Differential expression of novel MicroRNAs from developing fetal heart of Gallus gallus domesticus implies a role in cardiac development

  • Sharad Saxena
  • Priyanka Mathur
  • Vaibhav Shukla
  • Vibha RaniEmail author


Heart development is a complex process regulated by multi-layered genetic as well epigenetic regulators many of which are still unknown. Besides their critical role during cardiac development, these molecular regulators emerge as key modulators of cardiovascular pathologies, where fetal cardiac genes’ re-expression is witnessed. MicroRNAs have recently emerged as a crucial part of signalling cascade in both development and diseases. We aimed to identify, validate, and perform functional annotation of putative novel miRNAs using chicken as a cardiac development model system. Novel miRNAs were obtained through deep sequencing of small RNAs extracted from chicken embryonic cardiac tissue of different developmental stages. After filtering out real pre-miRNAs, their expression analysis, potential target gene’s prediction and functional annotations were performed. Expression analysis revealed that miRNAs were differentially expressed during different developmental stages of chicken heart. The expression of selected putative novel miRNAs was further validated by real-time PCR. Our analysis indicated the presence of novel cardiac miRNAs that might be regulating critical cardiac development events such as cardiac cell growth, differentiation, cardiac action potential generation and signal transduction.


Fetal Cardiac Development Novel miRNAs Cardiovascular diseases Gene re-expression 



This study was supported by DST-SERB, Government of India (File No: EMR/2016/005914); and CSIR, Government of India (File No: 09/1132 (0004)/18-EMR-I).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

11010_2019_3618_MOESM1_ESM.tif (149 kb)
Supplementary material 1—Flowchart depicting the steps taken for identification and validation of novel miRNAs and generation of interaction network between enriched GO terms and KEEG pathways. (TIFF 148 kb)


