Expression of miR-145 and Its Target Proteins Are Regulated by miR-29b in Differentiated Neurons

  • Abhishek Jauhari
  • Tanisha Singh
  • Sanjay Yadav


MicroRNAs (miRNAs) are emerging as the most potential regulator of neuronal development. Recent studies from our lab and elsewhere have demonstrated a direct role of miRNAs in regulating neuronal differentiation and synaptogenesis. MicroRNA-145, a miRNA identified to regulate pluripotency of stem cells, downregulates the protein levels of reprogramming transcription factors (RTFs) like OCT4, SOX2, and KLF4 (cell, 137,647–658,2009). Studies have shown that miR-145 is multifunctional and crucial for fate determination of neurons. In our recently published study, we have identified a set of miRNAs including miR-145 and miR-29b families differentially expressed in SH-SY5Y cells exposed sequentially with retinoic acid + brain-derived neurotrophic factor (RA+BDNF) for differentiation into mature neurons (Mol Neurobiol (2016) doi: In the present study, we have identified the role of miR-29b in upregulation of miR-145, which is upregulated after exposure of RA+BDNF in a P53-dependent manner. In differentiating SH-SY5Y cells, expression of miR-29b downregulates expression of P85α, a P53 inhibitor, which results in upregulation of miR-145 and downregulation of RTF proteins. Ectopic expression of miR-145 and miR-29b in amounts equivalent to their endogenous expression has induced G1 phase cell cycle arrest. In conclusion, our studies have identified miR-29b as an upstream regulator of miR-145 and targets its RTF genes during differentiation of SH-SY5Y cells.


MicroRNAs Neuronal differentiation Reprogramming transcription factors (RTFs) P53,SH-SY5Y cells SOX2 KLF4 OCT4 NANOG miR-145 miR-29b 



brain-derived neurotrophic factor




hypoxanthine-guanine phosphoribosyltransferase


integrated density value




messenger RNA


polymerase chain reaction


retinoic acid


reprogramming transcription factor


relative quantification


reverse transcription




phosphate buffer saline


propidium iodide




polyvinylidene fluoride



Mr. Abhishek Jauhari is grateful to UGC, New Delhi, and Ms. Tanisha Singh is grateful to DST, New Delhi, for providing research fellowships. The technical assistance of Mr. B S Pandey and Mr. Puneet Khare is also gratefully acknowledged. The CSIR-IITR communication reference number is 3493.

Funding Information

Funding for the work carried out in the present study had been provided by the CSIR network project (miND).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

12035_2018_1009_MOESM1_ESM.pptx (7 mb)
ESM 1 (PPTX 7218 kb)
12035_2018_1009_MOESM2_ESM.docx (31 kb)
ESM 2 (DOCX 25 kb)


