Novel_miR218对山羊外周血单核细胞中SLAM受体表达的影响

doi:10.11843/j.issn.0366-6964.2018.11.015

摘要/Abstract

摘要：

为研究山羊源novel_miR218对小反刍兽疫病毒（PPRV）在淋巴细胞的主要受体——淋巴细胞信号活化分子（SLAM）表达水平的调节作用，通过荧光定量PCR法和Western blot技术检测PPRV疫苗毒N75-1株接种山羊外周血单核细胞（PBMCs）中SLAM和novel_miR218表达变化以及病毒增殖水平，然后检测novel_miR218模拟物（novel_miR218 mimic）和拮抗物（novel_miR218 inhibitor）转染山羊PBMCs中SLAM表达水平变化及对PPRV增殖的影响。结果表明，山羊PBMC感染PPRV后24 h （hpi）即可检测到细胞病变效应，特别在感染后48～72 hpi尤为明显；与对照组相比，PPRV感染组中SLAM受体mRNA表达水平在24 hpi极显著升高（P<0.01），随后逐渐降低，而novel_miR218表达水平在24 hpi极显著降低（P<0.01），随后逐渐升高，对照组中novel_miR218和SLAM的mRNA表达水平在各检测时间点未发现显著变化；感染组中SLAM受体的蛋白表达规律与其mRNA表达相似，而PPRV-N蛋白的表达水平呈现不断增加的趋势；novel_miR218 mimic转染组中SLAM和PPRV-N蛋白表达水平较对照组显著降低（P<0.05或P<0.01），且呈转染剂量依赖性，而novel_miR218 inhibitor转染组中SLAM和PPRV-N蛋白表达水平较对照组显著升高（P<0.05或P<0.01），且呈转染剂量依赖性。结果表明，PPRV感染山羊PBMCs中novel_miR218和SLAM表达水平具有相关性，细胞转染检测结果提示novel_miR218对SLAM受体的表达水平具有负调控作用并影响PPRV在山羊PBMCs中的复制水平。

Abstract:

In order to study the effect of novel_miR218 on the expression of signaling lymphocyte activation molecule (SLAM), the receptor of Peste des petits ruminants virus (PPRV) on goat lymphocytes, we detected the expression levels of SLAM, miR218 and viral loads in goat peripheral blood mononuclear cells (PBMCs) infected with PPRV vaccine virus N75-1 strain by using Western blot assay and quantitative PCR analysis. Then, we detected the levels of SLAM expression and viral loads in PPRV-infected goat PBMCs pre-transfected with miR218 mimic or miR218 inhibitor. The results showed that the cytopathic effect (CPE) can be detected in infected cells at 24 h post infection (hpi) with progressive increase in CPE at 48-72 hpi. In compared with the mock infected group, the expression fold of SLAM mRNA in PPRV infected group increased significantly at 24 hpi (P<0.01) followed by a gradually decreased levels, while the expression level of novel_miR218 decreased significantly at 24 hpi (P<0.01) and then gradually increased. In the uninfected control group, the expression level of novel_miR218 and SLAM mRNA showed no significant change at each detected time point. The expression pattern of SLAM protein in the infected group was similar to that of the mRNA, while an increased levels of PPRV-N protein was observed. The expression levels of SLAM and PPRV-N in miR218 mimic transfected group were significantly lower (P<0.05 or P<0.01) than that in the uninfected control group in a dose-dependent manner, while the expression levels of SLAM and PPRV-N in miR218 inhibitor transfected group were significantly higher (P<0.05 or P<0.01) than those in control group in a dose-dependent manner. This study indicated that the expression of SLAM was closely correlated with the miR218 expression in PPRV infected goat PBMCs, and the results of transfection of cells with miR218 mimic or inhibitor suggested that miR218 can negatively regulate the expression of SLAM and virus replication in PPRV infected PBMCs.

中图分类号:

S852.659.5

宋华杰, 王婷, 李珍, 王晶钰, 齐雪峰. Novel_miR218对山羊外周血单核细胞中SLAM受体表达的影响[J]. 畜牧兽医学报, 2018, 49(11): 2435-2441.

SONG Hua-jie, WANG Ting, LI Zhen, WANG Jing-yu, QI Xue-feng. The Effects of Novel_miR218 on the Expression of SLAM in Goat Peripheral Blood Mononuclear Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(11): 2435-2441.

