Study On The Molecular Biological Mechanism Of ERH Gene Affecting The Occurrence And Development Of Bladder Cancer

Background The occurrence and development of bladder urothelial carcinoma(BUC)are related to the excessive activation of proliferation genes,the decrease of apoptosis factor activity and the activation of metastasis-related genes.Objection To study the molecular biological role and mechanism of ERH gene in the occurrence and development of BUC.Methods In this study,we used UALCAN tools to analyze the TCGA database and referred to immunohistochemistry to analyze the tissue microarray to determine the expression of the ERH gene in BUC,and explored its relationship with prognosis.Then siRNA was used to knockdown the expression of ERH in BUC T24 cells,and then compared the cell proliferation and cloning ability of ERH normal cell(NC)group and ERH knockeddown(KD)group through MTT assay,BrdU assay,Celigo,and clone formation assay.Observe the apoptotic ability of cells through apoptosis and cycle experiments;observe the migration and invasion ability of cells through scratch experiment,transwell cell migration and invasion assaies to verify the role of ERH gene in BUC cells.Then we observed the effect of ERH gene on the tumorigenesis and metastasis ability of BUC cells in animals by subcutaneous tumor formation experiment and tail vein injection lung metastasis experiment in nude mice.Then,we use gene expression profile chip technology and followed-up bioinformatics analysis technology to explore the possible inference of the molecular biological mechanism of ERH’s functions.Finally,we performed functional rescue experiments on possible results to verify possible inferences.Results We found and verified that the ERH gene is highly expressed in BUC through the TCGA database.This high expression state is not related to the pathology,tumor grade,tumor size and other factors of BUC patients,but it is related to the patient’s prognosis.When ERH is relatively highly expressed,the prognosis of BUC patients was relatively poor,which proved that the ERH gene may be an independent predictor of the prognosis of BUC.We knockeddown the expression of ERH gene in BUC cells,and found that the proliferation ability of ERH KD group cells was significantly reduced.Knockdown of ERH gene can significantly promote cell apoptosis and block cells to S phase.Through the scratch assay,transwell cell migration and invasion experiments,we found that after ERH KD,the cell migration and invasion ability was significantly reduced.After ERH KD,the ability of nude mice to form tumors under the skin and to metastasize to the lungs by tail vein injection also decreased significantly.The results of the gene expression profile chip indicated that after the expression of the ERH gene was reduced,the expression of 344 genes was up-regulated and the expression of 254 genes was down-regulated compared with the ERH NC group.Bioinformatics analysis technology found that ERH genes may affect proliferation and apoptosis by influencing "apoptosis","cell cycle","Toll-like receptor","NF-κB" or "TGF-β" signal transduction pathways;Upstream analysis found that the possible upstream pathways of ERH gene are TNF and NK-κB.ERH gene may participate in BUC’s "cell death and survival" molecular network,and regulate TNF process through KITLG.Through molecular network regulation analysis,we found that a possible downstream gene of ERH is MYC gene,which promotes cell proliferation,migration and invasion by inhibiting MYC expression.Then,we confirmed through functional rescue experiments that after MYC gene overexpression,the proliferation,migration,and invasion functions of BUC T24 cells in the ERH KD group were partially restored.Conclusion ERH can affect the proliferation,migration and invasion functions of BUC cells by regulating the expression of the classic oncogene MYC,and ultimately promote the occurrence and development of malignant tumors.