Study On The Mechanism Of TNIP1 Gene In Peripheral Blood B Lymphocytes Of SLE Patients

Background Systemic lupus erythematosus (SLE) is a clinical complex autoimmune disease characterized by T and B cells abnormal activation, the formation of autoantibodies and immune complex deposition. So far, the exact etiology and pathogenesis of SLE has not been entirely elucidated. But generally accepted genetic factor may play a key role in the pathogenesis of SLE. Studies found that the main pathological mechanism of SLE is a large number of autoantibodies production and immune complex deposition in the organization that as result of T and B cells highly activated and abnormal proliferation. The B cells phenotypes, life, function and regulation of signal transduction and so on in SLE patients are exhibited abnormal. In recent years, genome-wide association study(GWAS) and candidate genes approach have confirmed that TNIP1 is the susceptible gene of SLE in Chinese Han population. As tumor necrosis factor-a-induced protein 3(TNFAIP3) of interacting protein, TNIP1 is synergistic and alone with TNFAIP3 gene to negative regulatory NF-κB signal pathway activation, thereby inhibit B cells overactive and apoptosis defect, may play a crucial role in occurence and development of SLE and other autoimmune diseasea.Objective 1. To study the mRNA expression of TNIP1 gene in SLE peripheral blood total lymphocytes and CD 19+B cells, as well as their relationship with SLE Disease Activity Index (SLEDAI) and TNIP1 expression level.2. Respectively, overexpression of TNIP1 gene that in SLE patients’ and normal controls’peripheral blood B cells, then detect its influence on B cells growth and survival, detect other genes mRNA expression in the B cells.3. Getting recombinant lentiviral that can effectively silence human B cells TNIP1 gene and investigates lentiviral-mediated TNIP1-siRNA silencing that influence on B cells growth and survival, study the relative expression of TLRs mRNA. Through the experimental research on the topic to explore TNIP1 gene potential mechanism and clinical significance in SLE patients’ B cells. Objective to investigate the role of TNIP1 gene regulation about molecular signaling pathways in immunological pathogenesis of SLE and disease activity factors provide new clues, meanwhile providing a new method for the treatment of diseases.Method 1. we collected 20 patients with SLE and 15 normal controls. The peripheral blood lymphocytes were isolated using Ficoll-Hypaque gradient centrifugation and using immunomagnetic beads separation CD 19+B cells. The mRNA was extracted from total lymphocytes and B cells, and then reverse transcription it for cDNA. We used the Real-time PCR to detect the expression of TNIP1 mRNA in total lymphocytes and B cells.2. Construction of TNIP1 retroviral expression vector, then chose 293T cells for virus packing and collection cells culture supernatant which rich in viral particles. Finally, we can add that cells culture supernatant to the CD 19+ B cells, then cells were collected after infection 48h. Using Real-time PCR to detect TNIP1 mRNA expression in B cells that were overexpressed and to evaluate the relative expression of TLR7 and TLR9 mRNA of Bcells; MTT assay TNIP1 gene overexpression whether impact the B cells growth and survival in vitro.3. By TNIP1-siRNA lentivirus transfect 293T then screen a highest inhibition efficiency of sequence to conduct virus package. Collecting cells culture supernatant and then infect SLE patients’ and normal controls’ B cells. Also using qRT-PCR technology to detect TNIP1 mRNA expression after RNA interference B cells’ TNIP1, to investigate TLR7 and TLR9 expression levels; by MTT experiment determine TNIP1 gene defect whether effect on the growth and survival of B cells.Results 1. The mRNAs expression level of TNIP1 gene in peripheral blood total lymphocytes of SLE patients was lower than the healthy controls, but TNIP1 mRNA expression level in SLE patients’B cells higher then normal control group; both the difference were statistically significant (p<0.01).2. TNIP1 retrovival recombinant expression vector was constructed successfully, and has proved it can well expressed in mammalian cells.3. Overexpressed respectively SLE patients and normal controls B cells TNIP1 gene then get TNIP1-pBABE expression plasmids infection group than the control group pBABE, TNIP1 mRNA expression was significantly upregulated. (P<0.01). Not only SLE patients but only healthy control, TNIP1-pBABE infected group compared with pBABE control group, TLR7 mRNA expression level decreased, but TLR9 mRNA levels increased. Both the difference were statistically significant (p<0.05). Whether it is over-expressing TNIP1 gene of SLE patients or normal controls, TNIP1-pBABE group compared with the control group pBABE, the relative growth number of cells is basically same.4. Effectively silence B cells TNIP1 gene of SLE patients and healthy controls can lead to TNIP1 mRNA expression significantly downregulated. TLR7 mRNA expression increased, but TLR9 mRNA expression significantly decreased, with statistical significance. RNA interference TNIP1 gene of B cells did not directed impact the cells growth and survival in vitro.Conclusion 1. TNIP1 gene abnormal expression suggestes that the gene may play an important regulation role in the pathogenesis of SLE.2.TNIP1 gene may be impact on the TLR mediated signaling pathway by regulating post-receptor signaling transduction, influence the expression of TLRs gene in B cells.4. TNIP1 gene did not affect the growth and survival of B cells in vitro.