Effect And Mechanism Of ST3GAL5 On Malignant Biological Behavior Of Bladder Cancer

Objective: To explore the expression level,clinicopathological characteristics and survival prognostic analysis of ST3GAL5 gene in bladder cancer.Methods:(1)Screen out target differential genes through multiple bladder cancer data sets in the GEO database.The gene expression profile and clinicopathological information of ST3GAL5 gene in bladder cancer patients’ tissues and cell lines were collected from multiple public and independent bioinformatics databases such as The Cancer Genome Atlas(TCGA),Genotype-Tissue Expression(GTEx),Gene Expression Omnibus(GEO),Oncomine,GENT2,CCLE,etc.The gene expression profile of ST3GAL5 in various cancers versus paired normal tissues,bladder cancer tissues versus normal bladder tissues,muscle-invasive bladder cancer(MIBC)tissues versus non-muscle-invasive bladder cancer(NMIBC)tissues,and high-grade bladder cancer tissues versus low-grade bladder cancer tissues were analyzed respectively;(2)The protein expression of ST3GAL5 in bladder cancer tissues versus paraneoplastic normal bladder tissue were detected by immunohistochemistry and Westernblot method,respectively;(3)The clinicopathological characteristics of ST3GAL5 gene in bladder cancer patients were evaluated by TCGA and GEO databases,respectively;(4)UniCox and Kaplan-Meier survival analyses were plotted to evaluate the prognostic relationship of ST3GAL5 gene in bladder cancer,and uni-and multi-Cox risk regression models were constructed to evaluate the independent influences of ST3GAL5 gene on overall survival and disease-specific survival in bladder cancer patients.Results:(1)Results from three bladder cancer datasets suggested that the ST3GAL5 gene can effectively serve as a differentially expressed gene for different types of bladder cancer.Multiple public bioinformatic databases had indicated that ST3GAL5 was significantly lower expressed in the majority types of cancer patient’s tissue and cancer cell lines,the gene expression level of ST3GAL5 was significantly downregulated in bladder cancer tissues,compared to normal bladder tissues,and more significantly downregulated in MIBC as well as high-grade bladder cancer;(2)Immunohistochemical staining showed that the protein expression of ST3GAL5 was mainly located in the cytoplasm and cell membrane,and the positive expression of ST3GAL5 was significantly lower in bladder cancer tissues than in paraneoplastic normal bladder tissues,with the expression in high-grade bladder cancer being significantly lower than in low-grade bladder cancer,and the expression in MIBC being significantly lower than in NMIBC(P < 0.05).Western-blot results showed that the protein expression of ST3GAL5 was significantly lower in cancer tissues than in normal bladder tissues,lower in high-grade lesions and MIBC compared to low-grade lesions and NMIBC,respectively(P < 0.05);(3)The lower expression level of ST3GAL5 in bladder cancer had higher histological grade,higher pathological stage,higher susceptibility to lymph node metastasis and higher mortality;(4)Kaplan-Meier survival analysis suggested that overall survival and disease-free survival were significantly lower in the lower ST3GAL5 expression of group than in the higher ST3GAL5 expression group(P < 0.05),and Cox risk proportional regression models showed that lower expression level of ST3GAL5 was an independent influence on overall survival and disease-free survival in bladder cancer patients(P < 0.05).Conclusions: ST3GAL5 gene may be a kind of oncogenic suppressor gene in bladder cancer and may serve as a potential predictive and prognostic marker for bladder cancer progression to myeloablative and high-grade invasion,and even as a molecular target for cancer therapy.Objective: To explore the effect of ST3GAL5 overexpression on the malignant biological behavior of bladder cancer cells,including proliferation,invasion,and metastasis.Methods:(1)A ST3GAL5 over-expressing lentiviral vector were constructed and packaged,human bladder cancer cell lines HT-1376 were selected for cell phenotype experiment,then q RT-PCR and Western-blot were used to measure the gene and protein expression of ST3GAL5 in the target cell lines;(2)CCK8,Ed U and colony formation assays were used to detect the functional effects of overexpression of ST3GAL5 on the in vitro proliferation of the bladder cancer,flow cytometry apoptosis