SPC25 Functions as a Prognostic-Related Biomarker, and Its High Expression Correlates with Tumor Immune Infiltration and UCEC Progression

Background : Most tumor tissues expressed spindle pole body component 25 ( SPC25 ), one of the four subunits of the NDC80 complex, at greater levels compared to surrounding normal tissues. According to earlier researches, this subunit strongly encouraged tumor cell proliferation and tumor growth, which resulted in worse prognoses in patients with hepatocellular, breast, lung, and prostate cancer. Precisely because SPC25 ’s role in uterine corpus endometrial carcinoma (UCEC) is understudied, we chose to concentrate on UCEC for gaining a more scientific and thorough understanding of SPC25 . Methods : Along with examining SPC25 ’s differential expression, prognostic significance, and biological function in UCEC, our research sought to clarify the underlying mechanism by which SPC25 influences the course of UCEC and patient prognosis from the viewpoints of methylation and immune infiltration. Results : We observed differential expression of SPC25 gene in different clinicopathological features of UCEC and identified SPC25 as a hazard factor for poorer overall survival (OS), disease-specific survival (DSS), and progress free interval (PFI) in UCEC, particularly in its multiple clinical subtypes. In addition, we also discovered that SPC25 and its co-expressed genes mostly engaged in biological processes and signal transduction routes linked to cell cycle and cell division in UCEC. After investigating SPC25 ’s methylation status, we discovered that patients with UCEC had elevated SPC25 expression and a poor prognosis due to hypomethylation of CpG sites in the SPC25 gene sequence. Finally, we investigated SPC25 ’s potential role in immunotherapy and discovered that SPC25 might alter the major immune cell infiltration levels in the tumor microenvironment (TME) by regulating the expression of immunoregulatory molecules and chemokines, which would be beneficial for SPC25 to control the progression of UCEC. Conclusions : In conclusion, SPC25 was a useful predictive biomarker as well as a possible therapeutic target for UCEC.


Introduction
Mitosis maintains the genetic properties of any multicellular organism throughout entire life cycle.Every time the mother cell divides, the chromosomes are evenly distributed among the daughter cells due to the action of the mitotic spindle [1].The NDC80 complex which is an extremely conserved kinetochore protein from yeast to humans, is an approximately 57 nm elongated heterotetramer consisting of NDC80 (NDC80P, HEC1, nuclear division cycle 80), NUF2 (NUF2P, NUF2 component of NDC80 kinetochore complex), SPC24 (SPC24P, spindle pole body component 24), and SPC25 (SPC25P, spindle pole body component 25).At its SPC24/SPC25 globular end, the complex connects the centromere-proximal inner kinetochore layer to the microtubule-binding outer kinetochore layer at its NDC80/NUF2 globular end [2,3].It is crucial that spindle microtubules attach to the chromosome kinetochores through the NDC80 complex during cell division [4].NDC80 has been considered as a major player in cancers' initiation, development, and spread in the last few years.NDC80 was abundantly expressed in various cancers and promoted tumor growth through the reduction of apoptosis and increasing cell cycle efficiency, resulting in poor survival and prognosis of cancer patients [5][6][7][8][9].SPC25 overexpression has been found in recent researches to enhance tumor growth and metastasis in hepatocellular carcinoma (HCC), as well as a worse prognosis for HCC patients [10][11][12][13].The same conclusion appears in the study of lung cancer [14][15][16], prostate cancer (PCa) [17,18], breast cancer (BC) [19], and neurodegeneration [20].SPC25 is also necessary for chromosomal alignment, spindle formation, and effective spindle checkpoint signaling during meiosis, according to research [21].
The number of deaths linked to endometrial cancer (EC) is rising, with an estimated 1-2% yearly increase in the disease's incidence.But no fresh medications were authorized for the treatment of EC.There are currently only two Food and Drug Administration (FDA)-approved medications for the treatment of EC, despite the fact that numerous medications are approved for the treatment of ovarian, fallopian tube, and primary peritoneal cancers.This highlights the need for novel therapies to treat advanced, recurrent, and metastatic EC [22].
Our research goal was to determine SPC25's differential expression, prognostic value, biological functions, and its potential in immune infiltration in uterine corpus endometrial carcinoma (UCEC) by providing a more systematic and comprehensive perspective.Thus, we looked into the associations between SPC25's expression and various clinicopathological features, clinical outcomes in various clinical subgroups, and coexpression network analyses in UCEC.Moreover, we focused on the methylation level of SPC25 in UCEC and its corresponding predictive significance, and explored the potential mechanism of SPC25 expression and methylation status in UCEC progression.
In our results, SPC25 was shown to be significantly associated with clinical stage, histologic grade, histological type, radiation therapy, tumor invasion, weight, primary therapy outcome, molecular subtype, immune subtype, and TP53 muation status.Additionally, elevated SPC25 expression might result in poorer overall survival (OS), diseasespecific survival (DSS), and progress free interval (PFI), especially in distinct clinical subgroups of UCEC.Furthermore, we also obtained the SPC25 co-expressed genes, which included 1981 positively correlated genes and 2213 negatively correlated genes.These genes primarily took part in chromosome segregation, DNA replication, cell cycle checkpoint, spindle organization, and negative regulation of cell cycle process.Co-expressed genes were enriched in Cell cycle, Spliceosome, DNA replication, RNA transport, and Oocyte meiosis.Most importantly, the majority of the CpG sites in the SPC25 DNA sequence were hypomethylated, which decreased patient survival and had a negative correlation with immunosuppressive state in the tumor microenvironment (TME).Therefore, it was hypothesized that hypomethylation of the SPC25 promoter caused the overexpression of SPC25 mRNA and protein, and this in turn decreased the expression of chemokines and their receptors, which reduced the aggregation of major immune cells to the tumor site and exerted antitumor immunity, ultimately worsening the disease and shortening the patient's survival time.
Based on these findings, we concluded that SPC25 would modify the composition and immunological processes of TME, which in turn might alter antitumor immunity and impact the course and outcomes of disease.So we speculated that SPC25 might be a significant prognostic biomarker and that SPC25 might work as a potential molecular biomarker to predict immunotherapy response or a novel anticancer immunotherapy target to achieve promising treatment outcomes in UCEC.

