Effects Of Sp110 Targeting JAK-STAT Signaling Pathway On Macrophage Function

Object: Tuberculosis(TB),caused by Mycobacterium tuberculosis(Mtb),poses a significant threat to human health.Sp110(Speckled 110)polymorphisms have been reported to be associated with TB risk in Sino-Tibetan populations,and GBP1(Guanylate Binding Protein-1)also has potential as a biomarker for TB.Interferon has been used in combination with first-line anti-TB drugs to treat TB for decades,but the function of Sp110 in regulating human macrophages in My cobacterium tuberculosis infection remains unknown,and the gene GBP1 has not been studied in depth.In the present study,we investigate the specific role and potential mechanisms of Sp110 and the pathogenesis of tuberculosis during the development of tuberculosis and investigate the role and clinical significance of GBP1 in tuberculosis.Methods:(1)GEO database(datasets GSE83456 and GSE34608)validates high expression of Sp110 m RNA in blood samples from pulmonary tuberculosis;Established a BCG-infected cell model to confirm the up-regulation of Sp110 expression after BCG infestation at the cellular level;Analyzed Sp110,chemokines(CXCL9,CXCL10),inflammatory factors(IL-6,TNF-α)and apoptosis-related genes(BAX and caspase-3 and BCL-2)expression in macrophages based on biochemical-single cell sequencing THP-1 cells could be differentiated into macrophages by fobolipids(PMA);To explore the possible interaction between Sp110 and STAT1;GSEA enriched and analyzed the signal pathways that Sp110 might participate in,so as to explore its immune function.(2)The three synthesized interfering fragment si RNAs and the constructed pc DNA3.1-Sp110 recombinant overexpression plasmid were transfected into macrophages by liposomes,and the interfering fragment sequences with the highest interfering efficiency were selected from the protein and gene levels,respectively,to detect the overexpression efficiency of the Sp110 gene and finally construct the Sp110 gene cell model.(3)To interfere with the expression of Sp110 gene,RT-q PCR and Western Blot detected the expression of chemokines,inflammatory factors and apoptosis-related genes in macrophages;flow cytometry observed the apoptosis;cell proliferation assay was performed to investigate the cell survival rate;To detect changes in the expression of the star molecule protein of the JAK-STAT signalling pathway.(4)To explore the expression of the related gene GBP1 in TB based on database expression matrix and clinical samples,and to analyse the signalling pathways it is involved in and its enrichment in immune cells.Results:(1)GEO database confirms significant high expression of Sp110 m RNA in bloodsamples from pulmonary tuberculosis;The BCG-infected cell model was established to confirm the up-regulation of Sp110 expression after BCG infestation at the cellular level;Sp110 was enriched in macrophages,and BCG-infected macrophages secreted chemokines CXCL9 and CXCL10 and inflammatory factors,which exerted pro-inflammatory effects in macrophages;there was a correlation between Sp110 and There is a correlation between SP110 and BCL-2 gene,which can promote the expression of apoptosis-related genes to induce apoptotic response;Sp110 may interact with STAT1 and exerts pro-inflammatory and pro-apoptotic immune defense effects through the JAK-STAT signaling pathway.(2)The experimental results revealed that the interference efficiency of si Sp110-1,si Sp110-2 and si Sp110-3 were 37%,41% and 69%.Respectively,and finally determined that si Sp110-3 was the best interference fragment sequence;the overexpression efficiency of pc DNA3.1-Sp110 recombinant plasmid on Sp110 was around 16-fold,which proved that the macrophage model of Sp110 gene was successfully established.(3)Overexpression of Sp110 promoted the expression of chemokines(CXCL9,CXCL10)and inflammatory factors(IL-6,TNF-α)as well as apoptosis-related genes(BAX and caspase-3),improved cell survival and activated the JAK-STAT signaling pathway,and the opposite results were obtained for the silencing of Sp110 gene;(4)GBP1 is closely related to STAT1,is expressed in elevated levels in both blood samples and TB specimens,is enriched in macrophages and plays an important role in the regulation of signalling pathways.Conclusion: 1.Bioinformatics analysis suggested that Sp110 was highly expressed in pulmonary tuberculosis macrophages,followed by molecular experiments to verify that Sp110 was enriched in macrophages through a BCG-infected macrophage model,that BCG infestation promoted chemokines and inflammatory factors to play a pro-inflammatory role,and that the expression levels of pro-apoptosis-related genes were increased,which in turn induced the development of an apoptotic response in macrophages;Finally,the above experimental results were further confirmed by the silencing/overexpression of Sp110.2.GBP1 was closely associated with the expression of STAT1,SAMD9 L and IFI44,was highly expressed in TB,was involved in the activation of signalling pathways such as IL-6-JAK-STAT3 and IL-2-STAT5,and was significantly upregulated in the immune enrichment fraction of M1 macrophages,activated dendritic cells and neutrophils.