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参考文献

[1] KUMAR P, TRIPATHI B N, SHARMA A K, et al. Pathological and immunohistochemical study of experimental peste des petits ruminants virus infection in goats[J]. J Vet Med B Infect Dis Vet Public Health, 2004, 51(4):153-159.
[2] TATSUO H, YANAGI Y. The morbillivirus receptor SLAM (CD150)[J]. Microbiol Immunol, 2002, 46(3):135-142.
[3] SEKI F, ONO N, YAMAGUCHI R, et al. Efficient isolation of wild strains of canine distemper virus in Vero cells expressing canine SLAM (CD150) and their adaptability to marmoset B95a cells[J]. J Virol, 2003, 77(18):9943-9950.
[4] PAWAR R M, RAJ G D, KUMAR T M A S, et al. Effect of siRNA mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro[J]. Virus Res, 2008, 136(1-2):118-123.
[5] ONO N, TATSUO H, TANAKA K, et al. V domain of human SLAM (CDw150) is essential for its function as a measles virus receptor[J]. J Virol, 2001, 75(4):1594-1600.
[6] VON MESSLING V, SVITEK N, CATTANEO R. Receptor (SLAM[CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus[J]. J Virol, 2006, 80(12):6084-6092.
[7] MANJUNATH S, MISHRA B P, MISHRA B, et al. Comparative and temporal transcriptome analysis of peste des petits ruminants virus infected goat peripheral blood mononuclear cells[J]. Virus Res, 2017, 229:28-40.
[8] LODISH H F, ZHOU B Y, LIU G, et al. Micromanagement of the immune system by microRNAs[J]. Nat Rev Immunol, 2008, 8(2):120-130.
[9] SCARIA V, HARIHARAN M, PILLAI B, et al. Host-virus genome interactions:Macro roles for microRNAs[J]. Cell Microbiol, 2007, 9(12):2784-2794.
[10] TENOEVER B R. RNA viruses and the host microRNA machinery[J]. Nat Rev Microbiol, 2013, 11(3):169-180.
[11] KUMAR N, MAHERCHANDANI S, KASHYAP S K, et al. Peste des petits ruminants virus infection of small ruminants:A comprehensive review[J]. Viruses, 2014, 6(6):2287-2327.
[12] STAAL S J, POOLE J, BALTENWECK I, et al. Targeting strategic investment in livestock development as a vehicle for rural livelihoods. Bill and Melinda gates foundation-ILRI knowledge generation project report[R]. Nairobi, Kenya:ILRI, 2009.
[13] BAILEY D, BANYARD A P, DASH P, et al. Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus[J]. Virus Res, 2005, 110(1-2):119-124.
[14] 蒙学莲,窦永喜,朱学亮,等. 小反刍兽疫病毒血凝素蛋白与受体蛋白SLAM的相互作用[J]. 畜牧兽医学报, 2014, 45(3):426-433.
MENG X L, DOU Y X, ZHU X L, et al. Interaction between hemagglutinin protein of Peste des Petits Ruminants virus and signalling lymphocyte activation molecule[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(3):426-433. (in Chinese)
[15] AYARI-FAKHFAKH E, GHRAM A, BOUATTOUR A, et al. First serological investigation of peste-des-petits-ruminants and Rift Valley fever in Tunisia[J]. Vet J, 2011, 187(3):402-404.
[16] SATO H, YONEDA M, HONDA T, et al. Morbillivirus receptors and tropism:Multiple pathways for infection[J]. Front Microb, 2012, 3:75.
[17] HASHIMOTO K, ONO N, TATSUO H, et al. SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein[J]. J Virol, 2002, 76(13):6743-6749.
[18] SCHNEIDER H, KAELIN K, BILLETER M A. Recombinant measles viruses defective for RNA editing and V protein synthesis are viable in cultured cells[J]. Virology, 1997, 227(2):314-322.
[19] FUJITA K, MIURA R, YONEDA M, et al. Host range and receptor utilization of canine distemper virus analyzed by recombinant viruses:Involvement of heparin-like molecule in CDV infection[J]. Virology, 2007, 359(2):324-335.
[20] ALBINA E, KWIATEK O, MINET C, et al. Peste des petits ruminants, the next eradicated animal disease?[J]. Vet Microbiol, 2013, 165(1-2):38-44.
[21] CAPOBIANCHI M R, GIOMBINI E, ROZERA G. Next-generation sequencing technology in clinical virology[J]. Clin Microbiol Infec, 2013, 19(1):15-22.
[22] KWIATEK O, ALI Y H, SAEED I K, et al. Asian lineage of peste des petits ruminants virus, Africa[J]. Emerg Infect Dis, 2011, 17(7):1223-1231.
[23] OHISHI K, ANDO A, SUZUKI R, et al. Host-virus specificity of morbilliviruses predicted by structural modeling of the marine mammal SLAM, a receptor[J]. Comp Immunol Microbiol Infect Dis, 2010, 33(3):227-241.
[24] PAWAR R M, DHINAKAR_RAJ G, BALACHANDRAN C. Relationship between the level of signaling lymphocyte activation molecule mRNA and replication of Peste-des-petits-ruminants virus in peripheral blood mononuclear cells of host animals[J]. Acta Virol, 2008, 52(4):231-236.
[25] FU Y X, ZHANG L, ZHANG F, et al. Exosome-mediated miR-146a transfer suppresses type I interferon response and facilitates EV71 infection[J]. PLoS Pathog, 2017, 13(9):e1006611.
[26] GAO L, GUO X K, WANG L H, et al. MicroRNA 181 suppresses porcine reproductive and respiratory syndrome virus (PRRSV) infection by targeting PRRSV receptor CD163[J]. J Virol, 2013, 87(15):8808-8812.
[27] BAERTSCH M A, LEBER M F, BOSSOW S, et al. MicroRNA-mediated multi-tissue detargeting of oncolytic measles virus[J]. Cancer Gene Ther, 2014, 21(9):373-380.
[28] LEBER M F, BOSSOW S, LEONARD V H, et al. MicroRNA-sensitive oncolytic measles viruses for cancer-specific vector tropism[J]. Mol Ther, 2011, 19(6):1097-1106.
[29] GEEKIYANAGE H, GALANIS E. MiR-31 and miR-128 regulates poliovirus receptor-related 4 mediated measles virus infectivity in tumors[J]. Mol Oncol, 2016, 10(9):1387-1403.
[30] VAN DRIEL B J, LIAO G X, ENGEL P, et al. Responses to microbial challenges by SLAMF receptors[J]. Front Immunol, 2016, 7:4.