miRNA在乌鳢水泡病毒感染月鳢细胞(SSN-1)中的表达变化及其调控作用
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摘要
为了探究microRNA(miRNA)对乌鳢水泡病毒(snakehead vesiculovirus, SHVV)感染月鳢细胞(striped snakehead cell, SSN-1)的影响,本研究对SHVV感染的SSN-1细胞及未感染的细胞进行miRNA高通量测序。分别用U6、β-actin和5S rRNA作为内参基因研究SHVV感染对细胞内22个高丰度(transcripts per million, TPM≥1 000) miRNA表达水平的影响,结果显示,U6基因作为内参时,SHVV感染对β-actin没有显著影响,但是5S rRNA显著上调表达,说明U6和β-actin基因适合作为内参基因研究SHVV感染对miRNA表达水平的影响。此外,我们研究了14种miRNA对SHVV增殖的影响。结果发现,miR-27a-3p、miR-26a-5p、miR-30e-3p等11种miRNA显著促进SHVV增殖,miR-150和miR-216b显著抑制SHVV增殖。进一步研究发现高丰度的miR-100-5p在SHVV感染SSN-1细胞早期上调表达,晚期下调表达。过表达miR-100-5p可以显著抑制SHVV的增殖,而抑制表达miR-100-5p可以显著促进SHVV的增殖。本研究为开发抗SHVV的核酸药物提供了理论基础。
In order to study the effects of miRNAs in stripped snakehead(SSN-1) cell line upon snakehead vesiculovirus(SHVV) infection, SHVV-infected and mock-infected SSN-1 cells were analyzed by miRNA highthroughput sequencing. The expression levels of twenty-two miRNAs with levels equal to or higher than 1 000 reads per million(TPM≥1 000) were determined by qRT-PCR with U6, β-actin, and 5 S rRNA as internal control.When U6 gene was used as internal control, no significant changes were observed for β-actin upon SHVV infection, while 5 S rRNA was upregulated in SSN-1 cells upon SHVV infection. The results showed that U6 gene or β-actin could be used as internal control genes for miRNAs in SSN-1 cells. Furthermore, fourteen miRNAs were selected and their effects on SHVV replication were analyzed. The results showed that eleven miRNAs(miR-27 a-3 p, miR-26 a-5 p, miR-30 e-3 p, etc.) promoted, whereas miR-150 and miR-216 b suppressed, SHVV replication.Next, we found that high-expression miR-100-5 p was upregulated at early stage of SHVV infection, while downregulated at later stage. Overexpression of miR-100-5 p exhibited significant suppression of SHVV replication, while inhibition of expression of miR-100-5 p promoted SHVV replication. Our findings presented valuable information for the study of molecular drugs against SHVV.
In order to study the effects of miRNAs in stripped snakehead(SSN-1) cell line upon snakehead vesiculovirus(SHVV) infection, SHVV-infected and mock-infected SSN-1 cells were analyzed by miRNA highthroughput sequencing. The expression levels of twenty-two miRNAs with levels equal to or higher than 1 000 reads per million(TPM≥1 000) were determined by qRT-PCR with U6, β-actin, and 5 S rRNA as internal control.When U6 gene was used as internal control, no significant changes were observed for β-actin upon SHVV infection, while 5 S rRNA was upregulated in SSN-1 cells upon SHVV infection. The results showed that U6 gene or β-actin could be used as internal control genes for miRNAs in SSN-1 cells. Furthermore, fourteen miRNAs were selected and their effects on SHVV replication were analyzed. The results showed that eleven miRNAs(miR-27 a-3 p, miR-26 a-5 p, miR-30 e-3 p, etc.) promoted, whereas miR-150 and miR-216 b suppressed, SHVV replication.Next, we found that high-expression miR-100-5 p was upregulated at early stage of SHVV infection, while downregulated at later stage. Overexpression of miR-100-5 p exhibited significant suppression of SHVV replication, while inhibition of expression of miR-100-5 p promoted SHVV replication. Our findings presented valuable information for the study of molecular drugs against SHVV.
引文
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[27] Yang L, Yang G, Zhang X B. The miR-100-mediated pathway regulates apoptosis against virus infection in shrimp[J]. Fish&Shellfish Immunology, 2014, 40(1):146-153.
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[34] Zhu X, He Z J,Hu Y W, et al. MicroRNA-30e*suppresses dengue virus replication by promoting NF-κBdependent IFN production[J]. Virus Research, 2014,8(8):e3088.
[35] Li Z H, Chen B B, Feng M, et al. MicroRNA-23b promotes avian leukosis virus subgroup J(ALV-J)replication by targeting IRF1[J]. Virus Research, 2015, 5:10294.