  1. 1.
    Srivastava D, Olson EN (2000) A genetic blueprint for cardiac development. Nature 407:221–226CrossRefGoogle Scholar
  2. 2.
    Abu-Issa R, Kirby ML (2007) Heart field: from mesoderm to heart tube. Annu Rev Cell Dev Biol 23:45–68CrossRefGoogle Scholar
  3. 3.
    Nandi SS, Mishra PK (2015) Harnessing fetal and adult genetic reprograming for therapy of heart disease. J Nat Sci 1:4–6Google Scholar
  4. 4.
    Barry SP, Davidson SM, Townsend PA (2008) Molecular regulation of cardiac hypertrophy. Int J Biochem Cell Biol 40:2023–2039CrossRefGoogle Scholar
  5. 5.
    Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297CrossRefGoogle Scholar
  6. 6.
    Fuller A, Qian L (2014) MiRiad roles for MicroRNAs in cardiac development and regeneration. Cells 3:724–750CrossRefGoogle Scholar
  7. 7.
    Rao PK, Toyama Y, Chiang HR et al (2009) Loss of cardiac microRNA-mediated regulation leads to dilated cardiomyopathy and heart failure. Circ Res 105:585–594CrossRefGoogle Scholar
  8. 8.
    Rustagi Y, Jaiswal HK, Rawal K et al (2015) Comparative characterization of cardiac development specific microRNAs: fetal regulators for future. PLoS ONE 10:e0139359CrossRefGoogle Scholar
  9. 9.
    Epstein JA (2010) Cardiac development and implications for heart disease. N Engl J Med 363:1638–1647CrossRefGoogle Scholar
  10. 10.
    Katz MG, Fargnoli AS, Kendle AP et al (2016) The role of microRNAs in cardiac development and regenerative capacity. Am J Physiol 310:H528–H541Google Scholar
  11. 11.
    Wittig J, Münsterberg A (2016) The early stages of heart development: insights from chicken embryos. J Cardiovasc Dev Dis 3:12CrossRefGoogle Scholar
  12. 12.
    Gao D, Middleton R, Rasko JEJ et al (2013) MiREval 2.0: a web tool for simple microRNA prediction in genome sequences. Bioinformatics 29:3225–3226CrossRefGoogle Scholar
  13. 13.
    Jiang P, Wu H, Wang W et al (2007) MiPred: classification of real and pseudo microRNA precursors using random forest prediction model with combined features. Nucleic Acids Res 35:W339–W344CrossRefGoogle Scholar
  14. 14.
    Gruber AR, Lorenz R, Bernhart SH et al (2008) The Vienna RNA websuite. Nucleic Acids Res 36:W70–W74CrossRefGoogle Scholar
  15. 15.
    Kozomara A, Griffiths-Jones S (2014) MiRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res 42:D68–D73CrossRefGoogle Scholar
  16. 16.
    Wong N, Wang X (2015) miRDB: an online resource for microRNA target prediction and functional annotations. Nucleic Acids Res 43:D146–D152CrossRefGoogle Scholar
  17. 17.
    Mi H, Muruganujan A, Thomas PD (2013) PANTHER in 2013: modeling the evolution of gene function, and other gene attributes, in the context of phylogenetic trees. Nucleic Acids Res 41:D377–D386CrossRefGoogle Scholar
  18. 18.
    Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57CrossRefGoogle Scholar
  19. 19.
    Shannon P, Markiel A, Ozier O et al (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504CrossRefGoogle Scholar
  20. 20.
    Varkonyi-Gasic E, Wu R, Wood M et al (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3:12CrossRefGoogle Scholar
  21. 21.
    Yang LH, Wang SL, Tang LL et al (2014) Universal stem-loop primer method for screening and quantification of microRNA. PLoS ONE 9:e115293CrossRefGoogle Scholar
  22. 22.
    Laganà A, Veneziano D, Spata T et al (2015) Identification of general and heart-specific miRNAs in sheep (Ovis aries). PLoS ONE 10:e0143313CrossRefGoogle Scholar
  23. 23.
    Ambros V, Bartel B, Bartel DP et al (2003) A uniform system for microRNA annotation. RNA 9:277–279CrossRefGoogle Scholar
  24. 24.
    Martinsen BJ (2005) Reference guide to the stages of chick heart embryology. Dev Dyn 233:1217–1237CrossRefGoogle Scholar
  25. 25.
    Xu H, Wang X, Du Z, Li N (2006) Identification of microRNAs from different tissues of chicken embryo and adult chicken. FEBS Lett 580:3610–3616CrossRefGoogle Scholar
  26. 26.
    Ji Z, Wang G, Xie Z et al (2012) Identification and characterization of microRNA in the dairy goat (Capra hircus) mammary gland by Solexa deep-sequencing technology. Mol Biol Rep 39:9361–9371CrossRefGoogle Scholar
  27. 27.
    Ge X, Zhang Y, Jiang J et al (2012) Identification of microRNAs in Helicoverpa armigera and Spodoptera litura based on deep sequencing and homology analysis. Int J Biol Sci 9:1–15CrossRefGoogle Scholar
  28. 28.
