To investigate the effects of small interfering RNA (siRNA) of HLA DRB1(*)0405 HLA-DRB1(*)0405 gene expression with plasmid-based siRNAs.
Plasmid expressing HLA-DRB1(*)0405-renilla fusion protein-siCHECK-2/HLA-DRB1(*)0405, 6 different short hairpin RNAs (shRNAs) targeting 6 19 bp nucleotide sequences of HLA-DRB1(*)0405 (siRNA1 approximately 6), and one shRNA targeting the control non-specific sequence (siRNAC) were designed and constructed. Human embryonic kidney cells of the line 293 were cultured and co-transfected by lipids some with the plasmid siCHECK-2/HLA-DRB1(*)0405 and one specific shRNA expressing vector transiently, and cells without shRNA-transfection were used as negative controls. The impact of RNAi on HLA-DRB1(*)0405 expression was analyzed by real time fluorescence quantification RT-PCR and luciferase test.
The expression of HLA-DRB1(*)0405 gene RNA of the 293 cells transfected with siRNA1, siRNA2, siRNA3, siRNA, and siRNA6 were down-regulated to 10.75%, 83.22%, 30.63%, 48.54%, and 89.92% that of the control group with the inhibition rates of 89.25%, 16.78%, 69.37%, 51.46%, and 10.08% respectively. However, no significant downregulation was showed in the cells transfected with siRNA4 and siRNAC. The 293 cells transfected with siRNA1 and siRNA 3 showed a significant downregulation of the protein expression of HLA-DRB1(*)0405 gene with the inhibitory rates of 6.70% and 36.85% respectively; however, the cells transfected with siRNA2, siRNA4, siRNA5, and siRNA6 did not show a significant downregulation.
The significant inhibition of HLA-DRB1(*)0405 gene expression by siRNA suggests a therapeutic approach in rheumatoid arthritis: to use RNA interference (RNAi) to inhibit the abnormal immune reaction mediated by HLA-DRB1 in rheumatoid arthritis.