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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 杨倬等 /mir373 和 mir542-5p 调控肠道病毒 71 型在人横纹肌肉瘤细胞中的复制 June 25, 2014, 30(6): Chin J Biotech, All rights reserved 医学与免疫生物技术 943 mir373 mir542-5p 71 杨倬 1, 田波 ,. mir373 mir542-5p 71. 生物工程学报, 2014, 30(6): Yang Z, Tien P. mir373 and mir542-5p regulate the replication of Enterovirus 71 in rhabdomyosarcoma cells. Chin J Biotech, 2014, 30(6): 摘要 : 研究发现 micrornas (mirnas) 可以参与调控病毒在宿主细胞内感染和复制的过程为了揭示 mirnas 是否参与肠道病毒 71 型 (Enterovirus 71, EV71) 的感染与复制, 研究了 mirnas 对 EV71 病毒在宿主细胞内复制的影响构建 mirnas 靶基因筛选系统, 在双荧光素酶报告体系的 pmir 载体插入病毒基因, 如果插入的基因序列能被细胞内的 mirnas 靶向调控, 报告基因的表达将发生变化实验发现 EV71 病毒 5 -UTR 基因可能是 mirnas 的作用靶标随后利用 mirnas 在线分析软件预测并验证可能作用于 5 -UTR 基因片段的 mirnas 为了研究 mirnas 分子对 5 -UTR 基因的调控作用是否可以体现在 EV71 病毒的复制过程中, 在人横纹肌肉瘤 (Rhabdomyosarcoma, RD) 细胞中转染 mirnas mimics, 利用 Western blotting 和 real-time PCR 实验检验 EV71 病毒的复制和表达情况实验结果表明,miR373 和 mir542-5p 可以通过作用于 EV71 病毒 5 -UTR 基因从而抑制病毒在 RD 细胞中的复制和表达细胞内 mir373 和 mir542-5p 可以调控 EV71 在宿主细胞中的复制过程研究 EV71 病毒与宿主 mirnas 的相互作用机制为进一步阐明 EV71 病毒感染与复制机理奠定了基础 : mirnas, 肠道病毒 71 型, 人横纹肌肉瘤细胞, 病毒复制 Received: November 1, 2013; Accepted: January 8, 2014 Supported by: National Natural Science Foundation of China (No ). Corresponding author: Zhuo Yang. Tel: ; zhuoer8623@163.com (No )

2 944 ISSN CN /Q Chin J Biotech June 25, 2014 Vol.30 No.6 mir373 and mir542-5p regulate the replication of Enterovirus 71 in rhabdomyosarcoma cells Zhuo Yang 1, and Po Tien 2 1 National Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing , China 2 Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing , China Abstract: MicroRNAs (mirnas) play an important role in infection and replication of virus in host cells. To identify cellular mirnas involved in the host response to enterovirus 71 (EV71) infection, we examined mirnas effects on the replication of EV71 in rhabdomyosarcoma cells. We constructed target gene of mirnas screening system. 3 untranslated region (UTR) dual luciferase reporter analysis was used to identify putative mirna targets in the EV71 virus genome. First, 13 segments of EV71 virus genomes were inserted to the pmir vector and the luciferase expression were assayed to identify the target gene of putative mirna. The reporter gene expression of the cells transfected with the vector containing 5 -UTR was significantly downregulated. Then we screened the mirnas that may target to 5 -UTR using online analysis programs. Furthermore, Western blotting and real-time PCR test were performed to investigate the effect of mirnas on viral replication. The study showed that mir373 and mir542-5p could suppress the replication of EV71 virus through binding to the 5 -UTR gene. Cellular mirnas could regulate the replication of EV71 virus in host cells, and our paper should report the role of mir373 and mir542-5p in this regulation for the first time. Our findings supported the notion that the cellular mirnas might be essential in the host-pathogen interactions. Keywords: mirnas, Enterovirus 71, rhabdomyosarcoma cells, virus replication mirnas RNA mrna mrna mirnas pri-mirna pri-mirna Drosha mirnas premirna pre-mirna Dicer RNA mirnas mirnas RNA (RISC) mrna mrna [1] mirnas [2-3] 2005 Charles-Henri Lecellier mir32 mrna Ⅰ (PFV-I) [4] 2008 Henke Chang mir122 (HCV) 5 RNA (Huh-7 ) (

