments were performed in compliance with the ARRIVE guidelines and the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, or the National Research Council's Guide for the Care and Use of Laboratory Animals. Approval was granted by the Ethics Committee of Dalian University of Technology.

Consent to participate: Informed consent was obtained from all individual participants included in the study.

Consent to publish: Not applicable.

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Identification of an oncogenic role of centromere protein M in non-small cell lung cancer via CDC20/MYBL2/Wnt signals

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Cell lines and chemical reagents

Clinical specimens

Cell proliferation assay

Colony formation assay

Cell apoptosis assay

Small interfering RNA (siRNA)

Western blotting

Immunostaining

Animal protocols

Statistical analysis

Results

Upregulated CEMPM was found in NSCLC tissues and correlated with poor prognosis

CENPM promoted cell proliferation and tumorigenic potential in NSCLC

CENPM promoted NSCLC development through Wnt/β-catenin signaling

CENPM upregulated CDC20 and MYBL2 to mediate Wnt/β-catenin signaling activation

CENPM promoted NSCLC progression in vivo

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1