The Regulation Of Micro RNA On Th17 Cells In Patients With Primary Biliary Cirrhosis

Primary biliary cirrhosis(PBC) is a chronic cholestatic liver disease characterized by the antimitochondrial antibodies(AMA) in the serum and progressive destruction of small intrahepatic bile ducts, developing to liver cirrhosis , liver fibrosis , and eventually liver failure. The morbidity of PBC is increasing persistently in recent years, rising up to 1/1000 in over middle-aged,40 years ,women in North American. There is not systematically epidemic data with primary biliary cirrhosis, however, the morbility of primary biliary cirrhosis is increasing according to the growing reports documented and growing number of patient coming to hospital. At present, there is no effectively curative therapy for PBC. Ursodeoxycholic acid(UDCA) is known as the most effective drug for PBC, it can only provide symptomatic relief of cholestasis but not block disease course. At late stage of PBC, the only selection is liver transplantation. Due not to clearing the pathogenesis of primary biliary cirrhosis, we deeply research the etiopathogenesis with primary biliary cirrhosis to seek the critical pathway of regulation and make the cause of disease and the key regulatory point understood, it will not only have the value of scholarship, but also it will bring about gigiant prospect and social benefits.PBC has been considered as classical organ-specific autoimmune disease. Similar to other autoimmune diseases, there is an intense humoral and cellular response to the intracytoplastic antigen. The auto-antigens in PBC are members of the 2-oxo-acid dehydrogenase enzyme complexs(2-OADC) located on the inner mitochondrial membrane, the main among them is the pyruvate dehydrogenase E2 complex(PDC-E2), anti- PDC-E2 is positive in the serum of approximately 90-95% patients with PBC. The etiopathogenesis of PBC is incompletely defined. There is growing evidence that the genetic and environmental factors play a critical role in the pathogenesis of PBC. Molecular mimicry and xenobiotics may be involved in the initiation of diseases. Molecular mimicry between microbial agents and self-antigens might induce autoimmune diseases. Indeed, the role of molecular mimicry has been evaluated in CD4+ and CD8+T cells in the pathogenesis of PBC, and it has been shown that these T cells react to the peptides derived from several kinds of microbial agents. Despite considerable researches confirm that autoreactive T cells are involved in the pathogenesis of PBC, the underlying molecular mechanisms remain to be elicited.Recently ,a novel subset CD4+ T cells, called Th17 cell , which secreted mainly IL17 , was discovered and is discriminated from Th1 and Th2 cells. Many study showed it played a greatly important role in immune induced inflammation, especially in autoimmune disease, rejection of transplantation, tumor disease. In mice mode of experimental allergic encephalitis (EAE) , researchers found that if Th17 cells were transferred into mice, it was an more effective methods to induced EAE than Th1 cells, they also found that the mice was sever with IL17 injected to mice. Conversely, IL-17-/- mice was relieved the progression of EAE. From now on, many specialists observed that IL-23 deficiency mice is improved in EAE, and IL-6 deficiency mice and TGF-βR deficiency mice was induced tolerance in EAE. Later, scientists found RORγt was an important transcription factor in the course of Th17-inducing. We found that IL-17 mRNA was higher in peripheral blood mononuclear cells (PBMCs) with primary biliary cirrhosis than healthy control and the level of serum IL-17 was also increased than healthy control, moreover, compared healthy control, IL-17-producing CD4+ T cells was also higher. All these data demonstrated that Th17 cells may played an critical role in the pathogenesis or progression of primary biliary cirrhosis.MicroRNA (miRNA) are 22-nucleotide single-stranded regulatory RNA molecules found in nematodes, plants, and animals. The mature miRNA leads to target mRNA degradation or inhibits its translation following transcription via complete or incomplete complementary binding to the target mRNA. MiRNA participates in various biological processes such as cell proliferation, apoptosis, and metabolism of lipids by regulating target mRNA expression. The latest research demonstrated that some miRNAs are closely correlated with occurrence, development and prognosis of some diseases, which can be adopted as diagnostic and prognostic index for disease. Recent study found that bacterial lipopolysaccharide (LPS) and viral double stranded RNA can both induce the expression of miR-155. miR-155 gene knockout mice have compromised immune