An Omics Study Of Overexpression Of OVO-like Zinc Finger Transcription Factors 2 In Breast Cancer Cells Based On RNA-seq And ChIP-seq

Introduction:Breast cancer is a malignant tumor that occurs frequently in women,and the prognosis of patients with clinical treatment method is currently poor.OVOL2 is a transcription factor.The previous research of the research group found that OVOL2 has an inhibitory effect on the malignant behavior of most tumors.In this study,transcriptome sequencing experiments(RNA-seq)and chromatin immunoprecipitation sequencing experiments(ChIP-seq)were performed in MDA-MB-231 cells.Through bioinformatics analysis to find the most likely downstream targets of the OVOL2 gene,as well as the gene ontology and signal pathways that the differential genes caused by OVOL2 may participate in the differential changes,and find new research directions for the subject.Methods:1.By consulting the cell database,looking for breast cancer cell lines with low background expression of OVOL2.Through the lentivirus infection cell experiment,the MDA-MB-231 cells were re-overexpressed OVOL2,and the cell phenotype was observed to change.2.Real-time fluorescent quantitative PCR experiment and Western blotting experiment(Western blotting)were performed to confirm that the expression of OVOL2 in the prepared cell samples increased.The prepared breast cancer cell line was constructed and sequenced by RNA-seq experiment and ChIP-seq experiment.The ChIP sequencing platform adopts Illumina-NovaSeq 6000,and the paired-end sequencing(Paired-End)method is used to complete the sequencing.3.The Flag-OVOL2 group,IgG group,and Input group obtained from the ChIP-seq experiment were purified and library constructed,and the enriched DNA fragments were subjected to high-throughput sequencing experiments.The Flag-OVOL2 group and the IgG group were subjected to differential peak analysis to obtain The genes of interest may be combined in the genome of breast cancer MDA-MB-231 cell line,and these genes will be further analyzed by bioinformatics.The control group and the experimental group(overexpressing OVOL2)group of the RNA-seq experiment were compared and analyzed,and the difference genes obtained were analyzed by bioinformatics.4.Analyze the results of RNA-seq and ChIP-seq through Rstudio software,Cytoscape software,Cytohubba plug-in,STRING database,DAVID database,Venny website,in order to predict a series of gene ontology and signal pathways that OVOL2 may participate in in vivo.By consulting the pubmed literature,we are more interested in APLN and FSTL1.5.Use the Breast Cancer Gene-Expression Miner v4.5 database to analyze the correlation between O VOL2 and predicted downstream targets of interest.Results:1.Through lentivirus transfection of cells,it was observed that OVOL2 caused the phenotype of MDA-MB-231 cell line to change.2.The results of RNA-seq analysis showed that the experimental group(overexpressing OVOL2)and the control group(not overexpressing OVOL2)obtained a total of 545 differentially expressed genes with statistical significance(P<0.05,|Log2FC|≥1),which were up-regulated There are 316 differentially expressed genes and 229 down-regulated differentially expressed genes.3.RNA-seq results gene ontology and signal pathway analysis show that the differential genes in breast cancer cell line MDA-MB-231 are mainly enriched in the following gene ontology:tissue extracellular matrix,involved in the process of inflammation,and involved in the breakdown of collagen Metabolic process,participate in cell adhesion process,participate in the process of cell exosomes,participate in the process of tight junction of two cells,participate in the process of protein digestion and absorption,participate in the Rapl signaling pathway,and have an impact on the pathway of cell adhesion molecules.4.Three algorithms of DMNC,MNC,MCC in Cytoscape software analyze the results of RNA-seq differentially expressed genes.The central genes obtained by the intersection of the three algorithms are FSTL1,APOE,COL12A1,COL1A1,LEPRE1,STC2,SCG2,COL1A2,COL13A1,COL18A1,TNC,COL6A1,CDH2,COL5A1,IGFBP1,SDC2,FBN1,APOL1,PCSK9,APLN,C3,LTBP1,etc.5.The results of chromatin immunoprecipitation sequencing experiments showed that the Flag-OVOL2 group compared with the IgG group produced 4803 differentially expressed genes.These differential genes are mainly enriched in cell endoplasmic reticulum protein processing,cell cycle processes,MAPK signaling pathways,cancer-related pathways,steroid hormone receptor signaling pathways,regulating gene expression and epigenetics,RNA splicing,regulating mRNA Metabolic process etc.6.The results of RNA-seq and ChIP-seq experiments were jointly analyzed,and a total of 141 differential genes were obtained.The genes of interest were included by consulting the literature on pubmed.The genes of GPX8,NUCB2,IFI6,PCDH7,FBXO43,and STC2 were discussed.May be the downstream target of the biological role of OVOL2 gene in breast cancer.7.Correlation analysis results show that the negative correlation between OVOL2 and GPX8 and FSTL1 is more significant.Conclusion:The RNA-seq experiment and ChIP-seq experiment jointly analyzed 141 differential genes.OVOL2 may affect the malignant behavior of breast cancer MDA-MB-231 cells by regulating GPX8 and FSTL1,thereby affecting the prognosis and survival of breast cancer patients.The GO and KEGG analysis results of RNA-seq found that differential genes may be involved in cellular exosomal pathways.The GO and KEGG analysis results of ChIP-seq found that differential genes may be involved in the MAPK signaling pathway,cancer-related signaling pathways,etc.