[36] Zhang Q, Huang C, Yang Q, et al. MicroRNA-30c modulates type I IFN responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting JAK1[J]. The Journal of Immunology, 2016,196(5):2272-2282.
[37] Ge Y Y, Shi Q, Zheng Z Y, et al. MicroRNA-100promotes the autophagy of hepatocellular carcinoma cells by inhibiting the expression of mTOR and IGF-1R[J]. Virus Research,2014, 5(15):6218-6228.
[38] Lin Y, Deng W Y, Pang J K, et al. The microRNA-99family modulates hepatitis B virus replication by promoting IGF-IR/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy[J]. Cellular Microbiology,2017,19(5):e12709.
[39] Wang F Z, Weber F, Croce C, et al. Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication[J]. Journal of Virology, 2008, 82(18):9065-9074.
[2] Zheng Q L, Hou J, Zhou Y, et al. Type I IFN-inducible downregulation of microRNA-27a feedback inhibits antiviral innate response by upregulating siglecl/TRIM-27[J]. The Journal of Immunology, 2016, 196(3):1317-1326.
[3] Zhao Y, Srivastava D. A developmental view of microRNA function[J]. Trends in Biochemical Sciences,2007,32(4):189-197.
[4] Bartel D P. MicroRNAs:genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2):281-297.
[5] He L, Hannon G J. MicroRNAs:small RNAs with a big role in gene regulation[J]. Nature Reviews Genetics,2004,5(7):522-531.
[6] Pasquinelli A E. MicroRNAs and their targets:recognition, regulation and an emerging reciprocal relationship[J]. Nature Reviews Genetics, 2012, 13(4):271-282.
[7] Cullen B R. How do viruses avoid inhibition by endogenous cellular microRNAs?[J]. PLoS Pathogens, 2013,9(11):e1003694.
[8] Zhu B B, Ye J, Ashraf U, et al. Transcriptional regulation of miR-15b by c-Rel and CREB in Japanese encephalitis virus infection[J]. Scientific Reports, 2016, 6:22581.
[9] Kim G W, Lee S H, Cho H, et al. Hepatitis C virus core protein promotes miR-122 destabilization by inhibiting GLD-2[J]. PLoS Pathogens, 2016, 12(7):e1005714.
[10] Xu X P, Yuan J, Yang L W, et al. The Dorsal/miR-1959/cactus feedback loop facilitates the infection of WSSV in Litopenaeus vannamei[J]. Fish&Shellfish Immunology,2016, 56:397-401.
[11] Slonchak A, Shannon R P, Pali G, et al. Human MicroRNA miR-532-5p exhibits antiviral activity against west nile virus via suppression of host genes SESTD1and TAB 3 required for virus replication[J]. Journal of Virology, 2015, 90(5):2388-2402.
[12] Kaewkascholkul N, Somboonviwat K, Asakawa S, et al.Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection[J]. Developmental&Comparative Immunology, 2016, 60:191-201.
[13] Chen Z, Ye J, Ashraf U, et al. MicroRNA-33a-5p modulates Japanese encephalitis virus replication by targeting eukaryotic translation elongation factor 1A1[J].Journal of Virology, 2016, 90(7):3722-3734.
[14] Zhu B, Ye J, Nie Y, et al. MicroRNA-15b modulates Japanese encephalitis virus-mediated inflammation via targeting RNF125[J]. The Journal of Immunology, 2015,195(5):2251-2262.
[15] Zhang Q, Guo X K, Gao L, et al. MicroRNA-23 inhibitsPRRSV replication by directly targeting PRRSV RNA and possibly by upregulating type I interferons[J]. Virology, 2014,450-451:182-195.
[16] Ashraf U, Zhu B B, Ye J, et al. MicroRNA-19b-3p modulates Japanese encephalitis virus-mediated inflammation via targeting RNF11[J]. Journal of Virology, 2016,90(9):4780-4795.
[17] Qian J, Zhai A X, Kao W P, et al. Modulation of miR-122 on persistently Borna disease virus infected human oligodendroglial cells[J]. Antiviral Research, 2010,87(2):249-256.
[18] Zhai A X, Qian J, Kao W P, et al. Borna disease virus encoded phosphoprotein inhibits host innate immunity by regulating miR-155[J]. Antiviral Research,2013,98(1):66-75.
[19] Henke J I, Goergen D, Zheng J F, et al. microRNA-122stimulates translation of hepatitis C virus RNA[J]. The EMBO Journal,2008, 27(24):3300-3310.
