The Detection Of MiR-223-3p In PDC-E2 Reactive CD8~+T Cell And Its Mechanism Of Regulation On Cell Apotopsis

Primary biliary cholangitis(PBC)is a kind of chronic autoimmune disease(AID),the anti-mitochondrial antibody(AMA)which is the major autoantibody of PBC can be detected in the serum of 95% patients,the E2 subunit of the pyruvate dehydrogenase complex(PDC-E2)is the main autoantigen.In recent years,the incidence of PBC increased year by year,but there is no effective drug treatment,patients with late stage can progress to liver fibrosis even cirrhosis,so researching how autoantigen induces the PBC and looking for therapeutic targets can't be waited.There are a large number of studies which show that autoantigen reactive T cells participate in the morbidity in many common AID such as RA,SLE,et al.Similarly,autoantigen reactive T cells and PBC are inseparable,infiltration of large quantities of autoantigen reactive CD8~+ T cells and CD4+ T cells in the liver of patient with PBC is responsible for the injury of bile duct.There may be two pathway of injury,one is killing the bile duct cells directly through the Fas-fasL pathway,the other is damaging bile duct cells mainly through the secretion of cytokines such as perforin,granzyme and other toxic cytokines to cause the pathological changes of bile duct.The proliferation,survival and apoptosis of CD8~+ T cell may affect whether the damage happens and the degree of damage to the bile duct epithelial cells,thus affecting the occurrence and of development of PBC.With the mature of research technology of microRNA(miRNA),more and more studies have confirmed that miRNA participate in the occurrence and development of various AID.Several studies have confirmed the role of miRNA in the incidence of PBC.MiR-506 can damage the function of secretion of bile duct and is considered to be a potential therapeutic target,miRNA-21 can promote the formation of organs specific autoimmune cholangitis in the model of mice by regulating the secretion of proinflammatory cytokines.On the one hand,miRNA can affect a series of biological activities of CD8~+ T cell such as activation,differentiation,proliferation and apoptosis,on the other hand,mi RNA can adjust the effective function of CD8~+ T cell to influence the progression of AID.The research group has founded that the miRNAs expression of both peripheral blood mononuclear cells(PBMC)and T cells in PBC have changed and some miRNAs have a certain role in the pathogenesis of PBC.However,the expression of miRNA of autoreactive CD8~+ T cell and the regulatory mechanism of miRNA regulating cell apoptosis is not clear.In this study,we will detect the expression of miRNA in PDC-E2 reactive CD8~+ T cell in vitro,and further study the possible mechanism of miRNA in regulating the apoptosis.Part ?.The level of miR-223-3p in PDC-E2 reactive CD8~+ T cell decreased and the apoptosis of CD8 T cell decreasedWe first stimulate the PBMC from health people with PDC-E2 antigen peptide,then selecting CD8~+ T cell with magnetic beads that is PDC-E2 reactive CD8 cells.We then screen miRNA chip after extracting and identifing the RNA of cells and choice the miR-223-3p which decrease significantly in CD8~+ T cell considering the previous chip results of T cells in peripheral blood.At the same time,use RT-PCR to verify the change of miR-223-3p and the two results are the same.We found that the expression of miR-223-3p in PDC-E2 reactive CD8~+ T cell reduced and it is 0.47 times of the control group.Apoptosis of CD8~+ T cell was detected by using flow cytometry after the stimulation of PDC-E2 antigen peptide and we found the apoptosis PDC-E2 reactive CD8~+T cell decreased as the control group.Part ?.The effect of miR-223-3p on apoptosis in CD8~+T cellIn order to understand whether the downregulation of miR-223-3p in PDC-E2 reactive CD8~+T cell is related to apoptosis,we transfected miR-223-3p mimic or inhibitor in PBMC,then use PDC-E2 antigen peptide stimulation,sort CD8~+T cells and detected the apoptosis CD8~+T cell.We found there exist positive correlation between the expression of miR-223-3p in PDC-E2 reaction CD8~+T cell and apoptosis,the transfection of miR-223-3p mimic induces apoptosis of CD8~+T cell and transfection of miR-223-3p inhibitor inhibited apoptosis.The finds with results in the first part that miR-223-3p in PDC-E2 reactive CD8~+T cell decreased and its apoptosis reduced are consistent,thus wo can infer that miR-223-3p in PDC-E2 reactive CD8~+T cell cells can regulate cell apoptosis.Part ?.miR-223-3p promote the apoptosis by targeting IGF-1RWe predict target genes of miR-223-3p by multiple sites and select IGF-1R which is related to apoptosis among all target genes.Then we verified the target from both the gene and protein level.In order to determine whether miR-223-3p regulate cell apoptosis by IGF-1R,we design and synthesis the siRNA of IGF-1R to silence IGF-1R and detect whether the apoptosis of CD8~+T cell changed.We found that the apoptosis of CD8~+T cell increased after siRNA transfection.The result is the same with the mimic group results in the second part.The results showed that miR-223-3p in PDC-E2 reactive CD8~+T cell had positive regulation on cell apoptosis by targeting IGF-1R.In summary,we found that the expression of mi R-223-3p in PDC-E2 reactive CD8~+T cell reduced and miR-223-3p can regulate the apoptosis of CD8~+T cell by targeting IGF-1R.This regulation may be related to that IGF-1R is involved in the apoptosis pathway,miR-223-3p may participate in the development of PBC in this regulation type.