  1. 1.
    Coolen M, Bally-Cuif L (2009) MicroRNAs in brain development and physiology. Curr Opin Neurobiol 19(5):461–470CrossRefPubMedGoogle Scholar
  2. 2.
    Petri R, Malmevik J, Fasching L, Åkerblom M, Jakobsson J (2014) miRNAs in brain development. Exp Cell Res 321(1):84–89CrossRefPubMedGoogle Scholar
  3. 3.
    Singh T, Jauhari A, Pandey A, Singh P, B Pant A, Parmar D, Yadav S (2014) Regulatory triangle of neurodegeneration, adult neurogenesis and microRNAs. CNS Neurol Disord Drug Targets 13(1):96–103CrossRefPubMedGoogle Scholar
  4. 4.
    Stiles J, Jernigan TL (2010) The basics of brain development. Neuropsychol Rev 20(4):327–348CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Pandey A, Singh P, Jauhari A, Singh T, Khan F, Pant AB, Parmar D, Yadav S (2015) Critical role of the miR-200 family in regulating differentiation and proliferation of neurons. J Neurochem 133(5):640–652CrossRefPubMedGoogle Scholar
  6. 6.
    Jauhari A, Singh T, Pandey A, Singh P, Singh N, Srivastava AK, Pant AB, Parmar D et al (2016) Differentiation induces dramatic changes in miRNA profile, where loss of dicer diverts differentiating SH-SY5Y cells toward senescence. Mol Neurobiol:1–10Google Scholar
  7. 7.
    Le MT, Xie H, Zhou B, Chia PH, Rizk P, Um M, Udolph G, Yang H et al (2009) MicroRNA-125b promotes neuronal differentiation in human cells by repressing multiple targets. Mol Cell Biol 29(19):5290–5305CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Makeyev EV, Zhang J, Carrasco MA, Maniatis T (2007) The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol Cell 27(3):435–448CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Purvis JE, Karhohs KW, Mock C, Batchelor E, Loewer A, Lahav G (2012) p53 dynamics control cell fate. Science 336(6087):1440–1444CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Quadrato G, Di Giovanni S (2012) Gatekeeper between quiescence and differentiation: p53 in axonal outgrowth and neurogenesis. Int Rev Neurobiol 105:71–89CrossRefPubMedGoogle Scholar
  11. 11.
    Xu N, Papagiannakopoulos T, Pan G, Thomson JA, Kosik KS (2009) MicroRNA-145 regulates OCT4, SOX2, and KLF4 and represses pluripotency in human embryonic stem cells. Cell 137(4):647–658CrossRefPubMedGoogle Scholar
  12. 12.
    Roshan R, Shridhar S, Sarangdhar MA, Banik A, Chawla M, Garg M, Singh VP, Pillai B (2014) Brain-specific knockdown of miR-29 results in neuronal cell death and ataxia in mice. RNA 20(8):1287–1297CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Yang G, Song Y, Zhou X, Deng Y, Liu T, Weng G, Yu D, Pan S (2015) MicroRNA-29c targets β-site amyloid precursor protein-cleaving enzyme 1 and has a neuroprotective role in vitro and in vivo. Mol Med Rep 12(2):3081–3088CrossRefPubMedGoogle Scholar
  14. 14.
    Ripa R, Dolfi L, Terrigno M, Pandolfini L, Savino A, Arcucci V, Groth M, Tozzini ET et al (2017) MicroRNA miR-29 controls a compensatory response to limit neuronal iron accumulation during adult life and aging. BMC Biol 15(1):9CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ouyang YB, Xu L, Lu Y, Sun X, Yue S, Xiong XX, Giffard RG (2013) Astrocyte-enriched miR-29a targets PUMA and reduces neuronal vulnerability to forebrain ischemia. Glia 61(11):1784–1794CrossRefPubMedGoogle Scholar
  16. 16.
    Hébert SS, Horré K, Nicolaï L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, Kauppinen S, Delacourte A et al (2008) Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer’s disease correlates with increased BACE1/β-secretase expression. Proc Natl Acad Sci 105(17):6415–6420CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Yadav S,Pandey A,Shukla A, Talwelkar SS,Kumar A, Pant AB,Parmar D, (2011) miR-497 and miR-302b regulate ethanol-induced neuronal cell death through BCL2 protein and cyclin D2. J Biol Chem 286(43):37347–37357CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Jin HY, Gonzalez-Martin A, Miletic AV, Lai M, Knight S, Sabouri-Ghomi M, Head SR, Macauley MS et al (2015) Transfection of microRNA mimics should be used with caution. Front Genet 6:340PubMedPubMedCentralGoogle Scholar
  19. 19.
    Jauhari A,Singh T, Singh P, Parmar D, Yadav S, (2018) Regulation of miR-34 family in neuronal development. Mol Neurobiol 55(2):936–945CrossRefPubMedGoogle Scholar
  20. 20.
    Pandey A, Jauhari A, Singh T, Singh P, Singh N, Srivastava AK, Khan F, Pant AB, Parmar D, Yadav S (2015) Transactivation of P53 by cypermethrin induced miR-200 and apoptosis in neuronal cells. Toxicol Res 4(6):1578–1586CrossRefGoogle Scholar
  21. 21.
    Anderson P, Kedersha N (2006) RNA granules. J Cell Biol 172(6):803–808CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Cherkasov V, Hofmann S, Druffel-Augustin S, Mogk A, Tyedmers J, Stoecklin G, Bukau B (2013) Coordination of translational control and protein homeostasis during severe heat stress. Curr Biol 23(24):2452–2462CrossRefPubMedGoogle Scholar