SPC25 mRNA and Protein Expression Differences between Endometrial Cancer and Normal Tissues
In our study, we investigated the levels of SPC25 mRNA expression in tissue samples of endometrial cancer and endometrial hyperplasia using a UCEC dataset (GSE106191) that was retrieved from the gene expression omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/).The ggplot2 package [3.3.6] was utilized for data visualization, while the stats package [4.2.1] and car package [3.1-0] were employed for statistical analysis.The protein levels of SPC25 in UCEC were then determined using the university of alabama at birmingham cancer data analysis portal (UALCAN) database (http://ualcan.path.uab.edu/) [23][24][25], an internet application that allows users to analyze the cancer genome atlas (TCGA) and gene transcription data online.We also explored the SPC25 protein expression in endometrial cancer tissues via the Human Protein Atlas (HPA) (https://proteinatlas.org/)[26,27] which contains the human gene expression profile information for both normal and malignant tissues protein levels.Statistics were considered significant when p < 0.05.
We gathered tumor tissues and their paired adjacent normal tissues from 12 patients with endometrial carcinoma who received surgical treatment at the Department of Gynecology of the Second Hospital of Lanzhou University from March 2023 to June 2023.Prior to surgery, no additional therapies were administered to any of the 12 patients.This study was conducted in line with the tenets of the Helsinki Declaration and was approved by the Ethics Committee at the Second Hospital of Lanzhou University.Each patient voluntarily gave their informed permission.SPC25 protein expression was detected by Western blotting assay.In brief, lysing both normal and tumor tissues in RIPA (R0010, Solarbio, Beijing, China) lysis solution containing PMSF (P0100, Solarbio, Beijing, China) for 30 minutes allowed for the extraction of all tissue proteins.Following the protein solution's separation using 10% SDS-PAGE (P1200, Solarbio), the resultant product was subsequently transferred to PVDF membranes.After blocking the PVDF (YA1700, Solarbio) membranes for 30 minutes at room temperature with a blocking solution (P0252, Beyotime, Shanghai, China), they were incubated with primary antibodies for the whole overnight duration at 4 • C. Next day, secondary antibodies were incubated on the membranes for 2 hours at room temperature.Immunoreactive signals were recognized utilizing enhanced chemiluminescence (ECL, PE0010, Solarbio).The primary antibodies utilized were mouse anti-human SPC25 monoclonal antibody (26 kDa; 1:500; TA806559S, OriGene, Jiangsu, China) and mouse anti-human GAPDH monoclonal antibody (35.9 kDa; 1:2000; TA802519S, OriGene).

Relationships between SPC25 Expression and a Variety of Clinical Traits in UCEC
Firstly, we utilized the "Clinical" and "Subtype" modules of TISIDB (an integrated repository portal for tumorimmune system interactions) website (http://cis.hku.hk/TISIDB/) [28] to observe the correlations between SPC25 expression level and cancer stages, tumor grades, molecular subtypes, and immune subtypes of UCEC.Then, the Xi-antao Academic website (https://www.xiantao.love/)[29] provided box plots for SPC25 expression levels of individuals with various clinical features in UCEC.The RNA-seq data and linked clinical data were retrieved from the TCGA database (https://portal.gdc.cancer.gov/) in level 3 HTSeqfragments per kilobase per million (FPKM) format, translated to transcripts per million reads (TPM) format, and analyzed after log2 conversion.For statistical analysis and data visualization, the ggplot2 package [3.3.6] was utilized, along with the stats package [4.2.1] and car package [3.1-0].To detect two groups of data, the Shapiro-Wilk normality test, Kruskal-Wallis test, and Multiple hypothesis test (Dunn's test) with Bonferroni's method to adjust significant level were employed.Ultimatly, in order to verify and strengthen the previous findings, we uesd the UALCAN database to analyze the connection between SPC25 gene expression and clinicopathological characteristics in UCEC.A statistically significant p-value was defined as one less than 0.05.

