The Role And Mechanism Of PAX5 In The Pathogenesis And Prognosis Of B Cell Neoplastic Disease

Objective At present,due to a better understanding of biological characteristics of acute B lymphocytic leukemia and improvement of treatment,children and adults both have a significant higher CR rate,however,a considerable number of patients prone to be recurrent,especially adults.About 30% of the cases has been involved in PAX5 mutations,though there is no further investigation of the effects of PAX5 on adult disease,but studies have shown that COX analysis of PAX5 deletion can be an independent risk factor for DFS in children.In this study,we constructed a PAX5 haploid knockout cell model with the Raji cell line using gene editing techniques.By observing the biological function changes in this cell model,possible mechanism of PAX5 in B cell tumor would be explored.Methods In this study,we used the CRISPR-cas9 gene editing tool to construct the tool plasmid by analyzing the human PAX5 gene functional region,targeting specific exons,selecting the appropriate loci.The plasmid was transfected into human Burkitt lymphoma cell line Raji,and the gene knockout was achieved by using the targeted binding of g RNA and the cleavage effect of cas9 protein.After cell monoclonal sorting and a series of sequencing work,the eligible cell lines were selected.To observe its biological effects in vivo and in vitro,and to explore the possible mechanism of PAX5 deficiency in tumor development.Results A CRISPR-cas9 tool plasmid with strong cleavage effect on specific sites of PAX5 was constructed and more than 20 Raji cell clones with PAX5 haploid mutations were obtained.PAX5 mRNA and protein levels were decreased in mutant clones.Compared with the wild type,the Raji cell line of PAX5 haploid mutation had no significant changes in basal apoptosis,decreased proliferation ability,delayed cell cycle progression,reduced sensitivity to the drug,and reduced tumorigenicity in vivo.Conclusions The Raji cell model with clear genetic background and homologous control was successfully constructed by CRISPR-cas9,a third-generation gene editing technique.The method was simple and reliable.PAX5+/-Raji cell clone compared with the wild type,proliferation slowed down,slow progress of the cycle,it may due to the knockout of PAX5,promoting part of the cells into the dormant state,the tumor cells' ability of drug resistance increases,to avoid attacking from conventional chemotherapy drugs,become the source of recurrence and metastasis,leading to worse outcome.