  23. 23.
    Farny NG, Kedersha NL, Silver PA (2009) Metazoan stress granule assembly is mediated by P-eIF2α-dependent and independent mechanisms. RNA 15(10):1814–1821CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Riback JA, Katanski CD, Kear-Scott JL, Pilipenko EV, Rojek AE, Sosnick TR, Drummond DA (2017) Stress-triggered phase separation is an adaptive, evolutionarily tuned response. Cell 168(6):1028–1040. e1019CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Fenn AM, Smith KM, Lovett-Racke AE, Guerau-de-Arellano M, Whitacre CC, Godbout JP (2013) Increased micro-RNA 29b in the aged brain correlates with the reduction of insulin-like growth factor-1 and fractalkine ligand. Neurobiol Aging 34(12):2748–2758CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Kole AJ, Swahari V, Hammond SM, Deshmukh M (2011) miR-29b is activated during neuronal maturation and targets BH3-only genes to restrict apoptosis. Genes Dev 25(2):125–130CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Park S-Y, Lee JH, Ha M, Nam J-W, Kim VN (2009) miR-29 miRNAs activate p53 by targeting p85α and CDC42. Nat Struct Mol Biol 16(1):23–29CrossRefPubMedGoogle Scholar
  28. 28.
    Gil-Perotin S, Haines JD, Kaur J, Marin-Husstege M, Spinetta MJ, Kim KH, Duran-Moreno M, Schallert T et al (2011) Roles of p53 and p27 Kip1 in the regulation of neurogenesis in the murine adult subventricular zone. Eur J Neurosci 34(7):1040–1052CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Zheng H, Ying H, Yan H, Kimmelman AC, Hiller DJ, Chen A-J, Perry SR, Tonon G et al (2008) p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation. Nature 455(7216):1129–1133CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Meletis K, Wirta V, Hede S-M, Nistér M, Lundeberg J, Frisén J (2006) p53 suppresses the self-renewal of adult neural stem cells. Development 133(2):363–369CrossRefPubMedGoogle Scholar
  31. 31.
    Armesilla-Diaz A, Bragado P, Del Valle I, Cuevas E, Lazaro I, Martin C, Cigudosa J, Silva A (2009) p53 regulates the self-renewal and differentiation of neural precursors. Neuroscience 158(4):1378–1389CrossRefPubMedGoogle Scholar
  32. 32.
    Lookeren Campagne MV, Gill R (1998) Tumor-suppressor p53 is expressed in proliferating and newly formed neurons of the embryonic and postnatal rat brain: Comparison with expression of the cell cycle regulators p21Waf1/Cip1, p27Kip1, p57Kip2, p16Ink4a, cyclin G1, and the proto-oncogene bax. J Comp Neurol 397(2):181–198CrossRefPubMedGoogle Scholar
  33. 33.
    Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K (2009) Modulation of microRNA processing by p53. Nature 460(7254):529–533CrossRefPubMedGoogle Scholar
  34. 34.
    Barros R, Pereira D, Callé C, Camilo V, Cunha AI, David L, Almeida R, Dias-Pereira A et al (2016) Dynamics of SOX2 and CDX2 expression in Barrett’s mucosa. Dis Markers 2016:1–7CrossRefGoogle Scholar
  35. 35.
    Zou G, Liu T, Guo L, Huang Y, Feng Y, Huang Q, Duan T (2016) miR-145 modulates lncRNA-ROR and Sox2 expression to maintain human amniotic epithelial stem cell pluripotency and β islet-like cell differentiation efficiency. Gene 591(1):48–57CrossRefPubMedGoogle Scholar
  36. 36.
    Morgado AL, Rodrigues CM, Solá S (2016) MicroRNA-145 regulates neural stem cell differentiation through the Sox2–Lin28/let-7 signaling pathway. Stem Cells 34(5):1386–1395CrossRefPubMedGoogle Scholar
  37. 37.
    Ozen M, Karatas OF, Gulluoglu S, Bayrak OF, Sevli S, Guzel E, Ekici ID, Caskurlu T et al (2015) Overexpression of miR-145–5p inhibits proliferation of prostate cancer cells and reduces SOX2 expression. Cancer Investig 33(6):251–258CrossRefGoogle Scholar
  38. 38.
    Liu X, Huang J, Chen T, Wang Y, Xin S, Li J, Pei G, Kang J (2008) Yamanaka factors critically regulate the developmental signaling network in mouse embryonic stem cells. Cell Res 18(12):1177–1189CrossRefPubMedGoogle Scholar
  39. 39.
    Nolan K, Mitchem MR, Jimenez-Mateos EM, Henshall DC, Concannon CG, Prehn JH (2014) Increased expression of microRNA-29a in ALS mice: functional analysis of its inhibition. J Mol Neurosci 53(2):231–241CrossRefPubMedGoogle Scholar
  40. 40.
    Annis RP, Swahari V, Nakamura A, Xie AX, Hammond SM, Deshmukh M (2016) Mature neurons dynamically restrict apoptosis via redundant pre-mitochondrial brakes. FEBS J 283:4569–4582CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Developmental Toxicology Laboratory, Systems Toxicology and Health Risk Assessment GroupCSIR- Indian Institute of Toxicology Research (CSIR-IITR)LucknowIndia
  2. 2.Academy of Scientific and Innovative Research (AcSIR)New DelhiIndia
  3. 3.Department of Biochemistry, School of Dental SciencesBabu Banarasi Das UniversityLucknowIndia

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