Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells

Autoři: Xiangqin Ji aff001;  Penglei Jiang aff001;  Juntao Luo aff001;  Mengjia Li aff001;  Yajing Bai aff001;  Junfang Zhang aff001;  Bingshe Han aff001
Působiště autorů: Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai, China aff001;  National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China aff002;  International Research Center for Marine Biosciences at Shanghai Ocean University, Ministry of Science and Technology, Shanghai, China aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article


MicroRNAs (miRNAs) play vital roles in various biological processes under multiple stress conditions by leading to mRNA cleavage or translational repression. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the target genes of known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, both miR-100-3p inhibitor and miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. Further study showed that miR-100-3p and miR-16b could regulate inversely the expression of their target gene (atad5a, cyp2ae1, lamp1, rilp, atxn7, tnika, btbd9), and that overexpression of miR-100-3p disturbed the early embryonic development of zebrafish. In summary, the present data show that miRNAs are closely involved in cold acclimation in zebrafish ZF4 cells and provide information for further understanding of the roles of miRNAs in cold acclimation in fish.

Klíčová slova:

cDNA libraries – Gene expression – MicroRNAs – RNA extraction – RNA sequencing – Small nucleolar RNA – Thermal stresses – Zebrafish


1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–97. doi: 10.1016/s0092-8674(04)00045-5 14744438.

2. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nature reviews Genetics. 2004;5(7):522–31. doi: 10.1038/nrg1379 15211354.

3. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. Widespread changes in protein synthesis induced by microRNAs. Nature. 2008;455(7209):58–63. doi: 10.1038/nature07228 18668040.

4. Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome research. 2009;19(1):92–105. doi: 10.1101/gr.082701.108 18955434; PubMed Central PMCID: PMC2612969.

5. Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell. 2006;126(6):1203–17. doi: 10.1016/j.cell.2006.07.031 16990141.

6. Pal AS, Kasinski AL. Animal Models to Study MicroRNA Function. Advances in cancer research. 2017;135:53–118. doi: 10.1016/bs.acr.2017.06.006 28882225; PubMed Central PMCID: PMC5860655.

7. Soyano K, Mushirobira Y. The Mechanism of Low-Temperature Tolerance in Fish. Advances in experimental medicine and biology. 2018;1081:149–64. doi: 10.1007/978-981-13-1244-1_9 30288709.

8. López-Olmeda JF, Sánchez-Vázquez FJ. Thermal biology of zebrafish (Danio rerio). Journal of Thermal Biology. 2011;36(2):91–104. doi: 10.1016/j.jtherbio.2010.12.005

9. Malek RL, Sajadi H, Abraham J, Grundy MA, Gerhard GS. The effects of temperature reduction on gene expression and oxidative stress in skeletal muscle from adult zebrafish. Comparative biochemistry and physiology Toxicology & pharmacology: CBP. 2004;138(3):363–73. doi: 10.1016/j.cca.2004.08.014 15533794.

10. Eremina M, Rozhon W, Poppenberger B. Hormonal control of cold stress responses in plants. Cellular and molecular life sciences: CMLS. 2016;73(4):797–810. doi: 10.1007/s00018-015-2089-6 26598281.

11. Feng R, Sang Q, Zhu Y, Fu W, Liu M, Xu Y, et al. MiRNA-320 in the human follicular fluid is associated with embryo quality in vivo and affects mouse embryonic development in vitro. Scientific reports. 2015;5:8689. doi: 10.1038/srep08689 25732513; PubMed Central PMCID: PMC4346788.

12. Yang Y, Zhang X, Su Y, Zou J, Wang Z, Xu L, et al. miRNA alteration is an important mechanism in sugarcane response to low-temperature environment. BMC genomics. 2017;18(1):833. doi: 10.1186/s12864-017-4231-3 29084515; PubMed Central PMCID: PMC5661916.

