原发性胆汁性肝硬化患者外周血T细胞microRNA表达谱及其调控机制的研究
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摘要
本研究旨在探讨原发性胆汁性肝硬化中T细胞microRNA(miRNA)表达谱及其调控机制。首先采用芯片和定量PCR技术分析并验证原发性胆汁性肝硬化中T细胞miRNA的表达谱;然后培养自身抗原PDC-E2特异性T细胞,研究特定miRNA对其自身反应性的调控作用;接着建立原发性胆汁性肝硬化小鼠模型,研究特定miRNA对小鼠PBC样病变的调控作用;最后通过生物信息学预测和生物学实验验证,探讨特定miRNA可能的靶基因及其作用位点。结果表明,与正常对照组相比,PBC患者T细胞中有23个miRNA低表达,仅有2个miRNA高表达;miR-1et-7b、miR-17-5p、miR-20a和miR-346表达可抑制自身抗原特异性T细胞增殖和IFN-γ、TNF-α的分泌,而miR-129和miR-451可促进细胞增殖和IFN-γ、TNF-α的分泌;在小鼠中输入Ad-miR-17-5p或Ad-miR-346可延缓由poly I:C诱发产生的PBC样病变;miR-17-5p、miR-346的靶基因分别为TNFRSF21、BCL-6,作用位点分别为TNFRSF21 3'UTR区335-342位和BCL-63'UTR区576-583及893-900位。
To explore the RNA mechanism of PBC,we focus on the microRNA(miRNA) profile in the peripheral blood T cells of PBC and study on the action mechanism of miRNA in PBC.We first explored changes in the miRNA expression profile in the peripheral blood T cells of PBC patients by microarray and real-time PCR,then investigated the regulatory effects of some miRNA abnormally expressed in PBC on the proliferation of PDC-E2-specific autoreactive T cells.Subsequently,the effects of miRNA on the pathology of PBC disease were studied in animal models.Finally,miRNA target genes were predicted by bioinformatics and the effect of miRNA on the function of target genes was determined.The results showed that Twenty-three miRNA in the array were found to be expressed in low levels and two miRNA had high levels of expression in the peripheral blood T cells of patients with PBC.The proliferation and secretion of IFN-γ,TNF-αof autoantigen-specific T cells were inhibited by miR-let-7b,miR-17-5p,miR-20a,miR-346 and promoted by miR-129,miR-451.Injecting poly I:C into syngenic wild-type mice produced PBC-like pathological changes,which were alleviated following injection of Ad-miR-17-5p or Ad-miR-346.The target gene of miR-17-5p,miR-346 is TNFRSF21, BCL-6.The interaction sites are TNFRSF21 3' UTR 335-342,BCL-6 3' UTR 576-583 and 893-900.
To explore the RNA mechanism of PBC,we focus on the microRNA(miRNA) profile in the peripheral blood T cells of PBC and study on the action mechanism of miRNA in PBC.We first explored changes in the miRNA expression profile in the peripheral blood T cells of PBC patients by microarray and real-time PCR,then investigated the regulatory effects of some miRNA abnormally expressed in PBC on the proliferation of PDC-E2-specific autoreactive T cells.Subsequently,the effects of miRNA on the pathology of PBC disease were studied in animal models.Finally,miRNA target genes were predicted by bioinformatics and the effect of miRNA on the function of target genes was determined.The results showed that Twenty-three miRNA in the array were found to be expressed in low levels and two miRNA had high levels of expression in the peripheral blood T cells of patients with PBC.The proliferation and secretion of IFN-γ,TNF-αof autoantigen-specific T cells were inhibited by miR-let-7b,miR-17-5p,miR-20a,miR-346 and promoted by miR-129,miR-451.Injecting poly I:C into syngenic wild-type mice produced PBC-like pathological changes,which were alleviated following injection of Ad-miR-17-5p or Ad-miR-346.The target gene of miR-17-5p,miR-346 is TNFRSF21, BCL-6.The interaction sites are TNFRSF21 3' UTR 335-342,BCL-6 3' UTR 576-583 and 893-900.
