Mechanism Of LncRNA PVT1/miR-30e-5p Regulation Of CD4~+ T Cells Imbalance In SLE

Background: Systemic lupus erythematosus(SLE)is a multifactorial autoimmune disease(AID)of unknown etiology involving immune intolerance to endogenous nuclear substances,increased levels of autoreactive B cells,and chronic inflammation leading to systemic autoimmune and organ.The disease is characterized by the production of auto-reactive antibodies against self-antigens.The prevalence of SLE is high and racially variable,ranging from 12 to 39 per 100,000 worldwide,with a prevalence of about 70 per 100,000 in China and up to 113 per 100,000 women.Studies have shown that T cells in SLE have abnormal primary biochemical signaling pathways leading to altered gene regulation/expression,which impairs T cell immune effector functions and compromises the immune regulatory role of T cells on B cell clones,leading to activation and proliferation of polyclonal B cells,resulting in excessive production of natural and pathological antibodies by B cells.Abnormal T cell signaling,activation and differentiation are associated with changes in cytokine production that can contribute to the initiation and persistence of SLE.Imbalance of helper T cells(T helper cells,Th),such as Th1,Th2,Th17 and Treg,is involved in the development of SLE and is associated with disease activity.Therefore,understanding the pathophysiological mechanisms of abnormal T-cell function in SLE is important for the diagnosis and targeted therapy of patients.Objective:1.To investigate the gene expression of lnc RNA PVT1/mi R-30e-5p in peripheral blood of SLE patients;2.To confirm the correlation between PVT1 and Th1/Th2 and Th17/Treg imbalance in peripheral blood of SLE patients;3.To construct si-PVT1 and PVT1-OE in spontaneous lupus-like model mice MRL/lpr and observing the effect of PVT1 on lupus phenotype;4.To verify the differential expression of lnc RNA PVT1 and mi RNA-30e-5p in MRL/lpr mice;5.To investigate the regulatory relationship of PVT1 on Th1/Th2 and Th17/Treg imbalance in MRL/lpr mice.Methods:1.40 MRL/lpr mice were randomly divided into 4 groups: SLE model group,SLE+Lenti-Ctrl group(Ctrl),SLE+Lenti-si-PVT1 group(si-PVT1)and SLE+Lenti-PVT1(PVT-OE)group,10 mice in each group.2.The gene expression of Lnc RNA PVT1 and mi RNA-10e-5p in peripheral blood of SLE patients and healthy controls(HCs)was detected by quantitative real-time polymerase chain reaction(QRT-PCR)technique.The gene expression of Lnc RNA PVT1,mi RNA-10e-5p,T-bet,GATA3,ROR γ t and Foxp3 in peripheral blood of si-PVT1 and PVT1-OE MRL/lpr lupus mice was detected.3.The proportions of Th1/Th2/Th17/Treg cells in the peripheral blood of SLE patients and HCs,and the proportions of Th1/Th2/Th17/Treg cells in the peripheral blood of si-PVT1 and PVT1-OE MRL/lpr lupus mice were detected by flow cytometry.4.Enzyme-linked immunosorbent assay(ELISA)was used to detect IL-2/IL-4/IL-17/TGF-β concentrations in the sera of SLE patients and HCs,as well as IL-2/IL-6/IL-17/TGF-β concentrations in the sera of si-PVT1 and PVT1-OE MRL/lpr lupus mice.5.24-hour urinary protein levels in MRL/lpr lupus mice were measured by Coomassie blue staining.Results:1.QRT-PCR for gene expression in peripheral blood of SLE patients showed that gene expression of lnc RNA PVT1 was significantly higher and gene expression of mi R-30e-5p was significantly lower in peripheral blood of SLE patients compared with HCs.2.The results of flow cytometry showed a significantly higher percentage of Th2 cells,a significantly higher percentage of Th17 cells,and a lower percentage of Th1 and Treg cells but the difference was not significant in the peripheral blood of SLE patients compared to HCs.3.ELISA results showed that IL-2 was significantly lower,IL-4 was significantly higher,IL-17 was significantly higher and TGF-β was significantly lower in the peripheral blood of SLE patients compared with HCs.4.Compared with the Ctrl,si-PVT1 MRL/lpr mice showed significantly lower24-hour urine protein,significantly lower anti-ds DNA antibody content,significantly lower lnc RNA PVT1 gene expression,and significantly higher mi RNA-30e-5p gene expression.Compared with the Ctrl,PVT1-OE MRL/lpr mice showed significantly higher 24-hour urine protein,significantly higher anti-ds DNA antibody content,significantly higher lnc RNA PVT1 gene expression,and significantly lower mi RNA-30e-5p gene expression.5.Compared with the Ctrl,si-PVT1 MRL/lpr mice,the proportion of Th1 and Treg cells was significantly increased,the proportion of Th2 and Th17 cells was significantly decreased,and the Th1/Th2 ratio was significantly increased and the Th17/Treg ratio was significantly decreased.Compared with the Ctrl,PVT1-OE MRL/lpr mice,the proportion of Th1 and Treg cells was significantly decreased,the proportion of Th2 and Th17 cells was significantly increased,and the ratio of Th1/Th2 was significantly decreased and the ratio of Th17/Treg was significantly increased.6.Compared with the Ctrl,si-PVT1 MRL/lpr mice had significantly higher gene expression of T-bet and FOXP3 and significantly lower gene expression of GATA3 and RORγt.Compared with the Ctrl group,PVT1-OE MRL/lpr mice had significantly lower gene expression of T-bet and FOXP3 and significantly higher gene expression of GATA3 and RORγt.7.Compared with the Ctrl,the concentrations of IL-2 and TGF-β was significantly increased and the expression of IL-17 and IL-6 was significantly decreased in si-PVT1MRL/lpr mice.Compared with the Ctrl,the concentrations of IL-2 and TGF-β was significantly decreased and the expression of IL-17 and IL-6 was significantly increased in PVT1-OE MRL/lpr mice.Conclusion:Our data demonstrate that PVT1 expression is increased specifically in female SLE patients,that targeting PVT1 affects Th1/Th2 and Th17/Treg homeostasis,and that lnc RNA PVT1/mi RNA-30e-5p may play an important role in the regulation of immune responses and validation in SLE through the competing endogenous RNAs(ce RNAs)mechanism.