Survival Prognosis Analysis in Different UCEC Clinical Subgroups
First, we evaluated the diagnostic ability of SPC25 for UCEC utilizing the receiver operating characteristic curve (ROC curve) of the Xiantao Academic web.Utilizing the pROC program [1.18.0], the data underwent ROC analysis, and ggplot2 [3.3.6] was utilized to display the outcomes.Next, we investigated how SPC25 affected the OS, relapse free survival (RFS), DSS, and PFI of UCEC using online tools like Kaplan-Meier Plotter (http://kmplot.com/analysis/) [30] and Xiantao Academic.We further looked into the relationships between SPC25 expression and prognosis (OS, DSS, and PFI) in distinct UCEC clinical subgroups using Kaplan-Meier plots on the Xiantao Academic website [31].In this website, the median SPC25 expression was used as a cutoff value to classify groups (low expression group: 0-50%; high expression group: 50-100%).For the proportional hazards assumption test and fitting survival regression, the Survival package [3.3.1] was utilized.The gg-plot2 and survminer programs [3.3.6]provided visual representations of the findings.In the hypothesis test, the logrank test and cox regression were adopted.p value less than 0.05 means statistical significance.

SPC25 Coexpression Network Analyse in UCEC
we used two of three analytical modules in the Linke-dOmics database (http://linkedomics.org/login.php)[32]: LinkFinder and LinkInterpreter.We utilized LinkFinder to find the SPC25 coexpressed genes using the Pearson correlation test, and the findings were shown using a volcano plot, heat maps, and scatter diagrams.Subsequently, utilizing gene set enrichment analysis (GSEA), we applied LinkInterpreter to the GO_BP (gene ontology biological process) and KEGG (kyoto encyclopedia of genes and genomes) pathways of SPC25 and its coexpressed genes.As statistically significant, a p-value of less than 0.05 was considered.
Then, we opened the web page (https://cn.string-db.org/) of STRING (search tool for the retrieval of interacting genes/proteins) [33] to get SPC25 binding proteins that have been experimentally verified and we calculated the intersection of the two sets of data-SPC25 binding proteins and SPC25-coexpressed genes-using Draw Venn Diagram (https://bioinformatics.psb.ugent.be/webtools/Venn/).To make the results more convincing, we used the function of "Single gene coexpression heat map" in Xiantao Academic to verify the correlation between SPC25 and the genes in the cell cycle pathway network obtained from PathCards website (https://pathcards.genecards.org/)[34].Coexpression heat maps were produced using the ggplot2 package [3.3.6] to illustrate the analysis findings (ns, p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

DNA Methylation Status in the CpG Islands of the SPC25 Gene and Their Prognostic Value in UCEC
DNA hypermethylation at promoters can result in gene silence [35].To further pinpoint the mechanisms underlying the upregulation of SPC25 in UCEC, the methylation levels of SPC25 in the UCEC dataset were analyzed via the UALCAN database.In addition, we also compared the differential expression of SPC25 and its promoter methylation status in UCEC patients with various tumor grades (grades 1, 2, 3, and 4).Not only does DNA methylation during carcinogenesis affect gene expression, but also the prognosis of cancer patients [36].MethSurv (https://biit.cs.ut.ee/methsurv/) [37] is a website that uses TCGA data to perform survival analysis based on DNA methylation biomarkers.DNA methylation status of the SPC25 gene's CpG sites and the prognostic significance of these CpG sites in UCEC were explored in the UCEC-TCGA dataset using the MethSurv database.p value less than 0.05 means statistical significance.

Associations of SPC25 Expression and Its Methylation Status with Immune Cells, Immunomodulators, and Chemokines in UCEC
The TISIDB database (http://cis.hku.hk/TISIDB)[28] is a web server for the interaction analysis of the immune system and tumor that can help forecast the effectiveness of immunotherapy.In our work, the associations between SPC25 expression and its methylation status and immune cells, immunomodulators, and chemokines were evaluated by the Xiantao Academic and TISIDB websites.The gg-plot2 package [3.3.6] was used to illustrate the analysis findings, which was carried out with R programming language [4.2.1].