3 杨倬等 /mir373 和 mir542-5p 调控肠道病毒 71 型在人横纹肌肉瘤细胞中的复制 945 HEK-293 HeLa) mir122 [5-7] (Hand foot and mouth disease HFMD) 71 (Enterovirus 71) RNA (Picrornaviridae) (Enterovirus, EV), RD HFMD EV71 CVA16 RD (CPE) mirnas EV71 mirnas EV71 mirnas EV71 5 -UTR mir373 mir542-5p EV71 mir373 mir542-5p EV71 RD mirnas EV71 1 材料与方法 1.1 材料 EV71 Hubei-09 (C4 GenBank Accession No. GU434678) (Rhabdomyosarcoma, RD) pmir prl-cmv mirnas mir373 mir542-5p mimics mir-ribotm mimics Negative control SpeⅠ Hind Ⅲ T4 DNA Pyrobest DNA TaKaRa EV71 (MAB979) Millipore β-actin IgG RNA Trizol Invitrogen Real time PCR Promega Lipofectamine 2000 Invitrogen Dulbecco's modified eagle medium (DMEM) (FBS) Gibco BRL PCR Bio-Rad Real time PCR ABI 1.2 mirnas 靶基因筛选系统的建立 mirnas 3 -UTR pmir prl-cmv pmir 3 -UTR prl-cmv EV71 Hubei-09 (GenBank Accession No. GU ) EV71 cdna EV UTR VP4 VP2 VP3 VP1 2A 2B 2C 3A 3BC 3D1 3D2 3 UTR 5 -UTR 3 5 -UTR pmir 3 -UTR SpeⅠ/Hind cjb@im.ac.cn

4 946 ISSN CN /Q Chin J Biotech June 25, 2014 Vol.30 No.6 pmir-ev71-(5 UTR VP4 VP2 VP3 VP1 2A 2B 2C 3A 3BC 3D1 3D2 3 UTR 5 UTR-1 5 UTR-2 5 UTR-3) 1 表 1 EV71 病毒基因片段的 PCR 扩增引物 Table 1 Primer sequences of EV71 gene segments for PCR Gene Nucleotide location Sequence UTR Forward primer: GCACTAGT TTAAAACAGC CTGTGGGTTGC Reverse primer: GCAAGCTT CGCTTCGTGTTCA GCAGTATAAT VP Forward primer: GCACTAGT ATGGGCT CCCAGGTCTCC Reverse primer: GCAAGCTT CTTTAAGGGCGCTGCCATTT VP Forward primer: GCACTAGT TCTCCCTCTGCTGAAGCATGTG Reverse primer: GCAAGCTT CTGCGTAACTGC CTGCCTG VP Forward primer: GCACTAGT GGTTTCCCCACTGAATTGAAAC Reverse primer: GCAAGCTT TTGAATAGTGGCCGTTTGCA VP Forward primer: GCACTAGT GGGGACAGAGTG GCAGATGT Reverse primer: GCACGCGT GAGCGTAGTGATTGCCGTTC 2A Forward primer: GCACTAGT GGGAAATTCGGTCAGCAGTCT Reverse primer: GCAAGCTT CTGCTCCATCGCTTCCTCAT 2B Forward primer: GCACTAGT GGGGTATCTGATTACATCAAAGGTC Reverse primer: GCAAGCTT CTGTTTTTGTGCCATGGGAAT 2C Forward primer: GCACTAGT AGTG CCTCATGGCTAAAGAAGTT Reverse primer: GCACGCGT TTGAAAGAGTGCTTCTATAGTATTC 3A Forward primer: GCACTAGT GGACCCCCTAAATTCAGGCCT Reverse primer: GCAAGCTT CTGGAACCCGGCAAACAG 3B+3C Forward primer: GCACTAGT GGTGCTTACTCTGGAGCGC Reverse primer: GCAAGCTT TTGCTCGCTGGCAAAA TAAC 3D Forward primer: GCGAGCTC GGAGAGATCCAGTGGATGAAGC Reverse primer: GCACGCGT TAAGGCTTGCATCATATCCTGAG 3D Forward primer: GCACTAGT GTCCTGTGTGGTTCAGAGCTCT Reverse primer: GCAAGCTT AAATAACTCGAGCCAATTTCTTCTC 3 -UTR Forward primer: GCACTAGT ACTTACAGCTCAATGCTGAAC Reverse primer: GCAAGCTT GCTATTCTGGTTATAACAAATTTAC 5 -UTR Forward primer: GCACTAGT TTAAAACAGC CTGTGGGTTGC Reverse primer: GCAAGCTTCGTTACTAGGTTTCTCGAAGTAGTTAGCC 5 -UTR Forward primer: GCACTAGTTTCGAGAAACCTAGTAACGCCATTG Reverse primer: GCAAGCTTAGTTGCCCGTTACGACACACTGCCA 5 -UTR Forward primer: GCACTAGT CCAAAGGGCAGTGTGTCGTAAC Reverse primer: GCAAGCTT CGCTTCGTGTTCA GCAGTATAAT

