Application Of Nude Mouse Xenograft Model In Nude Mice In The Correlation Between TMBIM1 And Colon Cancer

Objective: This research investigates the application of the animal model in colon cancer and uses the experience of existing animal models to stimulate the role of TMBIM1 in the development of colon cancer.This research discusses the feasibility of establishing the animal model of TMBIM1,expecting to find a new marker of colon cancer,and provide a new finding for colon cancer research as well as the clinical treatment of humans and pets.Methods: By comparing the database of colon cancer cases with normal people,this research uses the GWAS meta-analysis to find potential colon cancer-related loci and compares the differential expression of genetic loci which screened in normal epithelial cell lines and colon cancer cell lines.Eventually,COLCA2 and TMBIM1,two more related locus genes,were selected for further research.Use q PCR to detect the difference in the expression of TMBIM1 and COLCA2 m RNA of cancer and adjacent tissues from colon cancer patients;use sh RNA theory to construct TMBIM1 and COLCA2 knockdown and knockout cell lines;use lentiviral packaging system to construct TMBIM1 and COLCA2 overexpressing cell lines.Through the establishment of stable cell lines,this research verifies the functional tests of TMBIM1 and COLCA2 in vitro,including the cell phenotypes of cell proliferation,migration,apoptosis,cycle.After finding the significant phenotype of TMBIM1,this research uses the stable cell line to establish a nude mouse tumorigenic model by subcutaneous injection and evaluate the nude mouse tumorigenic model.meanwhile,this survey performs transcriptome sequencing on the stable transgenic cell line of the TMBIM1 to find the different expression in genes and signaling pathways.Results: The GWAS meta-analysis screens 60 potential gene loci for colon cancer risk.Detection of TMBIM1 and COLCA2 transcriptome expression in NCM-460,HCT-116,SW620,LS-174 T,HT29,Lo Vo,SW480,HCT-8,SNU-C1 cell lines.The expression levels of TMBIM1 and COLCA2 in colon cancer cell lines are significantly lower than that of normal colon epithelial cell lines.The results of TMBIM1 and COLCA2 m RNA expression test in colon cancer and adjacent tissues which from 24 pairs tissues of 24 colorectal patients shows that the expression of TMBIM1 and COLCA2 are significantly lower than that in adjacent tissues.The immune fluorescence results of tissue sections in cancer and adjacent tissues indicate that the expression of TMBIM1 in cancer tissues is lower than that in adjacent tissues which is consistent with the m RNA results.The constructed TMBIM1 stable transformed cell line has a knockdown efficiency of 80% and an overexpression efficiency of 11.84 times than the vehicle;the COLCA2 knockdown efficiency is 80% and an overexpression efficiency of 104.46 times than that of the control.In the cell proliferation test in vitro,the cell proliferation and migration ability of TMBIM1 knockdown cell line is significantly higher than that of the control group,while the apoptosis level is lower than that of the control group;the results in the overexpressing cell line are consistent with the knockdown group,indicating that TMBIM1 has a potential tumor suppressor effect,but COLCA2 has no obvious phenotype.The results of the tumorigenesis test in vitro are that the TMBIM1 stable cell line is successfully used to establish a nude mouse tumorigenesis model and the volume and weight of the tumor formed by the experimental group and the control group were significantly different.Larger tumors were formed in nude mice injected with HCT-116 cells in the TMBIM1 knockdown group,while the overexpression group is smaller.In the TUNEL test of nude mouse tumor slides,the apoptosis in the knockdown group decreased,while the apoptosis in the overexpression group increased.The database prediction and test results verify that TMBIM1 plays an inhibitory role in the development of colon cancer.At the same time,the TMBIM1 knockdown group stabilizes the transcriptome sequencing results of the cell line: when the p-value is set to 1.2,there are 810 differentially expressed genes,of which 341 were up-regulated and 469 were down-regulated.By using the down-regulated genes to enrich signaling pathways,multiple signaling pathways are obtained,including Wnt signaling pathway which has been in the development of cancer in existing studies.The research shows that TMBIM1 may be an important link that has never been discovered in the development of colon cancer,and provides a theoretical basis for the future study of mouse colon cancer models.