Inhibition Of HLA-DRB1*0405 Gene Expression By SiRNA

The susceptibility of rheumatoid arthritis strongly related to the DRB1 allele subtypes of human leukocyte antigen. Many researches have demonstrated that the inhibition of DRB1 could avoid the abnormal immunoreaction. The significant inhibition by siRNA might suggest a therapeutic approach in rheumatoid arthritis.As people susceptive to in Asian RA are mostly born of DRB1*0405 gene, our research concentrated on siRNA aimed at HLA-DRB1*0405 gene in order to develop experiment basis for the new therapy. Plasmids to express renilla luciferase protein, 6 different shRNA targeting HLA-DRB1*0405 and a shRNA targeting the scramble sequence were designed and constructed. Human embryonic kidney cells 293 were co-transfected by lipidosome with psiCHECK-2/HLA-DRB1*0405 and one specific shRNA expression vector transiently, and cells without shRNA-transfection were used as the negative control. The impact of RNAi on HLA-DRB 1*0405 expression was analyzed by the real time fluorescence quantitative real-time PCR and the detection of renilla intensity.The research found that the mRNA of HLA-DRB 1*0405 gene in 293 cells was inhibited by siRNA, with a maximal inhibition rate of 89.25% detected by real-time fluorescence quantitative real-time PCR and 46.70% analyzed by the luciferase detection. According to the results, siRNA1 and siRNA3 were screened out with remarkable inhibition effect. We singled out RA patient with HLA-DRB 1*0405 allele subtype and separated peripheral blood mononuclear cells (PBMC) from blood. RNAi to DRB1*0405 gene on PBMC was studied and analyzed through Flow Cytometry. The positive rate of DRBl*0405 protein in the groups of siRNAl and siRNA3 is 60.45% and 54.67% separately, evidently lower than the scrambled siRNA group and the non-siRNA group.