5 杨倬等 /mir373 和 mir542-5p 调控肠道病毒 71 型在人横纹肌肉瘤细胞中的复制 mirnas ViTa mirnas miranda TargetScan mirnas MicroInspector mirnas mirnas rna22 RNA Hybrid RNA MicroInspector ViTa mirnas rna22 RNA Hybird RD 24 pmir prl-cmv Lipofectamine 2000 RD 30 h PBS μl 10 min r/min 2 min Promega pmir mirnas 1.3 Western blotting 检测 RD 10% 100 μg/ml 100 μg/ml DMEM 37 5% CO 2 80% 90% 2.5% RD DMEM r/min 5 min DMEM /ml 1 ml/ h mirnas mimics mir-ribotm mimics Negative control RD 6 h 10 μl TCID EV71 48 h 20 min r/min 10 min 15% SDS-PAGE PVDF 10% EV β-actin IgG ECL 1.4 Real-time PCR 检测 2.5% RD DMEM /ml 1 ml/ h mirnas mimics mir-ribotm mimics Negative control RD 6 h 10 μl TCID EV71 48 h Trizol RNA RNA 150 ng cdna Primer 5.0 EV71 β-actin Real-time PCR ( 2) Promega Real-Time PCR Real-time PCR min s 60 1 min 40 PCR 2 ΔΔCt 1.5 数据分析 x ±s t P<0.05 cjb@im.ac.cn

6 948 ISSN CN /Q Chin J Biotech June 25, 2014 Vol.30 No.6 2 结果 2.1 双荧光素酶报告系统的构建 pmir 3 -UTR prl-cmv 1 EV UTR VP4 VP2 VP3 VP1 2A 2B 2C 3A 3BC 3D1 3D2 3 UTR pmir 3 -UTR SpeⅠ/Hind Ⅲ pmir-ev71-(5 UTR VP4 VP2 VP3 VP1 2A 2B 2C 3A 3BC 3D1 3D2 3 UTR) mirnas 表 2 Real-time PCR 反应中用于扩增 EV 71 vp1 和 β-actin 的引物 Table 2 Primers used for detection of EV 71 vp1 and β-actin in Real-time PCR assay Gene name Forward primer sequence (5 3 ) Reverse primer sequence (5 3 ) EV71 vp1 AGTATGATTGAGACTCGGTG GCGACAAAAGTGAACTCTGC β-actin ACCCACACTGTGCCCATCTACGA GCCGTGGTGGTGAAGCTGTAGCC 图 1 双荧光素酶报告系统构建 Fig. 1 Construction of Luciferase dual reporter analysis system for selection of mirnas putative targets. Circular map of the pmir vector. The EV71 genes were inserted into the SpeⅠ/Hind sites. prl-cmv vector was used as control.

