Clinical Significance And Mechanism Of Action Of Pan-cancer Biomarker ZIC2 In Renal Cell Carcinoma

Part Ⅰ:Bioinformatics analysis of ZIC2 as a pan-cancer prognostic biomarkerPurposeZinc finger protein ZIC2,as a transcription factor,can interact with various DNA and proteins.Current research indicates that ZIC2 plays an oncogenic role in multiple cancers.This study systematically analyzed the expression and prognostic significance of ZIC2 in various cancer types(pan-cancer)based on multiple independent publicly available datasets.MethodsThis study explored multiple public databases,including Oncomine,The Cancer Genome Atlas(TCGA),Kaplan-Meier Plotter,and PrognoScan,to evaluate the expression difference of ZIC2 between pan-cancer samples and normal control samples.The relationship between ZIC2 expression and patient prognosis was then investigated using Kaplan-Meier curves and univariate Cox regression analysis.ResultsPublic data from Oncomine and TCGA databases showed that ZIC2 was expressed at higher levels in most cancer tissues compared to adjacent normal tissues(P<0.05).Public data from TCGA,Kaplan-Meier Plotter,and PrognoScan databases indicated that high expression of ZIC2 was significantly associated with poor prognosis in multiple cancers(P<0.05).ConclusionZIC2 gene expression may serve as a potential molecular biomarker for poor prognosis in pan-cancer and may have potential regulatory roles as an oncogene in tumor development and progression.Part Ⅱ:Expression and clinical significance of ZIC2 in renal cell carcinomaPurposeZIC2,as a transcription factor,has been found to be aberrantly expressed in tumors and closely associated with the biological behavior of tumors in recent years.In the previous study,ZIC2 has been proved to be an effective pan-cancer prognostic biomarker,especially with significant prognostic significance in renal cell carcinoma.Therefore,this study focused on exploring the expression and clinical significance of ZIC2 in renal cell carcinoma.MethodsA systematic analysis was performed on the expression and prognostic significance of ZIC2 in kidney cancer using TCGA,GEO,and Array Express datasets.Additionally,Western blot and immunohistochemical analysis were performed on renal cell carcinoma tissues and corresponding adjacent normal tissues collected from our center.Fisher’s exact test,chi-square test,Wilcoxon rank-sum test,independent sample t-test,paired sample ttest,and Pearson correlation analysis were used to evaluate the relationship between ZIC2 and clinical pathological features.Kaplan-Meier curve and receiver operating characteristic(ROC)curve were used to evaluate the impact of ZIC2 on prognosis.The independent prognostic factors were determined by univariate and multivariate Cox regression analysis.ResultsCompared to normal renal tissue,ZIC2 was significantly overexpressed in renal cancer,and high ZIC2 expression was associated with poorer prognosis.ROC curve analysis demonstrated that ZIC2 expression had a strong prognostic predictive capability.ZIC2 expression was also significantly correlated with clinical pathological features,such as age,tumor grade and tumor staging.Univariate and multivariate Cox regression analyses showed that ZIC2 expression was an independent predictor of prognosis in renal cancer.These results were largely validated in other external independent datasets.Western blot and immunohistochemical analysis of renal cancer and adjacent normal tissues in our center further confirmed the high expression of ZIC2 in renal cancer and its significant correlation with advanced pathological features of tumors.ConclusionThis study jointly confirmed the high expression of ZIC2 in renal cancer and its significant correlation with clinical pathological features and poor prognosis using TCGA,GEO databases,other external independent databases,and Western blot and immunohistochemical analysis of renal cancer and adjacent normal tissues in our center.ZIC2 may become a promising prognostic biomarker and a potential therapeutic target.Part Ⅲ:Biological functional validation of ZIC2 in renal cancer in vitro and in vivoPurposeZIC2 has been demonstrated as a pan-cancer prognostic biomarker,especially in renal cell carcinoma,in previous studies.However,the biological role of ZIC2 in renal cell carcinoma remains unknown.Therefore,this study aimed to explore the biological function of ZIC2 in RCC development through cell biology and animal experiments.MethodsFirst,functional