The Role Of B4GALNT2 On Proliferation,Invasion And Migration Of Breast Cancer Cells And Its Related Mechanism

BackgroundBreast cancer(BC)is the most common malignant tumor in women,and its incidence rate ranks among the top three among all the types of cancer internationally.According to the global cancer data in 2018,there were 18.1 million new cases of breast cancer and 9.6 million death due to BC in that year.The incidence of breast cancer ranks first as lung cancer,accounting for 11.6%(2.1 million)of the total cancer incidence.Breast cancer has the fourth highest mortality rate,behind lung cancer,liver cancer,and gastric cancer,accounting for 6.6%(0.63 million)of the total death due to cancer The Cancer Genome Atlas(TCGA)database is a publicly funded project that has created a comprehensive cancer genome atlas which contains more than 30 large populations of human tumors.TCGA can be applied to high-throughput genomic analysis.Through the expression analysis of differential germs in tumor and non-tumor tissues,new tumor-related biomarkers can be found for tumor diagnosis,prognosis,and as new therapeutic targets.B4GALNT2 is a blood group-associated antigen produced by vascular endothelial cells.Related research shows that B4GALNT2 is a newly discovered antigen for xenograft,which can change the susceptibility of Salmonella and affect the distribution of intestinal flora.However,the correlation between·B4GALNT2 gene and tumor malignancy has not been previously studied.Therefore,the purpose of this study was to explore the correlation between B4GALNT2 gene and the proliferation,migration and invasion of breast cancer cells and its related mechanisms.ObjectivesExplore the correlation between B4GALNT2 gene and the proliferation,invasion and migration of breast cancer cells and its related mechanisms.Methods1.Download the relevant data of breast cancer patients containing RNAseq through TCGA database and compare the RNAseq between breast cancer tissue and non-tumor tissue to determine the differential expression of B4GALNT2 gene in breast cancer tissue and non-tumor tissue.2.Detection of B4GALNT2 expression in 5 breast cancer cell lines(MDA-MB-231,HCC1937,T47D,MCF7,SK-BR-3)using rt-PCR to determine the cell model3.Construct sh-B4GALNT2 lentiviral vector based on the sequence information of B4GALNT2 gene,package sh-B4GALNT2 lentivirus,and transfect two cell lines of MDA-MB-231 and HCC1937.After 72 hours of infection of the target cells with the control and the target lentivirus,observe the cell transfection efficiency under a fluorescent microscope and the knockdown efficiency of B4GALNT2 gere was verified by rt-PCR.Follow-up cell function experiments and animals were performed when knockdown efficiency reached 50%,Western Blot was used to detect the expression level of B4GALNT2 after its knockdown.The effects of knockdown of B4GALNT2 on the proliferation,migration and invasion of breast cancer cells in vitro were examined by Celigo cell counting experiments,MTT proliferation experiments,apoptosis experiments,cell migration experiments,cell invasion experiments and cell cycle expperiments.4.MDA-MB-231 cells after knocking down the B4GALNT2 gene and control cells were injected subcutaneously into nude mice to construct a mouse tumor formation model.The tumor volume and tumor weigbt difference between the experimental group and the control group were compared to determine the effect of B4GALNT2 on breast cancer cell proliferation in vivo.5.Gene enrichment analysis ofB4GALNT2 gene in breast cancer patients,using GSEA algorithm to explore possible pathways related to B4GALNT2 gene.Results1.A total of 1094 breast cancer patients with RNAseq were obtained from the TCGA database,of which 106 were paired tissue(cancer and adjacent cancer)patients.Analysis of differential expression of genes between breast cancer tissues and non-tumor tissues revealed that B4GALNT2 was significantly overexpressed in cancer tissues(p<0.05).2.RT-PCR was used to detect the expression of B4GALNT2 in five breast cancer cell lines.The results were as follows:MDA-MB-231 and HCC1937 were the least expressed,and T47D and MCF7 were the most expressed.SK-BR-3 The expression level is intermediate,so MDA-MB-231 and HCC1937 were determined as the cell models.3.The transfection efficiency of the two cell lines was more than 90%,and the expression level of the gene was significantly reduced by RT-PCR,and the knockdown efficiency was more than 50%.After B4GALNT2 gene of cells were knocked down,Western Blot detected that the protein expression was significantly reduced,the cell proliferation and migration and invasion ability of the experimental group were reduced through Celigo cell counting and MTT proliferation experiments,migration and invasion experiments,and apoptosis experiments.Cell cycle experiments revealed that cell cycle was arrested in both cell lines(p<0.05).4.Tumor formation experiments in nude mice found that:compared with the control group,the volume and weight of the mice tumor decreased in the experimental group.5.Through gene enrichment analysis of B4GALNT2,it was found that the pathways that this gene may participate in regulation include negative regulation TORC1 signaling pathway,the POSITIVE-REGULATION-OF-SYNAPSE-ASSEMBLY signaling pathway,and the RESPIRATORY-SYSTEM-PROCESS signaling pathway.all of which are negatively correlated to the expression of B4GALNT2.Conclusions1.Compared with normal tissues,B4GALNT2 gene is highly expressed in breast cancer tissues.2.B4GALNT2 gene is positively correlated with the proliferation,migration and invasion ability of breast cancer cells.3.Through gene enrichment analysis of B4GALNT2,it was found that B4GALNT2 gene has a negative correlation with the negatively regulated TORO1 signaling pathway,which indicates that a possible mechanism that B4GALNT2 promotes the proliferation,invasion and migration of breast cancer cells is via activating the TORC1 signaling pathway.