systems, similar symptom as human autoimmune diseases, and are less able to resist infection of bacteria such as saimonella . Further study has found that miR-155 can negatively regulate c-Maf, a gene indispensable to the functions of T cells. Therefore, microRNA may play a significant role in the functional regulation of T cells. However, its specific mechanisms are still subject to further study. We found that many micro RNA was differentiated expression from earlier micro RNA profile with primary biliary cirrhosis.On account of the recent and above research, we propose some doubts that whether micro RNA can regulate the immune function and how micro RNA regulate immune function in primary biliary cirrhosis, moreover, whether micro RNA can regulate the functions of Th17 cells, how micro RNA invoved with Th17. In order to resolve these doubts, three parts of investigations were included in our experiments: Firstly, we predicted the target of which micro RNA is IL-17 by means of bio-informatics theory, and the micro RNA is also changed in our earlier micro RNA profile. Secondly, the culture of Th17 cells. Finally, we testify the the role of micro RNA on Th17 with primary biliary cirrhosis and observed the changed functions of transfectioned Th17 by pri-miR and anti-miR.Part I The micro RNA to act on IL-17 predicted by bio-infomaticsIn the course of research, people found there is a relatively innate regulations of actions between miRNA and its target genes and the regulations can be drawn in a proper program to predict the target gene of the miRNA. Of course, these software is good to us and also is harmful, so it is necessary that the results need to verify. We select three most authority and most common software from many software to predict which microRNA can regulate IL-17 mRNA.The results showed that IL-17 maybe is a candidate gene of four micro RNAs from the profile of miRNA with primary biliary cirrhosis. To further compare the differentiate expression of these four micro RNAs on na?ve CD4+ T cells, memory CD4+ T cells, effector CD4+ T cells and Jurkat cells, we found that miR-26a and miR-30d have a differentiate expression on na?ve CD4+ T cells, memory CD4+ T cells, effector CD4+ T cells . MiR-30d also has a weak expression on Jurkat cells. Due to the greatly differentiate expression on na?ve CD4+ T cells, memory CD4+ T cells, effector CD4+ T cells of miR-26a, we considered miR-26a as a key point to observe the consistency as predicted by software.To verify the regulatory of miR-26a on IL 17A , we cultured Jurkat cells(a type of human T lymphocyte cell line) and make Jurkat cells tranfectioned by the gene of IL 17A and pri-miR-26a.The results showed that the fluorescence of transfectioned Jurkat cells is gradiently decreasing , which testify the regulatory of miR-26a on IL 17A. Part II The culture and purification of Th17 cellsThrere are many protocols of human Th17 cells stimulation and culture recently. The main elements are co-stimulation molecules and which type of T lymphocyte, lesion region, CD4+ T cells from PBMCs or CD4+CD45RO+T cells. We separated peripheral blood mononuclear cells, sorted CD4+ T cells and induced monocyte-derivatize dendritic cells (DC). After DCs were incubated with mitomycin (MMC), DCs cultured with CD4+ T cells, plus antigen PDC-E2 163-176 and related cytokines stimulation. Finally, we sorted the cells which were captured by captured complexand stimulated by PMA plus ionomycin. IL-17 mRNA and RORγt mRNA was detected and the purity was verified by flow cytometry.The results demonstrated that the purified cells were expressed IL-17 mRNA and RORγt mRNA, Th17 cells were purified and the purity was up to 80%, which is remain lower than literature reports.PARTⅢThe mechanisms study of miR 26a on Th17 cells with primary biliary cirrhosisIn this part, We make Th17 cells with primary biliary cirrhosis transfected pre-miR 26a and anti-miR 26a, later, we examined the level of miR 26a and detected the level of IL-17 mRNA and RORγt mRNA, the level of IL-17 in supernatants, the proliferation of Th17 with stimulation and the level of the activation-induced apoptosis. We also analysised the activity of NF-κB and AP-1, finally, we tested the levels of variety of IL-17 promoter's histone acetylation.The results showed that compared normal control, tranfected pre-miR 26a cells had a significantly lower level of IL-17 mRNA and RORγt mRNA, decreased the activity of NF-κB and AP-1.The proliferation of transfeced ones was lower but not statistical significance, the level of activation-induced apoptosis was significantly increasing.The results of transfected anti-miR 26a cells were converse besides the proliferation of . transfeced ones was higher.