13. Zhen L, Guo W, Peng M, Liu Y, Zang S, Ji H, et al. Identification of cold-responsive miRNAs in rats by deep sequencing. J Therm Biol. 2017;66:114–24. doi: 10.1016/j.jtherbio.2017.03.005 28477904.

14. Ma C, Burd S, Lers A. miR408 is involved in abiotic stress responses in Arabidopsis. The Plant journal: for cell and molecular biology. 2015;84(1):169–87. doi: 10.1111/tpj.12999 26312768.

15. Tang Z, Zhang L, Xu C, Yuan S, Zhang F, Zheng Y, et al. Uncovering small RNA-mediated responses to cold stress in a wheat thermosensitive genic male-sterile line by deep sequencing. Plant physiology. 2012;159(2):721–38. doi: 10.1104/pp.112.196048 22508932; PubMed Central PMCID: PMC3375937.

16. Guo W, Lian S, Zhen L, Zang S, Chen Y, Lang L, et al. The Favored Mechanism for Coping with Acute Cold Stress: Upregulation of miR-210 in Rats. Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology. 2018;46(5):2090–102. doi: 10.1159/000489449 29723850.

17. He P, Wei P, Zhang B, Zhao Y, Li Q, Chen X, et al. Identification of microRNAs involved in cold adaptation of Litopenaeus vannamei by high-throughput sequencing. Gene. 2018;677:24–31. doi: 10.1016/j.gene.2018.07.042 30016670.

18. Yang R, Dai Z, Chen S, Chen L. MicroRNA-mediated gene regulation plays a minor role in the transcriptomic plasticity of cold-acclimated zebrafish brain tissue. BMC genomics. 2011;12:605. doi: 10.1186/1471-2164-12-605 22168751; PubMed Central PMCID: PMC3258298.

19. Spence R, Gerlach G, Lawrence C, Smith C. The behaviour and ecology of the zebrafish, Danio rerio. Biological reviews of the Cambridge Philosophical Society. 2008;83(1):13–34. doi: 10.1111/j.1469-185X.2007.00030.x 18093234.

20. Peterson RT, Nass R, Boyd WA, Freedman JH, Dong K, Narahashi T. Use of non-mammalian alternative models for neurotoxicological study. Neurotoxicology. 2008;29(3):546–55. doi: 10.1016/j.neuro.2008.04.006 18538410; PubMed Central PMCID: PMC2702842.

21. Han B, Li W, Chen Z, Xu Q, Luo J, Shi Y, et al. Variation of DNA Methylome of Zebrafish Cells under Cold Pressure. PloS one. 2016;11(8):e0160358. doi: 10.1371/journal.pone.0160358 27494266; PubMed Central PMCID: PMC4975392.

22. Jiang P, Hou Y, Fu W, Tao X, Luo J, Lu H, et al. Characterization of lncRNAs involved in cold acclimation of zebrafish ZF4 cells. PloS one. 2018;13(4):e0195468. doi: 10.1371/journal.pone.0195468 29634734; PubMed Central PMCID: PMC5892903.

23. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature methods. 2012;9(4):357–9. doi: 10.1038/nmeth.1923 22388286; PubMed Central PMCID: PMC3322381.

24. Stocks MB, Moxon S, Mapleson D, Woolfenden HC, Mohorianu I, Folkes L, et al. The UEA sRNA workbench: a suite of tools for analysing and visualizing next generation sequencing microRNA and small RNA datasets. Bioinformatics. 2012;28(15):2059–61. doi: 10.1093/bioinformatics/bts311 22628521; PubMed Central PMCID: PMC3400958.

25. Anders S, Huber W. Differential expression analysis for sequence count data. Genome biology. 2010;11(10):R106. doi: 10.1186/gb-2010-11-10-r106 20979621; PubMed Central PMCID: PMC3218662.

