A Preliminary Study Of TNFAIP3 Participating In The Mechanism Of Systemic Lupus Erythematosus

Objectives:1.To study the expression of TNFAIP3 gene in the peripheral blood and B lymphocytes of patients with SLE and normal;2.To study the effect of overexpression of TNFAIP3 gene in peripheral blood B lymphocytes of SLE patients and normal,and detect the effects of the gene on B cell receptor BAFF,Blimp-1 and TLR7/TLR9-NF-kappa B signaling pathway and downstream signal molecule IRF7;3.To study the effect of silencing TNFAIP3 gene in peripheral blood B lymphocytes of SLE patients and normal,and detect the effects of the gene on B cell receptor BAFF,Blimp-1 and TLR7/TLR9-NF-kappa B signaling pathway and downstream signal molecule IRF7;4.To investigate the potential mechanism of TNFAIP3 gene in patients with SLE in B cells and its clinical significance,and role of molecular signaling pathway related gene TNFAIP3 in the pathogenesis of SLE disease activity,disease factors to provide new clues and new ideas for the treatment of diseases.Methods:1.We collected 45 cases of SLE blood and 35 cases of normal blood,using Ficoll-Hypaque density gradient centrifugation to centrifuge peripheral blood lymphocytes and immunomagnetic separation of CD19+ B lymphocytes;then the separated cells were RNA extraction,and reverse transcription cDNA,finally the application of qRT-PCR technology in total peripheral blood lymphocytes and B cells to detect expression of TNFAIP3;2.We constructed a retroviral expression vector TNFAIP3-pEZ-Lv201 containing TNFAIP3 gene.We used 293T cells for virus packaging,and collected virus supernatant cell culture supernatants,then added to pre selected CD19+B cells and collected cells after 48h.Then we detected TNFAIP3 mRNA expression in B cells that were overexpressed and the expression level of BAFF,Blimp-1 and TLR7,TLR9 and downstream signal molecule IRF7 by qRT-PCR;3.We screened the TNFAIP3-shRNA fragments with stronger interference efficiency,packaged them with lentivirus,collected cell culture supernatants,infected SLE patients and normal CD19+B cells,and then collected cells after 48h infection.Then we detected TNFAIP3 mRNA expression in B cells that were silenced and the expression level of BAFF,Blimp-1 and TLR7,TLR9 and downstream signal molecule IRF7 by qRT-PCR.Results:1.The mRNA expression level of TNFAIP3 gene in peripheral blood total lymphocytes and B cells of SLE was lower than the healthy controls,both the difference were statistically significant(P<0.01);2.TNFAIP3 retrovival recombinant expression vector was constructed successfully,and has proved it can well expressed in mammalian cells;3.Overexpression of TNFAIP3 gene was observed in B cells of SLE and the normal.The overexpression level of TNFAIP3 mRNA in over expressing plasmid infection group(TNFAIP3-pEZ-Lv201)was significantly higher than that in the control group(pEZ-Lv201),and there was a significant difference(P<0.01).In normal and SLE B cells,overexpression plasmid BAFF and Blimp-1 infection group,the expression levels of mRNA were significantly lower than the control group,and the difference was significant(P<0.01),and the overexpression plasmid infection group TLR7,TLR9 mRNA and TRAF6 ubiquitination closely related downstream signal molecules IRF7 mRNA expression levels were significantly lower than the self control group,and the difference was significant(P<0.01);4.Through effective silencing of TNFAIP3 gene in B cells of SLE and normal,it resulted in a significant down-regulation of TNFAIP3 mRNA expression,while the interference of TNFAIP3 gene in B cells resulted in the expression of BAFF and Blimp-1 mRNA in B cells,which was significantly higher than that in the control group.The expression level of TLR7?TLR9 and IRF7 mRNA were significantly higher than that of the control group,and the difference was significant(P<0.01)Conclusions:1.The abnormal expression of TNFAIP3 gene suggests that the gene may be one of the pathogenesis of SLE by regulating the maturation and differentiation of B cells;2.TNFAIP3 gene can inhibit the activation of TLR7/TLR9-NF-kappa B signaling pathway through the effect of ubiquitination and deubiquitination.When the TNFAIP3 gene is mutated or defective,A20 can be used as a ubiquitin for TRAF6 and activation of the downstream NF-kappa B pathway;3.The overexpression of TNFAIP3 gene significantly inhibited the up regulation of TLR7/9 expression,and vice versa.This suggests that NF-kappa B regulates the expression of TLR7/9,and NF-kappa B may increase the inflammatory damage induced by A20 in the silent state by positive feedback mechanism.