Statistical Analysis
Examining the SPC25 expression differences between normal (or hyperplasia) and malignant tissues was done Utilizing the Wilcoxon rank sum test (Mann-Whitney U test), Paired Samples T test, and Independent Sample T test.The Kruskal-Wallis test, Dunn's test, Wilcoxon rank sum test, Welch one-way ANOVA test, Games-Howell test, and Independent-Sample T Test were utilized to explore the association between SPC25 expression and clinicopathological features.The area under the curve (AUC) acquired from the time-dependent ROC curve analysis was employed to assess the SPC25's UCEC diagnosis accuracy.Additionally, utilizing the Kaplan-Meier (KM) curve, which employs the Log-rank test and Cox regression, the potential predictive significance of SPC25 expression in UCEC patients with different clinical pathological features was evaluated.Pearson Correlation Analysis was utilized to search for gene sets that correlated positively and negatively with SPC25 expression in UCEC and conduct gene set enrichment analysis (GSEA) analysis.The expression association between SPC25 and genes relevant to the cell cycle pathway network as well as immunological checkpoints in UCEC was examined using Spearman association Analysis.The influence of each SPC25 gene DNA methylation site's methylation status on the UCEC patients' prognosis was examined using the likelihood ratio test.To investigate the relationship between SPC25 expression and the enrichment of 24 different types of immune cells in UCEC TME, Spearman Correlation Analysis was employed.To compare the immune cell enrichment scores of the high and low SPC25 expression groups, the Wilcoxon rank sum test was employed.At p < 0.05, statistical significance was established.

SPC25 was Upregulated in UCEC
We discovered that SPC25 was highly expressed in UCEC (Fig. 1A,B).We also verified that SPC25's expression was considerably higher in UCEC than in matched nearby normal tissues (Fig. 1C).Additionally, the GSE106191 dataset's endometrial cancer tissues showed greater levels of SPC25 expression than the endometrial hyperplasia tissues (p < 0.05) (Fig. 1D).Results from the UALCAN database and immunohistochemistry (IHC) staining further indicated that SPC25's protein expression was increased in endometrial cancer tissues (Fig. 1E,F).The outcomes of Western blotting on 12 matched pairs of tumor and surrounding tissues confirmed once more that endometrial cancer had much greater levels of SPC25 protein than healthy tissues (Fig. 1G,H).

SPC25 was Linked to a Variety of Clinical Features in UCEC
The results of TISIDB indicated that the expression level of SPC25 was closely related to the cancer stages (p = 1.35 × 10 −6 ), tumor grades (p = 1.89 × 10 −30 ), molecular subtypes (p = 2.44 × 10 −24 ), and immune subtypes (p = 2.05 × 10 −22 ) of UCEC (Fig. 2A1-A4, respectively).We also used Xiantao Academic to present the relationships between SPC25 and various clinical features in UCEC, and discovered that SPC25 was expressed at a greater level in patients with stage III, histologic grade 3, histological type of serous, radiation therapy (yes), and tumor invasion (%) (≥50) while it was expressed at a lower level in patients with weight >80 and primary therapy outcome (CR) (Fig. 2B1-B7, respectively).Then, using the UAL-CAN database, we looked at SPC25 expression in UCEC with various clinical features and discovered that SPC25 expression was substantially varied in race, age, weight, menopause status, stage, histological subtypes, and TP53 mutation status of UCEC (Fig. 3A-G, respectively).

SPC25 Prognostic Value in UCEC
The area under the ROC curve was 0.986 (>0.9), demonstrating SPC25's excellent accuracy in predicting UCEC and its positive diagnostic impact on this condition (Fig. 4A).Additionally, we discovered that the OS, RFS, DSS, and PFI of UCEC patients with high SPC25 expression were worse (Fig. 4B-F).So we then looked at the relationships between SPC25 and prognosis (OS, DSS, and PFI) in several UCEC clinical subgroups.Increased SPC25 expression was associated with a lower OS in most clinical subgroups, including subgroup of race (white), age >60, height >160, weight >80, body mass index (BMI) >30, menopause status (post), clinical stage (stage III), histological type (endometrioid), diabetes (No), hormones therapy (No), radiation therapy (No), and surgical approach (minimally invasive) (Fig. 5A-L

SPC25 Coexpression Network Correlated with the Cell Cycle
Then, utilizing the LinkedOmics, STRING, Draw Venn Diagram, PathCards, and Xiantao Academic online tools, we investigated the SPC25 coexpression network to confirm SPC25's possible role in tumor tissue, using UCEC as an example to demonstrate its possible impact.In UCEC, 1981 genes (dark red dots) were substantially positively connected to SPC25, whereas 2213 genes (dark green dots) were inversely proportional to SPC25 (FDR, 0.01) (Fig. 8A).In addition, Supplementary Table 1 included a list of all SPC25 coexpressed genes.The top 50 genes that were positively and negatively linked with SPC25 were exhibited on heat maps (Fig. 9A,C).The top 10 genes that were positively and negatively linked with SPC25 were exhibited on scatter diagrams (Fig. 9B,D The GO_BP and KEGG analysis of SPC25 coexpressed genes were then determined using GSEA.We looked into the GO biological process categories and discovered that SPC25 and its coexpressed genes were mostly involved in chromosome segregation, DNA replication, cell cycle checkpoint, spindle organization, and negative regulation of cell cycle process.Then, we carried out KEGG pathway analysis, which revealed that SPC25 coexpressed genes were prominent in Cell cycle, Spliceosome, DNA replication, RNA transport, and Oocyte meiosis (Fig. 10 and Supplementary Table 2).
We obtained 29 SPC25 interacting proteins from STRING, and Venn diagram showed that BUB1 and NDC80 were both SPC25 interacting proteins and SPC25 coexpressed genes (Fig. 8B,C).The heat map showed that SPC25 was significantly correlated with the expression of genes in the cell cycle pathway network, which further indicated that SPC25 was highly likely to participate in the biological processes and signal transduction pathways of cell cycle, DNA replication, and chromosome separation (Fig. 11 and Supplementary Table 3).

