Functional Study Of Dip2B In Tumor Growth And Mouse Model Development Using CRISPR-Cas9 Mediated Base Editing

Cancer is a group of diseases characterized by uncontrolled cell proliferation that ultimately invades and disrupts the function of vital organs,causing death.Cancer is a complex disease that can originate from different tissues and progress through multiple stages during its development.Disco-interacting protein 2 homolog B（Dip2B）is a member of Dip2 family encoded by Dip2b gene.Dip2B has been reported to regulate murine epithelial KIT+progenitor cell expansion and differentiation epigenetically via exosomal mi RNA targeting during salivary gland organogenesis.Recent evidence suggests that SNPs in the coding region of DIP2B and its promoter are associated with an increased risk of colorectal cancer（CRC）development.DIP2B was suggested to involve in regulating autophagy in pancreatic ductal adenocarcinoma.Similarly,genome wide-expression studies identified DIP2B as fusion partner with Neuregulin-1 in non-small-cell lung carcinoma or with Erb-B2 Receptor Tyrosine Kinase 2（ERBB2）in urothelial bladder tumors.However,DIP2B is poorly understood and its role in cancer development and progression remains unclarified.In this thesis,to gain a better understanding of Dip2B role in tumor growth in vivo,Dip2b^tm1a/+heterozygous knockout mouse model was used to investigate the growth of B16F10 tumor cells and their metastasis.Results show that Dip2B deficiency significantly promoted tumor growth in vivo and metastasis most likely by immune dysfunction and reduction of macrophage and CD8⁺T lymphocytes infiltration into tumor microenvironment.Next,in chapter 4,we investigated the transcriptome of Dip2B-deficient mouse embryonic lung fibroblasts（MELFs）isolated from E14.5 embryos by RNA-Seq.Expression profiling identified 1369 and 1104 differentially expressed genes（DEGs）from Dip2b^-/-and Dip2b^+/-MELFs in comparisons to wild-type(Dip2b^+/+).Functional clustering of DEGs revealed that many gene ontology terms belong to membrane activities such as‘integral component of plasma membrane’,and‘ion channel activity’,suggesting possible roles of Dip2B in membrane integrity and membrane function.KEGG pathway analysis revealed that multiple metabolic pathways are affected in Dip2b^-/-and Dip2b^+/-when compared to Dip2b^+/+MELFs.These include‘protein digestion and absorption’,‘pancreatic secretion’and‘steroid hormone synthesis pathway’.These results suggest that Dip2B may play important roles in metabolism.Molecular function analysis shows transcription factors including Hox-genes,b HLH-genes,and Forkhead-genes are significantly down-regulated in Dip2b^-/-MELFs.These genes are critical in embryo development and cell differentiation.In addition,Dip2B-deficient MELFs demonstrated a reduction in cell proliferation and migration,and an increase in apoptosis.All results indicate that Dip2B plays multiple roles in cell proliferation,migration and apoptosis during embryogenesis and may participate in control of metabolism.Here,we provide valuable information for further understanding of the function and regulatory mechanisms of Dip2B.In chapter 5,we show that many human genetic diseases arise from point mutations.These genetic diseases can theoretically be corrected through gene therapy.However,gene therapy in clinical application is still far from mature.Nearly half of the pathogenic single-nucleotide polymorphisms（SNPs）are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing.The most current base editing methods are developed by David Liu’s lab.These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require a donor DNA sequences for homologous recombination.These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency,but has not been confirmed in mice.Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo.Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine（C）to uridine（U）.In this study,BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene.High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection.Sequencing results showed that expected base changes were obtained with high precision and efficiency（56.25%）.There are no off-targeting events identified in predicted potential off-target sites.Results confirm BE4 system can work in vivo with high precision and efficacy,and has great potentials in clinic to repair human genetic mutations.