    Islam MT, Ferdous AS, Najnin RA et al (2015) High-throughput sequencing reveals diverse sets of conserved, nonconserved, and species-specific miRNAs in jute. Int J Genom 2015:125048Google Scholar
  29. 29.
    Kim GH (2013) MicroRNA regulation of cardiac conduction and arrhythmias. Transl Res 161:381–392CrossRefGoogle Scholar
  30. 30.
    Yang B, Lin H, Xiao J et al (2007) The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2. Nat Med 13:486–491CrossRefGoogle Scholar
  31. 31.
    Liu X, Zhang Y, Du W et al (2016) MiR-223-3p as a novel MicroRNA regulator of expression of voltage-gated K + channel Kv4.2 in acute myocardial infarction. Cell Physiol Biochem 39:102–114CrossRefGoogle Scholar
  32. 32.
    Yang WM, Jeong HJ, Park SW, Lee W (2015) Obesity-induced miR-15b is linked causally to the development of insulin resistance through the repression of the insulin receptor in hepatocytes. Mol Nutr Food Res 59:2303–2314CrossRefGoogle Scholar
  33. 33.
    Motohashi N, Alexander MS, Shimizu-Motohashi Y et al (2013) Regulation of IRS1/Akt insulin signaling by microRNA-128a during myogenesis. J Cell Sci 126:2678–2691CrossRefGoogle Scholar
  34. 34.
    Min KH, Yang WM, Lee W (2018) Saturated fatty acids-induced miR-424–5p aggravates insulin resistance via targeting insulin receptor in hepatocytes. Biochem Biophys Res Commun 503:1587–1593CrossRefGoogle Scholar
  35. 35.
    Ono K, Igata M, Kondo T et al (2018) Identification of microRNA that represses IRS-1 expression in liver. PLoS ONE 13:e0191553CrossRefGoogle Scholar
  36. 36.
    DeBosch BJ, Muslin AJ (2008) Insulin signaling pathways and cardiac growth. J Mol Cell Cardiol 44:855–864CrossRefGoogle Scholar
  37. 37.
    Jia G, Whaley-Connell A, Sowers JR (2018) Diabetic cardiomyopathy: a hyperglycaemia- and insulin-resistance-induced heart disease. Diabetologia 61:21–28CrossRefGoogle Scholar
  38. 38.
    Boucher J, Kleinridders A, Ronald Kahn C (2014) Insulin receptor signaling in normal and insulin-resistant states. Cold Spring Harb Perspect Biol 6:a009191CrossRefGoogle Scholar
  39. 39.
    Chen Z, Ding L, Yang W et al (2017) Hepatic activation of the FAM3C-HSF1-CaM pathway attenuates hyperglycemia of obese diabetic mice. Diabetes 66:1185–1197CrossRefGoogle Scholar
  40. 40.
    Park J, Ahn S, Jayabalan AK et al (2016) Insulin signaling augments eIF4E-dependent nonsense-mediated mRNA decay in mammalian cells. Biochim Biophys Acta 1859:896–905CrossRefGoogle Scholar
  41. 41.
    Tian Y, Cohen ED, Morrisey EE (2010) The importance of Wnt signaling in cardiovascular development. Pediatr Cardiol 31:342–348CrossRefGoogle Scholar
  42. 42.
    Li M, Hu X, Zhu J et al (2014) Overexpression of miR-19b impairs cardiac development in zebrafish by targeting ctnnb1. Cell Physiol Biochem 33:1988–2002CrossRefGoogle Scholar
  43. 43.
    Ye X, Hemida MG, Qiu Y et al (2013) MiR-126 promotes coxsackievirus replication by mediating cross-talk of ERK1/2 and Wnt/β-catenin signal pathways. Cell Mol Life Sci 70:4631–4644CrossRefGoogle Scholar
  44. 44.
    Han P, Li W, Lin CH et al (2014) A long noncoding RNA protects the heart from pathological hypertrophy. Nature 514:102–106CrossRefGoogle Scholar
  45. 45.
    Tian J, An X, Niu L (2017) Role of microRNAs in cardiac development and disease. Exp Ther Med 13:3–8CrossRefGoogle Scholar
  46. 46.
    Ling TY, Wang XL, Chai Q et al (2017) Regulation of cardiac CACNB2 by microRNA-499: potential role in atrial fibrillation. BBA Clin 7:78–84CrossRefGoogle Scholar
  47. 47.
    Rienks M, Barallobre-Barreiro J, Mayr M (2018) The emerging role of the ADAMTS family in vascular diseases. Circ Res 123:1279–1281CrossRefGoogle Scholar
  48. 48.
    Liu R, Correll RN, Davis J et al (2015) Cardiac-specific deletion of protein phosphatase 1β promotes increased myofilament protein phosphorylation and contractile alterations. J Mol Cell Cardiol 87:204–213CrossRefGoogle Scholar
  49. 49.
    Hoeflich A, David R, Hjortebjerg R (2018) Current IGFBP-related biomarker research in cardiovascular disease: we need more structural and functional information in clinical studies. Front Endocrinol 9:388CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Sharad Saxena
    • 1
  • Priyanka Mathur
    • 1
  • Vaibhav Shukla
    • 2
  • Vibha Rani
    • 1
    Email author
  1. 1.Transcriptome Laboratory, Centre for Emerging Diseases, Department of BiotechnologyJaypee Institute of Information TechnologyNoidaIndia
  2. 2.Manipal School of Life SciencesManipal Academy of Higher EducationManipalIndia

Personalised recommendations