Methylation Status of the SPC25 Gene Was Connected with the Prognosis of UCEC Patients
In contrast to normal samples, UCEC tissues had considerably reduced levels of SPC25 DNA methylation (Fig. 12A).The higher the tumor grade, the lower the methylation degree (Fig. 12B).Utilizing the Met-Surv tool, it was possible to examine the SPC25 gene's DNA methylation levels as well as its CpG islands' prognostic significance.The outcomes revealed 12 methylated CpG islands: cg05868191, cg06350524, cg06580318,  cg13605690, cg22278106, cg15237047, cg04949346, cg17942426, cg07224215, cg20609092, cg06971765, and cg14465028 among which only cg05868191 was hypermethylated (Fig. 12C).Furthermore, methylation levels of four CpG islands, namely, cg22278106, cg15237047, cg07224215, and cg20609092 were associated with the prognosis of UCEC patients (p < 0.05) (Fig. 12D-G and Table 1).Specifically, when compared to individuals with greater methylation levels in SPC25 CpG sites, UCEC patients with lower levels of SPC25 methylation in the cg22278106 and cg15237047 CpG islands had a better OS rate.Conversely, as opposed to individuals with greater levels of CpG methylation in SPC25, UCEC patients with lower levels of SPC25 methylation in the cg07224215 and cg20609092 CpG islands had a worse OS rate.

SPC25 was Linked to Infiltration Levels and Checkpoint-Related Genes of Immune Cells in UCEC
Considering that tumor-infiltrated lymphocyte cells play a significant role in cancer development and affect patient prognosis and that SPC25 might be a potential oncogene in UCEC, we next examined whether SPC25 was related to the immune infiltration degree in UCEC.Our finding suggested that mRNA expression levels of SPC25 had a significantly positive association with Th2 cells, T helper cells, Tgd, Tcm, and Th1 cells.On the contrary, SPC25 expression was negatively correlated with Tfh, T cells, Treg, CTL, Th17 cells, mast cells, NK cells, eosinophils, neutrophils, iDC, pDC, and NK CD56bright cells (Fig. 13A-N).We collected these immune cell s' markers from the TISIDB website, and investigated the ex-pression association between SPC25 and these markers using the Xiantao academic web, which further verified the results above (Supplementary Table 4).Furthermore, we found that the expression levels of SPC25 had a significant correlation with immune checkpoints in UCEC, such as TNFRSF14, TNFSF4, CD244, LAG3, ICOS, CD40LG, ADORA2A, CD276, CD80, LGALS9, TNFSF14, ICOSLG, TMIGD2, PDCD1LG2, HHLA2, TNFSF18, TNFSF9, TN-FRSF8, CD27, TNFRSF25, VSIR, TNFRSF4, CD40, TN-FRSF18, and CD274 (Fig. 13O).