26. John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human MicroRNA targets. PLoS biology. 2004;2(11):e363. doi: 10.1371/journal.pbio.0020363 15502875; PubMed Central PMCID: PMC521178.

27. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature protocols. 2009;4(1):44–57. doi: 10.1038/nprot.2008.211 19131956.

28. Huang DW, Sherman BT, Tan Q, Kir J, Liu D, Bryant D, et al. DAVID Bioinformatics Resources: expanded annotation database and novel algorithms to better extract biology from large gene lists. Nucleic acids research. 2007;35(Web Server issue):W169–75. doi: 10.1093/nar/gkm415 17576678; PubMed Central PMCID: PMC1933169.

29. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8. doi: 10.1006/meth.2001.1262 11846609.

30. He Y, Huang CX, Chen N, Wu M, Huang Y, Liu H, et al. The zebrafish miR-125c is induced under hypoxic stress via hypoxia-inducible factor 1alpha and functions in cellular adaptations and embryogenesis. Oncotarget. 2017;8(43):73846–59. doi: 10.18632/oncotarget.17994 29088751; PubMed Central PMCID: PMC5650306.

31. Wang X, Liu XS. Systematic Curation of miRBase Annotation Using Integrated Small RNA High-Throughput Sequencing Data for C. elegans and Drosophila. Frontiers in genetics. 2011;2:25. doi: 10.3389/fgene.2011.00025 22303321; PubMed Central PMCID: PMC3268580.

32. Koh W, Sheng CT, Tan B, Lee QY, Kuznetsov V, Kiang LS, et al. Analysis of deep sequencing microRNA expression profile from human embryonic stem cells derived mesenchymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of hepatic nuclear factor 4 alpha. BMC genomics. 2010;11 Suppl 1:S6. doi: 10.1186/1471-2164-11-S1-S6 20158877; PubMed Central PMCID: PMC2822534.

33. Yu Z, Li N, Jiang K, Zhang N, Yao LL. MiR-100 up-regulation enhanced cell autophagy and apoptosis induced by cisplatin in osteosarcoma by targeting mTOR. European review for medical and pharmacological sciences. 2018;22(18):5867–73. doi: 10.26355/eurrev_201809_15913 30280766.

34. Zheng Y, Tan K, Huang H. Long noncoding RNA HAGLROS regulates apoptosis and autophagy in colorectal cancer cells via sponging miR-100 to target ATG5 expression. Journal of cellular biochemistry. 2019;120(3):3922–33. doi: 10.1002/jcb.27676 30430634

35. Braga TV, Evangelista FCG, Gomes LC, Araujo S, Carvalho MDG, Sabino AP. Evaluation of MiR-15a and MiR-16-1 as prognostic biomarkers in chronic lymphocytic leukemia. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2017;92:864–9. doi: 10.1016/j.biopha.2017.05.144 28599250.

36. Thiebaut F, Rojas CA, Almeida KL, Grativol C, Domiciano GC, Lamb CR, et al. Regulation of miR319 during cold stress in sugarcane. Plant, cell & environment. 2012;35(3):502–12. doi: 10.1111/j.1365-3040.2011.02430.x 22017483.

37. Shin SC, Ahn DH, Kim SJ, Pyo CW, Lee H, Kim MK, et al. The genome sequence of the Antarctic bullhead notothen reveals evolutionary adaptations to a cold environment. Genome biology. 2014;15(9):468. doi: 10.1186/s13059-014-0468-1 25252967; PubMed Central PMCID: PMC4192396.

38. Berthelot C, Clarke J, Desvignes T, William Detrich H III, Flicek P, Peck LS, et al. Adaptation of Proteins to the Cold in Antarctic Fish: A Role for Methionine? Genome biology and evolution. 2019;11(1):220–31. doi: 10.1093/gbe/evy262 30496401; PubMed Central PMCID: PMC6336007.