enrichment analysis was performed to explore the potential biological functions of ZIC2 in Hallmark and KEGG pathways between the ZIC2 low expression group and the high expression group in the TCGA dataset.The expression of ZIC2 in commonly used human renal cell carcinoma cell lines was detected by Western blot.Stable cell lines with ZIC2 knockdown and overexpression were established using lentiviral vectors.The effects of ZIC2 on renal cell carcinoma were evaluated by CCK-8 assay,clone formation assay,EdU assay,flow cytometry,scratch assay,Transwell assay,detection of epithelial-mesenchymal transition markers,as well as subcutaneous tumorigenesis and lung metastasis experiments in nude mice.ResultsFunctional enrichment analysis showed that high expression of ZIC2 was significantly enriched in "KEGG CELL CYCLE","KEGG DNA REPLICATION","HALLMARK G2M CHECKPOINT",and "HALLMARK EPITHELIAL MESENCHYMAL TRANSITION".Cellular experiments showed that ZIC2 significantly promoted the proliferation,colony formation,invasion,migration,and epithelial-mesenchymal transition of renal cell carcinoma cells,and regulated the cell cycle at G2/M phase.Animal experiments demonstrated that ZIC2 significantly enhanced the subcutaneous tumorigenicity and lung metastasis of nude mice.ConclusionThis part of the study confirmed through cellular and animal experiments that ZIC2 could promote the malignant biological behavior of renal cell carcinoma and may serve as an important regulatory factor in the development and progression of renal cell carcinoma.This further suggests that ZIC2 may be a potential research target for precise treatment of renal cell carcinoma in the future.Part Ⅳ:Downstream mechanisms of ZIC2 in promoting malignant phenotype in renal cancerPurposePrevious studies have shown that the pan-cancer biomarker ZIC2 is particularly significant in the prognosis of renal cancer.Further cellular and animal experiments have confirmed that ZIC2 can promote the malignant phenotype of renal cancer.However,the underlying mechanisms remain unknown.This study aims to explore the specific downstream mechanisms through which ZIC2 promotes the occurrence and development of renal cancer.MethodsFirstly,bioinformatics analysis was employed to explore the downstream signaling pathways activated by ZIC2,which were then validated through Western blot.Subsequently,potential target genes regulated by ZIC2 and specific binding sites were predicted using transcription factor-related databases,followed by validation through chromatin immunoprecipitation(ChIP)experiments and dual-luciferase reporter gene assays.Additionally,bioinformatics methods will be utilized to analyze the correlation between ZIC2 and tumor immune infiltration,tumor microenvironment,tumor immune cycle,and tumor immune checkpoint gene expression.Building upon an established eightgene immune gene prognosis model in bladder cancer,the potential transcriptional regulatory relationship between ZIC2 and these eight genes will be explored.ResultsFunctional enrichment analysis revealed that high expression of ZIC2 significantly enriched the AKT/mTOR signaling pathway,which was confirmed by Western blot.Through bioinformatics analysis combined with the prediction by GTRD database,Cistrome Data Browser database,and JASPAR database,ZIC2 may act as a transcriptional activator by binding to the UBE2C promoter to promote UBE2C transcription.This prediction was further validated through chromatin immunoprecipitation(ChIP)experiments and dual-luciferase reporter gene assays.Furthermore,functional enrichment analysis indicated a significant association between ZIC2 and immune-related pathways.Further bioinformatics analysis revealed correlations between ZIC2 and tumor immune infiltration,tumor microenvironment,tumor immune cycle,and tumor immune checkpoint gene expression.Combining the established eight-gene immune-related risk prediction model in bladder cancer,it was found that ZIC2 potentially regulates three immune genes(AHNAK,ADRB2,KITLG).ConclusionThis study,through bioinformatics analysis combined with prediction from transcription factor-related databases and cellular experiments,confirmed that ZIC2 functions as a transcriptional activator of the UBE2C gene,thereby activating the AKT/mTOR signaling pathway to promote the malignant phenotype of renal cancer.Additionally,ZIC2 may potentially regulate three immune-related genes(AHNAK,ADRB2,KITLG),thereby influencing tumor immunity in renal cancer.