experiments were used to detect the effect of overexpression of ST3GAL5 on the apoptosis of bladder cancer cell,and scratch assay,Transwell metastasis assay and Transwell invasion assay were used to detect the functional effects of overexpression of ST3GAL5 on the in vitro invasion and metastasis of the bladder cancer cell.Results:(1)The ST3GAL5 overexpression lentiviral vector was successfully constructed,and after stable infection with HT-1376 cells,q RT-PCR and Western-blot showed that the gene and protein expression levels of ST3GAL5 in the lentiviral overexpression group were lower than those in the negative control group(P < 0.05);(2)The results of CCK-8,Ed U and clone formation assays all suggested that the proliferation ability of ST3GAL5 overexpression group was significantly decreased compared with the negative control group(P < 0.05),the results of flow cytometric apoptosis experiments indicatee that the ST3GAL5 overexpression group has a tendency to induce bladder cancer cell apoptosis compared with the negative control group scratch(P < 0.05),and the results of scratch,Transwell transfer and Transwell invasion assays all suggested that the invasion and transfer ability of ST3GAL5 overexpression group was significantly increased compared with the negative control group(P < 0.05).Conclusions: ST3GAL5 has a tumor suppressor effect in bladder cancer cells,and ST3GAL5 can inhibit the proliferation,invasion and metastasis ability of bladder cancer cells.Objective: To explore the potential molecular mechanism of ST3GAL5 in inhibiting the proliferation,invasion,and metastasis of bladder cancer cells.Methods:(1)By analyzing the gene expression matrix of two large sample bladder cancer datasets from TCGA and GEO database,the spearman correlation coefficients between ST3GAL5 gene and all co-expressed genes in bladder cancer were calculated,then gene set enrichment analyses(GSEA)model of KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway and Wiki-Pathways signaling pathway was constructed and validated using the R language;(2)Bioinformatic Meta-analysis of ST3GAL5 and significant positively correlated co-expressed genes for biological function,signaling pathways,disease ontology and Protein-Protein Interactions(PPI)networks were analyzed by multiple bladder cancer datasets;(3)By analyzing the gene expression matrix of two large sample bladder cancer datasets from TCGA and GEO database,the median expression of ST3GAL5 gene was used for grouping.Then the differential gene expression analysis and gene enrichment analysis of the two groups were carried out.At the same time,the key factor changes of the related signaling pathways were identified based on TCGA-BLCA RNA-seq data;(4)q RT-PCR and Western blot technology were used to verify the classical proliferation and metastasis related pathway factors.Results:(1)The signaling pathway results of GSEA showed that ST3GAL5 and its coexpressed genes could exert the ability to inhibit bladder cancer cell proliferation,invasion,and metastasis by activating PPAR signaling pathway as well as inhibiting PI3K/AKT signaling pathway;(2)Bioinformatics Meta-analysis results of multi-omics bladder cancer dataset suggested that ST3GAL5 and the significant positive co-expression genes in bladder cancer was associated with a variety of metabolic biological functions in bladder cancer,while disease ontology also suggested the association with bladder cancer.The PPI network could identify the PPAR signaling pathway related proteins;(3)Differential gene expression was analyzed by comparing the different ST3GAL5 median expression groups in TCGABLCA and GEO data set.Enrichment analysis showed that PPAR signaling pathway was significantly up-regulated and PI3K/AKT signaling pathway was significantly downregulated.And the results of the disease ontology enrichment analysis suggest that there is a significant negative correlation with bladder cancer-related diseases;(4)The results of q RTPCR and Western-blot showed that the PPAR signaling pathway key related factors in bladder cancer increased significantly after ST3GAL5 overexpression(P < 0.05),while the PI3K/AKT signaling pathway related factors decreased significantly(P < 0.05).Conclusions: ST3GAL5 can inhibit the proliferation,invasion,and metastasis of bladder cancer cells by activating the PPAR signaling pathway and inhibiting related molecules in the PI3K/AKT signaling pathway.