[20] Liu X D, Wen Y, Hu X Q, et al. Breaking the host range:mandarin fish is susceptible to a vesiculovirus derived from snakehead fish[J]. Journal of General Virology,2015, 96(4):775-781.
[21] Wang W W, Asim M, Yi L Z, et al. Abortive infection of snakehead fish vesiculovirus in ZF4 cells was associated with the RLRs pathway activation by viral replicative intermediates[J]. International Journal of Molecular Sciences,2015, 16(3):6235-6250.
[22] Wang P, Hou J, Lin L, et al. Inducible microRNA-155feedback promotes typeⅠIFN signaling in antiviral innate immunity by targeting suppressor of cytokine signaling 1[J]. The Journal of Immunology, 2010, 185(10):6226-6233.
[23] Liu X D, Tu J G, Yuan J F, et al. Identification and characterization of microRNAs in snakehead fish cell line upon snakehead fish vesiculovirus infection[J]. International Journal of Molecular Sciences, 2016, 17(2):154.
[24] Mullokandov G, Baccarini A, Ruzo A, et al. Highthroughput assessment of microRNA activity and function using microRNA sensor and decoy libraries[J].Nature Methods, 2012, 9(8):840-846.
[25] Selitsky S R, Baran-Gale J, Honda M, et al. Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C[J]. Scienti-fic Reports, 2015, 5:7675.
[26] Wang Z,Zhu F. MicroRNA,100 is involved in shrimp immune response to white spot syndrome virus(WSSV)and Vibrio alginolyticus infection[J]. Scientific Reports,2017, 7:42334.
[27] Yang L, Yang G, Zhang X B. The miR-100-mediated pathway regulates apoptosis against virus infection in shrimp[J]. Fish&Shellfish Immunology, 2014, 40(1):146-153.
[28] Lu M, Zhang P J, Li C H, et al. MiR-31 modulates coelomocytes ROS production via targeting p105 in Vibrio splendidus challenged sea cucumber Apostichopus japonicus in vitro and in vivo[J]. Fish&Shellfish Immunology, 2015,45(2):293-299.
[29] Li G X, Zhao Y L, Wen L, et al. Identification and characterization of microRNAs in the spleen of common carp immune organ[J]. Journal of Cellular Biochemistry,2014, 115(10):1768-1778.
[30] Nicoli S, Knyphausen C P, Zhu L J, et al. miR-221 is required for endothelial tip cell behaviors during vascular development[J]. Developmental Cell, 2012, 22(2):418-429.
[31]项荣,梁龙,刘艳平.病毒miRNA与免疫逃逸[J].中国细胞生物学学报,2012, 34(9):924-930.Xiang R, Liang L, Liu Y P. Viral miRNAs and immune avasion[J]. Chinese Journal of Cell Biology, 2012, 34(9):924-930(in Chinese).
[32] Yan Y, Guo C Y, Ni S W, et al. Singapore grouper iridovirus(SGIV)encoded SGIV-miR-13 attenuates viral infection via modulating major capsid protein expression[J]. Virus Research, 2015, 205:45-53.
[33] Chen W J, Yi L Z, Feng S S, et al. Characterization of microRNAs in orange-spotted grouper(Epinephelus coioides)fin cells upon red-spotted grouper nervous necrosis virus infection[J]. Fish&Shellfish Immunology, 2017, 63:228-236.
[34] Zhu X, He Z J,Hu Y W, et al. MicroRNA-30e*suppresses dengue virus replication by promoting NF-κBdependent IFN production[J]. Virus Research, 2014,8(8):e3088.
[35] Li Z H, Chen B B, Feng M, et al. MicroRNA-23b promotes avian leukosis virus subgroup J(ALV-J)replication by targeting IRF1[J]. Virus Research, 2015, 5:10294.
[36] Zhang Q, Huang C, Yang Q, et al. MicroRNA-30c modulates type I IFN responses to facilitate porcine reproductive and respiratory syndrome virus infection by targeting JAK1[J]. The Journal of Immunology, 2016,196(5):2272-2282.
[37] Ge Y Y, Shi Q, Zheng Z Y, et al. MicroRNA-100promotes the autophagy of hepatocellular carcinoma cells by inhibiting the expression of mTOR and IGF-1R[J]. Virus Research,2014, 5(15):6218-6228.
[38] Lin Y, Deng W Y, Pang J K, et al. The microRNA-99family modulates hepatitis B virus replication by promoting IGF-IR/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy[J]. Cellular Microbiology,2017,19(5):e12709.
[39] Wang F Z, Weber F, Croce C, et al. Human cytomegalovirus infection alters the expression of cellular microRNA species that affect its replication[J]. Journal of Virology, 2008, 82(18):9065-9074.