引文
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9. P Guglielmelli, L Tozzi, A Pancrazzi et al. MicroRNA expression profile in granulocytes from primary myelofibrosis patients. Exp Hematol 2007; 35(11):1708-18.
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14. S Shimoda, K Harada, H Niiro et al. Biliary epithelial cells and primary biliary cirrhosis: The role of liver-infiltrating mononuclear cells. Hepatology 2008.
15. S Shimoda, F Ishikawa, T Kamihira et al. Autoreactive T-cell responses in primary biliary cirrhosis are promflammatory whereas those of controls are regulatory. Gastroenterology 2006; 131(2):606-18.
16. E van Rooij, EN Olson. MicroRNAs: powerful new regulators of heart disease and provocative therapeutic targets. J Clin Invest 2007; 117(9):2369-76.
17. S Latinovic-Golic, M Walch, H Sundstrom, C Dumrese, P Groscurth, U Ziegler. Expression, processing and transcriptional regulation of granulysin in short-term activated human lymphocytes. BMC Immunol 2007; 8:9.
18. EJ Heathcote. Management of primary biliary cirrhosis. The American Association for the Study of Liver Diseases practice guidelines. Hepatology 2000; 31(4): 1005-13.
19. MM Kaplan, ME Gershwin. Primary biliary cirrhosis. N Engl J Med 2005; 353(12): 1261-73.
20. XS He, AA Ansari, WM Ridgway, RL Coppel, ME Gershwin. New insights to the immunopathology and autoimmune responses in primary biliary cirrhosis. Cell Immunol 2006;239(1):1-13.
21. H Kita, S Matsumura, XS He et al. Quantitative and functional analysis of PDC-E2-specific autoreactive cytotoxic T lymphocytes in primary biliary cirrhosis. J Clin Invest 2002; 109(9): 1231-40.
22. DJ Guarnieri, RJ Dileone. MicroRNAs: A new class of gene regulators. Ann Med 2008; 40(3): 197-208.
23. J Takamizawa, H Konishi, K Yanagisawa et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 2004; 64(11):3753-6.
24. O Slaby, M Svoboda, P Fabian, T Smerdova, D Knoflickova, M Bednarikova, R Nenutil, R Vyzula. Altered expression of miR-21, miR-31, miR-143 and miR-145 is related to clinicopathologic features of Colorectal cancer. Oncology 2007; 72(5-6):397-402.
25. J Kluiver, E Haralambieva, D de Jong, T Blokzijl, S Jacobs, BJ Kroesen, S Poppema, A van den Berg. Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt lymphoma. Genes Chromosomes Cancer 2006; 45(2): 147-53.
26. MV Iorio, M Ferracin, CG Liu et al. MicroRNA gene expression deregulation in human breast cancer. Cancer Res 2005; 65(16):7065-70.
27. GM Kasof, JJ Lu, D Liu, B Speer, KN Mongan, BC Gomes, MV Lorenzi. Tumor necrosis factor-alpha induces the expression of DR6, a member of the TNF receptor family, through activation of NF-kappaB. Oncogene 2001; 20(55):7965-75.
28. M Miozzo, C Selmi, B Gentilin et al. Preferential X chromosome loss but random inactivation characterize primary biliary cirrhosis. Hepatology 2007; 46(2):456-62.
29. H Miyakawa, A Tanaka, C Selmi et al. Serum reactivity against bacterial pyruvate dehydrogenase: increasing the specificity of anti-mitochondrial antibodies for the diagnosis of primary biliary cirrhosis. Clin Dev Immunol 2006; 13(2-4):289-94.
30. DP Bogdanos, H Baum, A Grasso et al. Microbial mimics are major targets of crossreactivity with human pyruvate dehydrogenase in primary biliary cirrhosis. J Hepatol 2004; 40(1):31-9.
31. T Dalmay. MicroRNAs and cancer. J Intern Med 2008; 263(4):366-75.
32. AJ Lowery, N Miller, RE McNeill, MJ Kerin. MicroRNAs as prognostic indicators and therapeutic targets: potential effect on breast cancer management. Clin Cancer Res 2008; 14(2):360-5.
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