Discussion
SPC25 is an significant kinetochore component that plays an indispensable role in normal mitosis.The lack of SPC25 results in mitotic abnormality, followed cell death and the loss of SPC25 also causes multiple spindle aberrations, including spindle elongation, multipolarity, and fracture.In the case where there is no SPC25, MAD1 and HEC1 fail to correctly localize at the kinetochores during mitosis [38].
Up to now, some scholars have published some research results on SPC25.For instance, SPC25 could serve as a potential tumor-promoting factor, a metastasis promoter, a useful prognostic indicator, and a new therapy target for HCC.SPC25 mRNA expression was shown to be elevated in HCC, and greater levels of SPC25 expression were linked to a worse prognosis.SPC25 accelerated the cell cycle, allowing HCC cells to proliferate in vitro and tumor growth in vivo.In vitro, knocking down or silencing of SPC25 led to a considerable reduction in HCC cell proliferation and metastasis, a marked inhibition of invasion and migration, and increased protein level of p53 pathway components.SPC25 might promote proliferation and metastasis of HCC via p53 or via activating the FAK/PI3K/AKT signaling pathway through ITGB4 [10][11][12][13].
SPC25 was considerably enhanced in lung adenocarcinoma (LUAD) tissue relative to normal lung tissue, increased cancer stem-like cell (CSC) properties and A549 cell invasion, and was an independent predictive factor for LUAD patients' poor OS and recurrence free survival (RFS).Additionally, lung cancer cell proliferation requires SPC25 homologs expressed in extracellular matrix (ECM) stiffening.In idiopathic pulmonary fibrosis (IPF-LC), because CADM1 and SPC25 were implicated in transforming growth factor-beta 1 (TGF-beta 1) signaling, gene mutations in these two genes resulted in reduced CADM1 expression and increased SPC25 expression in lung cancer cells, revealing TGF-beta 1-induced epithelialmesenchymal transition and cell proliferation [14][15][16].
In PCa, SPC25 was shown to be highly elevated.SPC25 knockdown decreased proliferation and accelerated apoptosis in PCa cells, resulting in a drop in the number of S phase cells and an increase in the number of G2/M phase cells.SPC25 also has several functional functions in regulating cell proliferation, apoptosis, invasion, transforming growth factor-beta signaling, and SUMOylation pathways in PCa, according to bioinformatic study.SPC25 was upregulated in advanced PCa, and PrC patients with higher SPC25 have lower OS than those with lower SPC25.SPC25+ cells developed considerably more tumorspheres in vitro culture than SPC25-cells, seemed to be more resistant to docetaxel-induced cell apoptosis, and created bigger tumors with a greater frequency after repeated adoptive transplantation than SPC25-cells [17,18].SPC25 level was higher in more aggressive BC cell subtypes, and BC patients had a worse prognosis when SPC25 was expressed to a greater extent.SPC25 increased BC cell proliferation, as evidenced by colony formation and CCK-8 experiment.In addition, treatment with DNA methyltransferase inhibitors and transcription factor inhibitors targeting SPC25 might improve prognosis in BC patients.The combination of the DNMT inhibitor 5-azacytidine and the HDAC inhibitor butyrate significantly reduced the abundance of CSC, blocked CSC tumori- genicity, attenuated breast tumor growth, and improved OS in the MMTV-Neu-Tg mouse model, likely because growth-promoting signaling molecules such RAD51AP1 and SPC25 were prevented when chromatin modifiers were inhibited [19].
However, as far as we know, there is currently no research that can fully assess the importance of SPC25 in UCEC.Therefore we made an effort to fill the research gap of SPC25 in UCEC.In our findings, SPC25 mRNA and protein expression were shown to be elevated in UCEC tissues and substantially connected with clinical stage, histologic grade, histological type, radiation therapy, tumor invasion, weight, primary therapy outcome, molecular subtype, immune subtype, and TP53 muation status in UCEC.Taken together, SPC25 was upregulated in UCEC and might act as a pivotal player in the carcinogenesis of UCEC.
Following that, we observed that increased SPC25 expression could lead to poorer OS, RFS, DSS, and PFI of UCEC patients.In addition, elevated SPC25 was associated with poorer OS, DSS, and PFI in a number of clinical subgroups of UCEC, yet cause a worse all of the OS, DSS, and PFI only in race (white), age >60, height >160, weight >80, BMI >30, menopause status (post), clinical stage (stage III), diabetes (No), and radiation therapy (No).It is of great importance that for the first time, we examined the relationships between SPC25 expression and various prognostic (OS, DSS, and PFI) in several clinical subgroups of UCEC.
SPC25 coexpression network analyses in UCEC pointed out that SPC25 regulated the cell cycle of tumor cells in the TME, which was in line with findings from other studies.
Tumorigenesis is significantly influenced by DNA methylation, a common epigenetic mechanism.Changes in the methylation status of several genes have been connected to the initiation, growth, and advancement of various malignancies [39,40].Consequently, we investigated the promoter methylation status of SPC25 in UCEC utilizing the UALCAN database.We discovered that the DNA methylation levels of SPC25 in cancer tissues were noticeably lower than those in healthy samples, indicating that SPC25's overexpression in UCEC was caused by a low degree of promoter methylation status.Our research also showed that high-grade UCEC commonly had lower methylation levels of SPC25, which might indicate that the pattern alteration of SPC25 methylation encouraged the advancement of UCEC.Besides, we looked at the association between the SPC25 gene's methylation levels and the prognosis of UCEC patients.UCEC patients who had hypomethylation at two CpG sites, including cg22278106 and cg15237047, had an excellent OS rate.However, UCEC patients' poor OS was linked to the hypomethylation of two CpG sites, namely cg07224215 and cg20609092.All four of these CpG sites saw a reduction in methylation.Our research yielded two contradicting results.Our hypothesis was that hypomeylation of the final two CpG sites had a greater impact on the prognosis of UCEC patients than did the first two.This suggested that the latter two CpG sites could be future therapeutic targets for reducing SPC25 expression and improving the UCEC patients' prognosis.In general, we discovered that the majority of methylation sites in the DNA sequences of SPC25 were hypomethylated in UCEC, and the degree of methylation was connected with patient outcomes.These findings revealed that SPC25 methylation levels functioned as an efficient predictive biomarker for UCEC, suggesting that SPC25 might play a crucial role in tumor progression.
