Advertisement

Cloning of Autophagy-Related MicroRNAs

  • Deniz Gulfem Ozturk
  • Muhammed Kocak
  • Devrim Gozuacik
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1854)

Abstract

Autophagy is a cellular survival pathway that is necessary for the degradation of cellular constituents such as long-lived proteins and damaged organelles. Conditions resulting in cellular stress such as starvation or hypoxia might activate autophagy. Being at the crossroads of various cellular response pathways, dysregulation of autophagy might result in pathological states including cancer and neurodegenerative diseases. Autophagy has also been shown to participate in stemness. MicroRNAs were introduced as novel regulators of autophagy, and accumulating results underlined the fact that they constituted an important layer of biological control mechanism on the autophagic activity.

MicroRNAs are protein noncoding small RNAs that control cellular levels of transcripts and proteins through posttrancriptional mechanisms. Novel miRNAs in human and mouse genomes are yet to be identified. Considering the emerging role of autophagy in health and disease, identification of novel autophagy-regulating miRNAs and determination of relations between miRNA expression and physiological and pathological conditions might contribute to a better understanding of mechanisms governing health and disease. High-throughput techniques were developed for miRNA profiling, yet for a thorough characterization and miRNA target determination, miRNA cloning remains as an important step. Here, we describe a modified miRNA cloning method for the characterization of novel autophagy-regulating miRNAs.

Keywords

Autophagy Cloning Identification miRNA Regulation 

References

  1. 1.
    Ravikumar B, Sarkar S, Davies JE, Futter M, Garcia-Arencibia M, Green-Thompson ZW et al (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev 90(4):1383–1435.  https://doi.org/10.1152/physrev.00030.2009 CrossRefGoogle Scholar
  2. 2.
    Gozuacik D, Kimchi A (2004) Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23(16):2891–2906.  https://doi.org/10.1038/sj.onc.1207521 CrossRefPubMedGoogle Scholar
  3. 3.
    Pan H, Cai N, Li M, Liu G, Belmonte J (2013) Autophagic control of cell stemness’. EMBO Mol Med 5(3):327–331.  https://doi.org/10.1002/emmm.201201999 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Liu F, Lee JY, Wei H, Tanabe O, Engel JD, Morrison SJ et al (2010) FIP200 is required for the cell-autonomous maintenance of fetal hematopoietic stem cells. Blood 116:4806–4814.  https://doi.org/10.1182/blood-2010-06-288589 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Salemi S, Yousefi S, Constantinescu MA, Fey MF, Simon H-U (2012) Autophagy is required for self-renewal and differentiation of adult human stem cells. Cell Res 22:432–435.  https://doi.org/10.1038/cr.2011.200 CrossRefPubMedGoogle Scholar
  6. 6.
    Vazquez P, Arroba AI, Cecconi F, de la Rosa EJ, Boya P, de Pablo F (2012) Atg5 and Ambra1 differentially modulate neurogenesis in neural stem cells. Autophagy 8:187–199.  https://doi.org/10.4161/auto.8.2.18535 CrossRefPubMedGoogle Scholar
  7. 7.
    Tra T, Gong L, Kao LP, Li XL, Grandela C, Devenish RJ et al (2011) Autophagy in human embryonic stem cells. PLoS One 6:e27485.  https://doi.org/10.1371/journal.pone.0027485 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Menendez JA, Vellon L, Oliveras-Ferraros C, Cufi S, Vazquez-Martin A (2011) mTOR-regulated senescence and autophagy during reprogramming of somatic cells to pluripotency: a roadmap from energy metabolism to stem cell renewal and aging. Cell Cycle 10:3658–3677.  https://doi.org/10.4161/cc.10.21.18128 CrossRefPubMedGoogle Scholar
  9. 9.
    Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6(5):376–385.  https://doi.org/10.1038/nrm1644 CrossRefPubMedGoogle Scholar
  10. 10.
    Gozuacik D, Akkoc Y, Ozturk DG, Kocak M (2017) Autophagy-regulating microRNAs and cancer. Front Oncol 7.  https://doi.org/10.3389/fonc.2017.00065
  11. 11.
    Barad O, Meiri E, Avniel A, Aharonov R, Barzilai A, Bentwich I et al (2004) Micro-RNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues. Genome Res 14(12):2486–2494CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT et al (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33(20):e179CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Lee RC, Ambros V (2001) An extensive class of small RNAs in Caenorhabditiselegans. Science 294(5543):862–864CrossRefPubMedGoogle Scholar
  14. 14.
    Lau NC, Lim LP, Weinstein EG, Bartel DP (2001) An abundant class of tinyRNAs with probable regulatory roles in Caenorhabditis elegans. Science 294(5543):858–862CrossRefPubMedGoogle Scholar
  15. 15.
    Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12(9):735–739CrossRefPubMedGoogle Scholar
  16. 16.
    Lu C, Meyers BC, Green PJ (2007) Construction of small RNA cDNA libraries for deep sequencing. Methods 43:110–117.  https://doi.org/10.1016/j.ymeth.2007.05.002 CrossRefPubMedGoogle Scholar
  17. 17.
    Gu W, Shirayama M, Conte D, Vasale J, Batista PJ, Claycomb JM et al (2009) Distinct argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline. Mol Cell 36:231–244.  https://doi.org/10.1016/j.molcel.2009.09.020 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Fu H, Tie Y, Xu C, Zhang Z, Zhu J, Shi Y, Jiang H, Sun Z, Zheng X (2005) Identification of human fetal liver miRNAs by a novel method. FEBS Lett 579(17):3849–3854.  https://doi.org/10.1016/j.febslet.2005.05.064 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Deniz Gulfem Ozturk
    • 1
  • Muhammed Kocak
    • 1
  • Devrim Gozuacik
    • 1
    • 2
  1. 1.Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics and Bioengineering ProgramSabanci UniversityIstanbulTurkey
  2. 2.Center of Excellence for Functional Surfaces and Interfaces for Nano Diagnostics (EFSUN)Sabanci UniversityIstanbulTurkey

Personalised recommendations