Effects Of DDI2 Gene On Proliferation And Migration Of Colorectal Cancer HT-29 Cells And Screening And Identification Of Genetically Identified Genes Of Recombinant Colorectal Cancer Candidate TGFBR2 3 'UTR

The worldwide incidence of colorectal cancer is rising, which has a serious effect on people’s physical and mental health. About 70% of colorectal cancer is sporadic colorectal cancer (sCRC). Owing to the indiscoverable characteristic on early stage and poor prognosis of sCRC, people pay more attention to the pathogenesis of sCRC in order to find new genetic biomarkes for early diagnosis of sCRC.Our Previous results of whole exome sequencing and related literature suggest DDI2 a possible candidate gene for sCRC. Up to now, there is no report about the relation between DDI2 and sCRC. Present study analyzed the DDI2 expression between Lovo, HT-29, colo320, sw480 colorectal cancer cell lines and normal cell lines NCM460 by western blot and RT-PCR, and builds expression vectors of wild type and mutant type of DDI2 gene (pIRES2, pCDNA3.1, pEGFP-C2). DDI2 gene expression vector was transfected in HT-29 cell line to study tumor cell proliferation, colony formation and cell migration. These studies might make a solid foundation for future mechanism research of DDI2 in sCRC.Protein expression analysis showed rare expression of DDI2 in cell lines (NCM460, colo320, sw480, Lovo, HT-29), and no significant difference, but mRNA were all expressed in above cell lines by RT-PCR assay, and show a significant difference from normal colon cell line NCM460.The proliferation rate, the number of colony forming and migration rate of HT-29 transfected with plRES2 vector significantly decreased compared with that transfected with empty vector (P<0.05). Conclusion:DDI2 genes may reduce proliferation and migration of colorectal cancer HT-29 cell lines, and may be involved in the process in sCRC. Further studies are needed to clarify this point.Colorectal cancer is one of the important malignant tumors, which seriously affect people’s life and health. colorectal cancer is easy to transfer and difficuLt to discover and treat in the early time. Therefore, it is essential to find genetic markers for early warning colorectal cancer risk. It was found that small RNA (microRNA, miRNA) can reguLate target gene expression levels through combination with the non-coding region of target genes. The 3’UTR region polymorphisms of tumor related genes may affect the occurrence and development of tumor through binding of special miRNA.In this subject, the binding miRNAs with 3’UTR of candidate genes were screened by consuLting relevant literature and the RegRNA, RNAhybrid websites. Finally, the rs11466537 polymorphisms of TGFBR2 gene was selected as the research object, which closely binded with hsa-miR-1193. Wild and mutant type pmirGLO vector which carried 300 bp flanking sequence of T alleleand A allele of rs11466537 site in TGFBR2 gene were builded respectively. Then hela cells were transfected with these two type of vectors, fluorescence value within them detected through dual luciferase assay method.The resuLts showed that fluorescence value of cells which carried wild type vector were decreased significantly (P<0.001) compared with the cells carried the mutant type. Conclusion:The 3’UTR of TGFBR2 rs11466537 site mutation affects the expression of TGFBR2 gene, which may provide the experimental basis for the further study of the relationship between TGFBR2 gene and colorectal cancer.