Initial theories presupposed that an effective immune response might have an antitumor impact, but cancer cells have evolved a number of mechanisms, such as impaired antigen presentation and the recruitment of immunosup-pressive cells, which encourage tumors to avoid the attack of immune cells.According to earlier research, immune infiltration can have an impact on a patient's prognosis, and tumor-infiltrating lymphocyte grade is an independent predictor of sentinel lymph node status in patients with tumors [41].In this study, SPC25 was shown to be substantially expressed in UCEC and linked to a poor outcome for those patients with UCEC.We also found that SPC25 expression had a clearly positive association with Th2 cells, T helper cells, and Th1 cells.Conversely, SPC25 was visibly negatively correlated with T cells, CTL, NK cells, neutrophils, iDC, and pDC.
The T helper cell-expressed TGF is a key player in the resistance mechanisms against cancer immunotherapy.An efficient tissue-level cancer defense response is elicited by targeted TGF-signaling inhibition in helper T cells, which can serve as a foundation for therapeutics focused on the cancer environment [42].Th2 cells can turn macrophages into tumor-promoting cells and change the immune response from a cytotoxic to a supportive function when they are present in the TME [43].Myeloma cell growth and function were markedly suppressed by CTL and Th1 cells, but Th2 cells promoted myeloma cell proliferation and cytokine secretion.In multiple myeloma, CTL and Th1 responses are advantageous and will result in tumor elimination after immunotherapy.In contrast, a Th2 response offers little protection and could even hasten the growth of tumors in vivo [44].Effector cells of the adaptive immune system are CD8 + CTL cells, which precisely identify and eliminate cancer cells through apoptosis that is mediated by perforin and granzyme [45].T cell depletion is a significant barrier to successful immunotherapy [46].Cytotoxic lymphocytes called NK cells may obliterate stressed cells even in the absence of an antigen presentation.NK cells identify and eradicate tumor cells through "missing-self" activation (loss of healthy cell markers) or "stress-induced" activation (gain of stressed cell markers) [47].Through triggering T cell responses against the tumor cells and directly lysing tumor cells, tumor-associated neutrophils perform a crucial anti-tumor function [48].By cross-presenting foreign antigens, dendritic cells (DCs) play a crucial role in CD8 + T cell activation and producing anti-tumor CD8 + T cell immunity [49].
Most findings state that the immunogenicity of the malignancies influences the effectiveness of immune checkpoint blockade [50].Low immunogenicity cancers tend to respond poorly to immune checkpoint-blocking treatment because T cell infiltration by malignancies is relatively small [51].In order to preserve self-tolerance and avoid autoimmune disease, immune checkpoint molecules normally downregulate activation signals from costimulatory molecules.This method, however, might be used by tumor cells to prevent T cells from becoming activated and functioning normally, which results in T-cell depletion, tumor immune escape, and aggressive tumor growth.In our finding, we confirmed that the SPC25 expression levels were evidently negatively correlated with immune stimulators, such as CD27, CD40LG, CD276, HHLA2, TMIGD2, TNFRSF4, TNFRSF14, TNFRSF18, TNFRSF25, and TN-FSF14.On the contrary, SPC25 was distinctly positively associated with immune inhibitors, including LAG3 and PDCD1LG2.
When it comes to the directed migration of immune cells, chemokines and their receptors are crucial [52][53][54].In this study, we foud that SPC25 was evidently negatively correlated with chemokines and their receptors: CCL14, CX3CL1, CCR3, CCR4, CCR5, CCR6, and CX3CR1, suggesting that High SPC25 expression might prevent immune cells from migrating into the TME.Studies show that CCL14's active form binds to CCR1, CCR3, and CCR5 to encourage the chemotaxis of monocytes, eosinophils, and T lymphocytes [55,56].The inflammatory receptor CCR4 stimulates the respiratory burst and phagocytosis of macrophages [57] and regulates the differentiation of M1/M2 macrophages [58].B cell integrin-mediated adhesion is regulated by CCR6 [59,60].White blood cell survival and NK cell activation are both regulated by CX3CR1 [61], which might explain how SPC25 regulates immune infiltration in UCEC.
These findings suggested the possibility of tumor immune escape and antitumor immunity being implicated in the carcinogenic mechanisms of UCEC.
Tumor immunogenicity and immune cells inside the TME will be impacted by dysregulated epigenome DNA methylation [62].We examined the connection between SPC25's methylation status and immune infiltration in order to decipher the mechanism by which SPC25 hypomethylation promotes the progression of UCEC.Our data showed that SPC25's methylation status was positively correlated with immune cells, immunostimulatory factors, chemokine and chemokine receptors while negatively connected with immunoinhibitors.This suggested that SPC25's hypomethylation might contribute to immunosuppression and promote tumor progression in UCEC, which could assist to explain the SPC25 low methylation status in high-grade UCEC tumors and poor prognosis of corresponding UCEC patients.Thus, SPC25 methylation might serve as an indicator of cancer immune infiltration and potential predictor of UCEC patient response to immunotherapeutic drugs.
In conclusion, the mRNA and protein of SPC25 were overexpressed in UCEC tumor tissues and associated with a variety of clinicopathological features.The expression and methylation of the SPC25 gene were related to the prognosis of UCEC patients.SPC25 expression levels and methylation status not only correlated with the extent to which various immune cell types were infiltrating the tumor, but also had a relationship with immunomodulators and chemokines, which might have an impact on how well immunotherapy works in UCEC patients.By modifying the expression of genes involved in cell cycle and immune response, SPC25 controlled the course of UCEC.Therefore, SPC25 was a valuable prognostic biomarker for UCEC, as well as a possible therapeutic target.However, further research is required to substantiate our findings.
However, even with our detailed and systematic research of SPC25 and cross-verification using many databases, we must admit that our current research has a number of potential limitations.
On the one hand, we explored SPC25 gene only using the public databases such as TCGA and GEO yet lacking actual clinical data.And there were variations in microarray and sequencing data from various databases, which could lead to systematic bias.Thus, further in vivo/in vitro verification experiments and follow-up multicenter, largesample, and prospective studies should be performed to provide precise verification and high-quality evidence in order to investigate if there's a link between SPC25 and patient survival, and to find more effective antitumor immunotherapy techniques.
On the other hand, although concluding that SPC25 expression was significantly correlated with immune cell infiltration and cancer patients' survival, we lacked direct evidence of SPC25 influencing prognosis despite its involvement in the regulation of immune cell infiltration.In other words, the processes by which SPC25 regulates the immune system as well as its precise route remain unknown and need further study.We also need sufficient and accurate clinical trial data to to determine the advantages of anti-SPC25 medicines in inhibiting tumor growth or improve patient prognosis.