7 杨倬等 /mir373 和 mir542-5p 调控肠道病毒 71 型在人横纹肌肉瘤细胞中的复制 EV71 病毒的 5 -UTR 基因片段是宿主细胞 mirnas 的作用靶点 pmir prl-cmv 24 RD 30 h Luciferase 2 pmir pmir-5 UTR mirnas 5 -UTR mirnas EV71 5 -UTR 3 pmir pmir-ev71-(5 UTR-1 5 UTR-2 5 UTR-3) 3 pmir-5 UTR-1 5 UTR-1 mirnas 2.3 靶向 5 UTR-1 基因片段的 mirnas 的预测 mirnas pmir-5 UTR-1 MicroInspector ViTa 5 -UTR-1 mirnas rna22 RNA (Hybrid online program) mir373 mir542-5p EV71 5 -UTR-1 mirnas 5 -UTR-1 3 mir373 mir542-5p5 5 -UTR mir373 和 mir542-5p 对 pmir-5 UTR-1 载体荧光素酶活性的影响 mir373 mir542-5p mimics mir-ribotm mimics Negative control pmir-5 UTR-1 prl-cmv RD 30 h Luciferase mir373 mir542-5p ( 4) mir373 mir542-5p EV71 5 -UTR-1 图 2 EV71 病毒 13 个基因片段重组 pmir 载体的相对荧光素酶活性比较 Fig. 2 Relative luciferase activity from the pmir reporter constructs carrying one of 13 fragments of EV71 and prl-cmv vector. Both prl-cmv and pmir vector were transfected into RD cell, 30 h after transfection,dual luciferase activity were assayed. pmir vector without gene inserted was used as control. n=3.*p<0.05. cjb@im.ac.cn

950 ISSN 1000-3061 CN 11-1998/Q Chin J Biotech June 25, 2014 Vol.30 No.6 图 3 EV71 病毒 5 -UTR 3 个亚基因片段重组 pmir 载体的相对荧光素酶活性比较 Fig.

Both prl-cmv and pmir vector were transfected into RD cell, 30 h after transfection, dual luciferase activity were assayed. pmir vector without gene inserted was used as control. n=3. *P<0.05.

Hybridization location mir-373 GAAGUGCUUCGAUUUGGGGUGU 155 176 mir-542-5p UCGGGGAUCAUCAUGUCACGAGA 159 187 图 4 mir373 和 mir542-5p 对 pmir-5 UTR-1 载体荧光素酶活性的影响 Fig.

8 950 ISSN CN /Q Chin J Biotech June 25, 2014 Vol.30 No.6 图 3 EV71 病毒 5 -UTR 3 个亚基因片段重组 pmir 载体的相对荧光素酶活性比较 Fig. 3 Relative luciferase activity from the pmir reporter constructs carrying fragments from the dissection of the 5 -UTR gene and prl-cmv vector. Both prl-cmv and pmir vector were transfected into RD cell, 30 h after transfection, dual luciferase activity were assayed. pmir vector without gene inserted was used as control. n=3. *P<0.05. 表 3 mir373 和 mir542-5p 序列及与 5 -UTR-1 基因片段的互补配对模式 Table 3 Model of hybridization between the mir373, mir542-5p and 5 UTR-1 mrna were predicted using RNA Hybrid software mirnas Sequence (5 3 ) Target Hybridization location mir-373 GAAGUGCUUCGAUUUGGGGUGU mir-542-5p UCGGGGAUCAUCAUGUCACGAGA 图 4 mir373 和 mir542-5p 对 pmir-5 UTR-1 载体荧光素酶活性的影响 Fig. 4 The relative luciferase activity from pmir-5 UTR-1 and prl-cmv vector in RD cells with overexpressing mir373 and mir542-5p. mir-ribotm mimics Negative control was used as control, n=3. *P< mir373 和 mir542-5p 调控 EV71 在 RD 细胞中病毒蛋白的表达 mir373 mir542-5p 5 -UTR-1 EV71 mir373 mir542-5p mimics mir-ribotm mimics Negative control 12 RD 48 h Western blotting EV71 β-actin EV71 5 mir373 mir542-5p EV71 RD 2.6 mir373 和 mir542-5p 调控 EV71 在 RD 细胞中病毒 mrna 的表达 mir373 mir542-5p EV71 mir373 mir542-5p mimics mir-ribotm mimics Negative control 12 RD 48 h Trizol

