The Effect Of Histone Monoubiquitination On T Cell Function

Objective:1.Construct RNF20-sh RNA lentiviral vector,target human peripheral blood acute T lymphocyte leukemia cells(jurkat cells),knock down the expression of RNF20 protein in cells,and screen out cell lines stably expressing RNF20-sh RNA.2.To explore the effect of RNF20 on gene repair,proliferation and immune function of human peripheral blood acute T lymphocyte leukemia cells.Methods:1.Search for the RNF20 gene sequence in Bank Gene,design a three-entry gene target interference sequence,connect to the p Lent-U6-GFP-Puro linear vector,transform DH5α competent cells,select the appropriate colonies by screening with relevant antibiotic LB solid medium.Transformants and positive clones were identified by PCR.2.The lentiviral vector was packaged by 293T-FT cells,Q-PCR was used to test the inhibition efficiency of the target protein,and the optimal lentiviral vector for inhibition efficiency was selected for bulk packaging amplification.3.p Lent-U6-GFP-Puro-sh RNF20 lentivirus infection and screening for T cells stably expressing the interfering gene.4.X-ray irradiation induced gene breakage damage,explore the RNF20 protein low expression of T cell gene repair and proliferation ability.5.The expression level of immune function-related genes in sh RNF20/Ctrl-jurkat cells was examined by transcriptome sequencing.Results:1.The RNF20-sh RNA lentiviral vector was successfully constructed,and the jurkat cells with high expression of the target gene were screened.2.sh RNF20-jurkat cells proliferate faster than sh Ctrl jurkat cells,and RNF20 expression maintains gene repair capacity.3.The expression levels of IL-2,γ-IFN,EOMES,TBX21 and FASLG in sh RNF20-jurkat cells were higher than those in sh Ctrl-jurkat cells,and RNF20 protein inhibited immune function of T cells.Conclusion:E3 ubiquitinated ligase RNF20 is involved in T cell gene damage repair,cell proliferation and immune process,its low expression hinders gene break repair,accelerates cell proliferation rate,and promotes immune function,which are consistent with the biological characteristics of cancer cells.This proves that RNF20 can be used as a research point for cancer prevention and treatment.