39. Long Y, Song G, Yan J, He X, Li Q, Cui Z. Transcriptomic characterization of cold acclimation in larval zebrafish. BMC genomics. 2013;14:612. doi: 10.1186/1471-2164-14-612 24024969; PubMed Central PMCID: PMC3847098.

40. Pinto R, Ivaldi C, Reyes M, Doyen C, Mietton F, Mongelard F, et al. Seasonal environmental changes regulate the expression of the histone variant macroH2A in an eurythermal fish. FEBS letters. 2005;579(25):5553–8. doi: 10.1016/j.febslet.2005.09.019 16213499.

41. Hu P, Liu M, Zhang D, Wang J, Niu H, Liu Y, et al. Global identification of the genetic networks and cis-regulatory elements of the cold response in zebrafish. Nucleic acids research. 2015;43(19):9198–213. doi: 10.1093/nar/gkv780 26227973; PubMed Central PMCID: PMC4627065.

42. Chen Z, Cheng CH, Zhang J, Cao L, Chen L, Zhou L, et al. Transcriptomic and genomic evolution under constant cold in Antarctic notothenioid fish. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(35):12944–9. doi: 10.1073/pnas.0802432105 18753634; PubMed Central PMCID: PMC2529033.

43. Sun X, Liu X, Wang Y, Yang S, Chen Y, Yuan T. miR-100 inhibits the migration and invasion of nasopharyngeal carcinoma by targeting IGF1R. Oncology letters. 2018;15(6):8333–8. doi: 10.3892/ol.2018.8420 29805566; PubMed Central PMCID: PMC5950178.

44. Wang Q, Yao J, Jin Q, Wang X, Zhu H, Huang F, et al. LAMP1 expression is associated with poor prognosis in breast cancer. Oncology letters. 2017;14(4):4729–35. doi: 10.3892/ol.2017.6757 29085473; PubMed Central PMCID: PMC5649640.

45. Porubsky PR, Meneely KM, Scott EE. Structures of human cytochrome P-450 2E1. Insights into the binding of inhibitors and both small molecular weight and fatty acid substrates. The Journal of biological chemistry. 2008;283(48):33698–707. doi: 10.1074/jbc.M805999200 18818195; PubMed Central PMCID: PMC2586265.

46. Mukai N, Nakayama Y, Ishi S, Murakami T, Ogawa S, Kageyama K, et al. Cold storage conditions modify microRNA expressions for platelet transfusion. PloS one. 2019;14(7):e0218797. doi: 10.1371/journal.pone.0218797 31269049.

47. Wang H, Zhang Y, Wu Q, Wang YB, Wang W. miR-16 mimics inhibit TGF-beta1-induced epithelial-to-mesenchymal transition via activation of autophagy in non-small cell lung carcinoma cells. Oncology reports. 2018;39(1):247–54. doi: 10.3892/or.2017.6088 29138833.

48. Liu GP, Wang WW, Lu WY, Shang AQ. The mechanism of miR-16-5p protection on LPS-induced A549 cell injury by targeting CXCR3. Artificial cells, nanomedicine, and biotechnology. 2019;47(1):1200–6. doi: 10.1080/21691401.2019.1593998 30957556.

49. Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proceedings of the National Academy of Sciences of the United States of America. 2005;102(39):13944–9. doi: 10.1073/pnas.0506654102 16166262; PubMed Central PMCID: PMC1236577.

50. Margiotta A, Progida C, Bakke O, Bucci C. Characterization of the role of RILP in cell migration. European journal of histochemistry: EJH. 2017;61(2):2783. doi: 10.4081/ejh.2017.2783 28735522; PubMed Central PMCID: PMC5460375.

51. Burke TL, Miller JL, Grant PA. Direct inhibition of Gcn5 protein catalytic activity by polyglutamine-expanded ataxin-7. The Journal of biological chemistry. 2013;288(47):34266–75. doi: 10.1074/jbc.M113.487538 24129567; PubMed Central PMCID: PMC3837167.

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