Comprehensive Analysis Of Prognostic Value Of NCAPD2 And Its Relationship With Immune Infiltrates In Skin Cutaneous Melanoma

Objective Skin cutaneous melanoma(SKCM)is the deadliest skin tumor with characteristics of high malignancy,early metastasis,high mortality,and poor prognosis.Therefore,there is an urgent need for new insights into the molecular mechanisms that lead to the occurrence and progression of SKCM and to promote the development of biomarker targets and novel therapeutic options to improve the survival rate of patients.Non-SMC complex subunit D2(NCAPD2)is not only involved in the aggregation and separation of chromosomes during cell division,but also plays an important role in the occurrence and development of a variety of cancers.At present,NCAPD2 has not been reported in melanoma.This study aims to explore the expression of NCAPD2 in cutaneous melanoma and clarify its relationship with prognosis,in order to explore its mechanism of action in cutaneous melanoma.Methods(1)Data on melanoma from several databases such as The Cancer Genome Atlas(TCGA),Gene Expression Profiling Interactive Analysis(GEPIA),Gene Expression database of Normal and Tumor tissues 2(GENT2),Tumor Immune Estimation Resource(TIMER)and Progno Scan were mined to derive NCAPD2 expression characteristics in melanoma and its impact on the prognosis of melanoma patients.Meanwhile,the immunohistochemistry of NCAPD2 protein in melanoma tissues was extracted from the Human Protein Atlas(HPA)database to verify its expression trend.(2)Univariate/ multivariate risk regression analysis was performed on NCAPD2 and clinical variables such as T-stage,N-stage,M-stage,age and pathological stage through the SKCM dataset in the TCGA database.In addition,the receiver operating characteristic curve(ROC)was plotted and the ability of NCAPD2 to differentiate between normal tissue and melanoma was analyzed according to the Area under curve(AUC).(3)A nomogram scale was constructed for NCAPD2 expression and clinical variables to predict 1-,3-and 5-year survival in melanoma patients.Cindex and calibration curves are used to assess the reliability of nomogram predictions.(4)To investigate the functional role of NCAPD2 in SKCM,the TCGA-SKCM dataset was divided into high and low expression groups based on the median NCAPD2 expression values and then subjected to Gene Expression Profiling Interactive Analysis(GSEA).(5)Differentially expressed genes associated with NCAPD2 in SKCM were explored using the Linked Omics website database.The top 200 positively related genes were analyzed using the STRING database and the Cytoscape tool to obtain the top 10 hub genes with the highest number of nodes.Gene Ontology(GO)analysis and survival analysis were performed on these hub genes using the Metascape and TIMER web databases.(6)The ESTIMATE algorithm was used to analyze three scores including the stromal score,immune score and estimate score in relation to overall survival(OS)and NCAPD2 expression(estimate score is the sum of stromal score and immune score).Further,the ss GSEA algorithm was used to analyze the relationship between NCAPD2 and the 24 immune cells in SKCM.(7)The TISIDB database was used to analyze the relationship between NCAPD2 expression and immunomodulators(immunoinhibitors,immunostimulators and MHC molecules)in SKCM.(8)Predicting the relevance of NCAPD2 to different functional states of melanoma cells via the Cancer SEA website.Next,NCAPD2 protein expression levels in human immortalized epidermal cells Ha Ca T and melanoma cell lines A375 and MV3 were measured using Western blotting.Gene expression silencing was performed by transfecting the corresponding si RNAs inside NCAPD2 high expressing A375 and MV3 melanoma cells.For the above treated cell lines and controls,the proliferation of melanoma cells was tested using the CCK-8assay as well as the migration and invasion of melanoma cells using the cell scratch assay and invasion assay.Result(1)NCAPD2 expression was significantly higher in cutaneous melanoma tissue than in normal tissue,and patients with high NCAPD2 expression had a poor prognosis,as analyzed by several biological databases.Immunohistochemistry in the Human Protein Atlas database also confirmed that NCAPD2 protein levels were higher in melanoma tissues than in normal tissues.(2)NCAPD2 is an independent prognostic factor for SKCM as well as having a high performance in diagnosing SKCM.(3)The C-index and calibration curve suggest that the nomogram scale has a high reliability in predicting survival in melanoma patients.(4)GSEA enrichment analysis revealed that the NCAPD2 high expression group was significantly enriched in cell cycle,DNA replication,MTOR signaling pathway,insulin signaling pathway,NOTCH signaling pathway and multiple cancer pathways.(5)GO analysis of these hub genes showed an association with cytokinesis activity,and all were associated with poor prognosis in SKCM.(6)Patients with high immune scores had better survival,suggesting that tumor immune infiltration favors SKCM survival.NCAPD2 expression was negatively correlated with the immune score and the level of infiltration of 15 immune cells in SKCM.(7)NCAPD2 expression is negatively correlated with immunomodulators.(8)Overexpression of NCAPD2 in melanoma cell lines and its downregulation inhibited proliferation,migration and invasion of A375 and MV3 cells in in vitro experiments.Conclusion This study shows that NCAPD2 can be used as a new marker for the diagnosis and prognosis of melanoma patients.Increased levels of NACPD2 expression were significantly associated with SKCM progression,potentially by promoting the tumor cell cycle,DNA replication,MTOR signaling pathway,insulin signaling pathway,NOTCH signaling pathway and inhibition of tumor immune cell infiltration.Therefore,NCAPD2 may be a new target for the treatment of cutaneous melanoma.