9 杨倬等 /mir373 和 mir542-5p 调控肠道病毒 71 型在人横纹肌肉瘤细胞中的复制 951 RNA cdna Real-time PCR mir373 mir542-5p EV71 mrna 6 mir373 mir542-5p EV71 mrna 47.59%±4.89% 61.41%±2.69% mir373 mir542-5p EV71 mrna RD Western blotting 3 讨论图 5 Western blotting 检测 mir373 和 mir542-5p 对 EV71 在 RD 细胞中病毒蛋白表达的影响 Fig. 5 Western blotting analysis of the effects of mir373 and mir542-5p on EV71 expression in RD cell. The lysates of total cells infected with EV71 were collected and analyzed for VP1 protein. Its decrease was determined using EV71 monoclonal antibody.the Beta-actin was used as an internal control and demonstrated equal loading of total protein. mir-ribotm mimics Negative control was used as control. 图 6 Real-time PCR 检测 mir373 和 mir542-5p 对 EV71 在 RD 细胞中病毒 mrna 表达的影响 Fig. 6 Real-time PCR analysis of the effects of mir373 and mir542-5p on EV71 mrna in RD cell. Cultured supernatants of RD cells infected with EV71were taken off at 48 h post-infection and viral mrna of the rest cells was extracted by means of TRIzol, and then subjected to a Real-time PCR relative quantification assay. The viral RNA transcripts level in control was set as 100%. mir-ribotm mimics Negative control was used as control. The data shown represent the mean percentage of RNA transcripts ± s from three independent experiments. *P < mirnas Epstein-Barr mir155 mir155 Epstein-Barr [6] EB LMP1 NF-κB mir146a mir146a EB [8] mirnas mirnas mirnas HIV HIV mirnas mir29a mir29b nef mir149 vpr mir378 env mir324-5p vif [9] mir32 Ⅰ (PFV-I) mirnas HCV mirnas Dicer [10] HIV-1 Tat cjb@im.ac.cn

10 952 ISSN CN /Q Chin J Biotech June 25, 2014 Vol.30 No.6 Dicer [11] RNA Haasnoot [12] Vero NS1 sirna Luciferase RNA mirnas HCMV mirul IE72 [13] HIV-1 CD4 CD28 Zheng [19] EV71 mir-296-5p mir-296-5p mirnas mir-23b Wen [20] mir-23b EV71 mir-23b EV71 3 UTR VP1 EV71 Li [21] mirna-548 IFN-λ1 3 UTR IFN-λ1 mirna-548 IL-2 IL-12 mirnas EV71 [14-15] mirnas Jopling [16] mir122 HCV 5 RNA Cui EV71 Hep2 EV71 mirnas [17] 64 mirna 2 (Gene ontology) EV71 mirna EV71 mirnas Ho mir-141 elf4e [18] RD EV71 PCR mirnas mir-141 mir-146a 14 mirnas mirna mirna-548 mirna-548 IFN-λ1 mir373 mir542-5p EV71 5 UTR EV71 EV71 REFERENCES [1] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004, 116(2): [2] Lynam-Lennon N, Maher SG, Reynolds JV. The roles of microrna in cancer and apoptosis. Biol Rev Camb Philos Soc, 2009, 84(1): [3] Schickel R, Boyerinas B, Park SM, et al. MicroRNAs: key players in the immune system, differentiation, tumorigenesis and cell death. Oncogene, 2008, 27(45): [4] Lecellier CH, Dunoyer P, Arar K, et al. A cellular microrna mediates antiviral defense in human cells. Science, 2005, 308(5721): [5] Henke JI, Goergen D, Zheng J, et al.

