Mechanism Study Of MiR-628-5p In Inhibiting The Progression Of Cholangiocarcinoma By Targeting BLM

Part ?: Screening of BLM gene and its effect on the progression of cholangiocarcinomaObjective: Cholangiocarcinoma(CCA)is one of the most common tumors of all gastrointestinal neoplasias.Due to its rapid progression and a high degree of malignancy,the prognosis of patients with cholangiocarcinoma is often poor.Therefore,possible biomarkers and drug targets need to be discovered urgently.To explore the pathogenesis of cholangiocarcinoma,we analyzed the genome profiles of cholangiocarcinoma samples in public databases and combined them with bioinformatics methods to find new key genes related to the occurrence,development,and prognosis of cholangiocarcinoma.Methods: the gene expression profile data of cholangiocarcinoma samples,adjacent tissues,and clinical data of tumor samples were obtained from The Cancer Genome Atlas(TCGA).Weighted gene co-expression network analysis(WGCNA)was used to find the similarity between differentially expressed genes in tumor samples,and the genes with highly similar expression patterns were grouped into the same gene module.After associating different gene modules with clinical traits,we identified the most relevant gene module for the progression of cholangiocarcinoma and analyzed all genes in this module by Gene Ontology and the KEGG pathway.Based on the results of PPI analysis and co-expression analysis,several candidate genes were identified.By verifying the expression and prognosis of these candidate genes in internal and external datasets,the hub gene was screened.Finally,according to the expression of hub gene in different samples,tumor samples were divided into high expression group and low expression group.Gene set enrichment analysis(GSEA)was used to explore the signaling pathways and mechanisms that hub gene may participate in the regulation of cholangiocarcinoma progression.Results: Based on 6219 differentially expressed genes between paracancerous and cholangiocarcinoma tissues,we used the WGCNA algorithm to distinguish 29 gene modules in the gene expression profile of tumor samples.Among them,the blue module was the most relevant gene module for clinical characteristics of cholangiocarcinoma.Gene ontology and KEGG pathway analysis of the genes in the blue module showed that the genes in the blue module were mainly involved in cell cycle regulation and cancer-related pathways.Five candidate hub genes BLM,GGH,RPS3,NUP107,and CCT2 identified in the blue module were verified in internal and external datasets,and a final hub gene BLM was obtained.GSEA results showed that BLM was involved in the regulation of cell cycle and cell proliferation in cholangiocarcinoma.Conclusion: BLM is closely related to the poor prognosis and malignant progression of cholangiocarcinoma,and it may be involved in the regulation of cell cycle and cell proliferation-related pathways.Part ?: Effect and mechanism of BLM on the biological behavior of cholangiocarcinomaObjective: To verify the expression of BLM using in vitro and in vivo experiments and explore its influence on the progression of cholangiocarcinoma.Methods: The m RNA and protein expression of BLM in cholangiocarcinoma cell lines QBC-939,RBE,and Hi BEC were detected by RT q PCR and WB respectively.Immunohistochemistry was used to detect the expression of BLM protein in cholangiocarcinoma tissue chips.BLM si RNA was transfected into QBC-939 and RBE cells.CCK-8 assay was used to detect the proliferation activity of transfected cells;Scratch test and Transwell test were used to detect the migration ability of cells.The changes in the cell cycle and apoptosis were detected by flow cytometry.Short hairpin RNA(sh RNA)plasmid was used to construct BLM low expression cell line,which was used to establish xenograft tumor model in nude mice.Results: BLM was highly expressed in cholangiocarcinoma tissues and cholangiocarcinoma cell lines.In vitro,down-regulation of BLM expression can inhibit the proliferation and migration of cholangiocarcinoma cells.Flow cytometry showed that knockdown of BLM expression led to G2 phase arrest of cholangiocarcinoma cells and increased the proportion of apoptotic cells.In nude mice xenograft tumor model,BLM knockdown inhibited tumor growth in nude mice.Conclusion: BLM can improve the proliferation and migration of cholangiocarcinoma cells,promote the progress of tumor cell cycle,inhibit the apoptosis of cholangiocarcinoma cells,and enhance the growth ability of tumor,thus promoting the progress of cholangiocarcinoma.Part ?: Prediction of miRNA targeting BLM and verification of its effect on BLM expressionObjective: As an important non-coding RNA in cells,miRNA is involved in regulating the expression of many genes.In this chapter,we identified the miRNA targeting BLM and explored its effect on BLM expression.Methods: The commonly used online bioinformatics databases miRDB,miRWalk,Tar Base,and Target Scan were used to predict the miRNAs targeting BLM,and the intersection of them was identified as predicted miRNA,namely miR-628-5p.We further predicted its binding site with BLM and detected the expression of miR-628-5p in the cholangiocarcinoma cell lines QBC-939,RBE,and normal bile duct epithelial cells Hi BEC by using RT-q PCR.The luciferase reporter gene plasmids carrying wild-type BLM 3'UTR region and mutant BLM 3'UTR region were constructed respectively,and the luciferase activity changes were observed when the mutant plasmid or wild-type plasmid were cotransfected with miR-628-5p mimic respectively.Mimic and inhibitor of miR-628-5p were transfected into cholangiocarcinoma cell lines to down-regulate and up-regulate the expression of miR-628-5p,and then we observed their effects on BLM expression.Results: Bioinformatics prediction analysis showed that miR-628-5p could target BLM.The expression of miR-628-5p in Hi BEC was significantly lower than that in QBC-939 and RBE.The luciferase activity of mutant plasmid was not affected by miR-628-5p mimic,while the luciferase activity of wild-type plasmid decreased significantly.After transfection with mimic or inhibitor of miR-628-5p,the expression of BLM protein and m RNA in cholangiocarcinoma cells decreased and increased,respectively.Conclusion: In cholangiocarcinoma,miR-628-5p can regulate the malignant progression of cholangiocarcinoma by targeting BLM.This new mechanism provides promise for the diagnosis and treatment of cholangiocarcinoma and is expected to be used to guide clinical targeted therapy and precise treatment in the future.