Conclusions
In conclusion, the discovery of SPC25 investigation in UCEC might provide a integrative understanding of its crucial function in tumor promotion and suppression, and add a comprehensive analytical basis for in-depth validation of molecular biology experiments, and even for future clinical applications of cancer therapy.

Fig. 1 .
Fig. 1.Spindle pole body component 25 (SPC25) mRNA and protein expression in uterine corpus endometrial carcinoma (UCEC).(A-C) SPC25 mRNA expression levels in endometrial cancer patients and matched adjacent normal samples.(D) SPC25 mRNA expression levels were upregulated in the endometrial cancer tissues compared to endometrial hyperplasia tissues in the GSE106191 dataset.(E,F) SPC25 protein expression levels based on the university of alabama at birmingham cancer data analysis portal (UALCAN) and Human Protein Atlas.(G,H) Utilizing Western blotting, the levels of SPC25 protein expression were confirmed.*p < 0.05; ***, p < 0.001; ****, p < 0.0001.

Fig. 8 .
Fig. 8. SPC25's co-expressed genes and interacting proteins.(A) Volcano plot obtained from the LinkedOmics database showing SPC25's co-expressed genes tested by Pearson test in UCEC cohort; (B) Protein-protein interaction network of SPC25 obtained from the STRING online tool; (C) Venn diagram showed the intersection result of SPC25's co-expressed genes and interacting proteins.

Fig. 9 .
Fig. 9. SPC25's positively and negatively co-expressed genes in UCEC analyzed by the LinkedOmics database.(A,C) the top 50 positively co-expressed (A) and negatively co-expressed (C) genes of SPC25 shown in heat maps; (B,D) the top 10 positively coexpressed (B) and negatively co-expressed (D) genes of SPC25 represented by scatter plots.A p value of less than 0.05 was considered statistically significant.

Fig. 10 .
Fig. 10.SPC25's gene set enrichment analysis (GSEA) analysis in UCEC cohort analyzed by the LinkedOmics database.(A) Bar chart of SPC25's gene ontology biological process (GO_BP) (biological process) analysis; (B) Bar chart of SPC25's kyoto encyclopedia of genes and genomes (KEGG) pathways.A false discovery rate (FDR) value of 0.05 or less was considered statistically significant.

Fig. 11 .
Fig. 11.The heat maps obtained from the Xiantao Academic web showing the correlation between SPC25 and the genes in the cell cycle pathway network queried from the PathCards website in UCEC.*, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 12 .
Fig. 12.DNA methylation levels in the SPC25 gene were associated with the prognosis of UCEC patients.(A) Promoter methylation level of SPC25 in normal tissues and primary UCEC tissues by the UALCAN database.(B) Promoter methylation level of SPC25 of various tumor grades in UCEC tissues by the UALCAN database.(C) The heat map of DNA methylation at CpG sites in the SPC25 gene by the MethSurv database.(D-G) The association between methylation levels of four CpG islands of the SPC25 gene and OS of UCEC patients: (D) cg22278106, (E) cg15237047, (F) cg07224215, and (G) cg20609092.

Fig. 13 .
Fig. 13.Correlation analysis of SPC25's expression and immune cells' infiltration level as well as immune checkpoints in UCEC conducted in the Xiantao Academic online tool.(A-N) The correlation between SPC25's expression and infiltration level of immune cells.(O) Expression correlation analysis of SPC25 and immune checkpoint-related genes.*p < 0.05; **p < 0.01; ***, p < 0.001.