11 杨倬等 /mir373 和 mir542-5p 调控肠道病毒 71 型在人横纹肌肉瘤细胞中的复制 953 MicroRNA-122 stimulates translation of hepatitis C virus RNA. EMBO J, 2008, 27(24): [6] Yin Q, McBride J, Fewell C, et al. MicroRNA-155 is an Epstein-Barr virus-induced gene that modulates Epstein-Barr virus-regulated gene expression pathways. J Virol, 2008, 82(11): [7] Chang J, Guo JT, Jiang D, et al. Block TM. Liver-specific microrna mir-122 enhances the replication of hepatitis C virus in nonhepatic cells. J Virol, 2008, 82(16): [8] Motsch N, Pfuhl T, Mrazek J, et al. Epstein-Barr virus-encoded latent membrane protein 1(LMP1) induces the expression of the cellular microrna mir-146a. RNA Biol, 2007, 4(3): [9] Hariharan M, Scaria V, Pillai B, et al. Targets for human encoded micrornas in HIV genes. Biochem Biophys Res Commun, 2005, 337(4): [10] Chen W, Zhang Z, Chen J, et al. HCV core protein interacts with Dicer to antagonize RNA silencing. Virus Res, 2008, 133(2): [11] Bennasser Y, Jeang KT. HIV-1 Tat interaction with Dicer: requirement for RNA. Retrovirology, 2006, 3: [12] Haasnoot J, de Vries W, Geutjes EJ, et al. The Ebola virus VP35 protein is a suppressor of RNA silencing. PLoS Pathog, 2007, 3(6): [13] Grey F, Meyers H, White EA, et al. A human cytomegalovirus-encoded microrna regulates expression of multiple viral genes involved in replication. PLoS Pathog, 2007, 3(11): [14] Bennasser Y, Le SY, Yeung ML, et al. HIV-1 encoded candidate micro-rnas and their cellular targets. Retrovirology, 2004, 1: [15] Couturier JP, Root-Bernstein RS. HIV may produce inhibitory micrornas (mirnas) that block production of CD28, CD4 and some interleukins. J Theor Biol, 2005, 235(2): [16] Jopling CL, Yi M, Lancaster AM, et al. Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science, 2005, 309(5740): [17] Cui LB, Guo XL, Qi YH, et al. Identification of micrornas involved in the host response to entervirus 71 infection by deep sequencing approach. J Biomed Biotechnol, 2010, 2010: [18] Ho BC, Yu SL, Chen JJ, et al. Enterovirus-induced mir-141 contributes to shutoff of host protein translation by targeting the translation initiation factore IF4E. Cell Host Microbe, 2011, 9(1): [19] Zheng Z, Ke X, Wang M, et al. Human microrna hsa-mir-296-5p suppresses enterovirus 71 replication by targeting the viral genome. J Virol, 2013, 87(10): [20] Wen BP, Dai HJ, Yang YH, et al. MicroRNA-23b inhibits enterovirus 71 replication through downregulation of EV71 VPl protein. Intervirology, 2013, 56(3): [21] Li Y, Xie J, Xu X, et al. MicroRNA-548 down-regulates host antiviral response via direct targeting of IFN-λ1. Protein Cell, 2013, 4(2): ( 本文责编陈宏宇 ) cjb@im.ac.cn

EV Allander [1] Human BocavirusHBoV 4 HBoV1-4 HBoV1 HBoV2-4 HBoV2 HBoV3 2 4 HBoV HBoV B19 973, : ( EV71 1 RNA mir-296 EV71 microrna mir

EV Allander [1] Human BocavirusHBoV 4 HBoV1-4 HBoV1 HBoV2-4 HBoV2 HBoV3 2 4 HBoV HBoV B19 973, : ( EV71 1 RNA mir-296 EV71 microrna mir 2008 3 EV71 19 16 84.2% EV71 EV71 RNA mir-296 EV71 EV71 HBoV1 NP IFN HBoV1 NS1 NS1-70 NF-κB HBoV1 VP2 RIG-I Journal of Immunology,Journal of Virology, ACS Nano, Biomaterials 30 8 6.83 Nature ACS NanoBiomaterialsJIVJI