Cell and Tissue Research

, Volume 376, Issue 2, pp 295–308 | Cite as

Oxidative stress modulates the expression of apoptosis-associated microRNAs in bovine granulosa cells in vitro

  • Md Mahmodul Hasan SohelEmail author
  • Bilal Akyuz
  • Yusuf Konca
  • Korhan Arslan
  • Serpil Sariozkan
  • Mehmet Ulas Cinar
Regular Article


Despite its essential role in ovulation, oxidative stress (OS) has been found to be cytotoxic to cells, while microRNAs (miRNAs) are known as a major regulator of genes involved in cellular defense against cytotoxicity. However, a functional link between OS and miRNA expression changes in granulosa cells (GCs) remains to be investigated. Here, we investigate the OS modulation of apoptosis-associated miRNAs and their biological relevance in bovine GCs. Following the evaluation of cell viability, accumulation of reactive oxygen species (ROS), cytotoxicity and mitochondrial activity, we used a ready-to-use miRNA PCR array to identify differentially regulated miRNAs. The results showed that exposure to 150 μM H2O2 for 4 h creates remarkable signs of OS in GCs characterized by more than 50% loss of cell viability, higher nuclear factor erythroid 2–related factor 2 (NRF2) nuclear translocation, significantly (p < 0.05) higher abundance of antioxidant genes, significantly (p < 0.001) higher accumulation of ROS, lower mitochondrial activity and a higher (p < 0.001) number of apoptotic nuclei compared to that of the control group. miRNA expression analysis revealed that a total of 69 miRNAs were differentially regulated in which 47 and 22 miRNAs were up- and downregulated, respectively, in stressed GCs. By applying the 2-fold and p < 0.05 criteria, we found 16 miRNAs were upregulated and 10 miRNAs were downregulated. Target prediction revealed that up- and downregulated miRNAs potentially targeted a total of 6210 and 3575 genes, respectively. Pathway analysis showed that upregulated miRNAs are targeting the genes involved mostly in cell survival, intracellular communication and homeostasis, cellular migration and growth control and disease pathways. Our results showed that OS modulates the expression of apoptosis-associated miRNAs that might have effects on cellular or molecular damages.


microRNA Oxidative stress Granulosa cells Apoptosis Signaling pathways 



The authors are indebted to Res. Asst. Mahmut Kaliber; Sebahattin Koknur, DVM; and Ali Ergin, DVM, for their assistance during sample collection.


This research was supported by the Erciyes University Scientific Research Projects Coordination Unit, Project No: FOA-2015-5655.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Statement on the welfare of animals

This article does not contain any studies with live animals performed by any of the authors.


The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary material

441_2019_2990_MOESM1_ESM.pdf (117 kb)
ESM 1 (PDF 117 kb)
441_2019_2990_MOESM2_ESM.pdf (71 kb)
ESM 2 (PDF 70 kb)


  1. Agarwal A, Gupta S, Sharma RK (2005) Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 3(28).
  2. Agarwal A, Aponte-Mellado A, Premkumar BJ et al (2012) The effects of oxidative stress on female reproduction: a review. Reprod Biol Endocrinol 10:49. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Al-Gubory KH, Fowler PA, Garrel C (2010) The roles of cellular reactive oxygen species, oxidative stress and antioxidants in pregnancy outcomes. Int J Biochem Cell Biol 42:1634–1650. CrossRefPubMedPubMedCentralGoogle Scholar
  4. Appasamy M, Jauniaux E, Serhal P et al (2008) Evaluation of the relationship between follicular fluid oxidative stress, ovarian hormones, and response to gonadotropin stimulation. Fertil Steril 89:912–921. CrossRefPubMedGoogle Scholar
  5. Ávila J, González-Fernández R, Rotoli D et al (2016) Oxidative stress in granulosa-lutein cells from in vitro fertilization patients. Reprod Sci 23:1656–1661. CrossRefPubMedGoogle Scholar
  6. Carletti MZ, Fiedler SD, Christenson LK (2010) MicroRNA 21 blocks apoptosis in mouse periovulatory granulosa cells1. Biol Reprod 83:286–295. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cheng Y, Liu X, Zhang S et al (2009) MicroRNA-21 protects against the H2O2-induced injury on cardiac myocytes via its target gene PDCD4. J Mol Cell Cardiol 47:5–14. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Colicchia M, Campagnolo L, Baldini E et al (2014) Molecular basis of thyrotropin and thyroid hormone action during implantation and early development. Hum Reprod Update 20:884–904. CrossRefPubMedGoogle Scholar
  9. Cui S-Y, Wang R, Chen L-B (2014) MicroRNA-145: a potent tumour suppressor that regulates multiple cellular pathways. J Cell Mol Med 18:1913–1926. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Donadeu FX, Schauer SN, Sontakke SD (2012) Involvement of miRNAs in ovarian follicular and luteal development. J Endocrinol 215:323–334. CrossRefGoogle Scholar
  11. Du J-Y, Wang L-F, Wang Q, Yu L-D (2015) miR-26b inhibits proliferation, migration, invasion and apoptosis induction via the downregulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 driven glycolysis in osteosarcoma cells. Oncol Rep 33:1890–1898. CrossRefPubMedGoogle Scholar
  12. Duffy DM, Stouffer RL (2003) Luteinizing hormone acts directly at granulosa cells to stimulate periovulatory processes: modulation of luteinizing hormone effects by prostaglandins. Endocrine 22:249–256. CrossRefPubMedGoogle Scholar
  13. Fasanaro P, D’Alessandra Y, Di Stefano V et al (2008) MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 283:15878–15883. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Fedail JS, Zheng K, Wei Q et al (2014) Roles of thyroid hormones in follicular development in the ovary of neonatal and immature rats. Endocrine 46:594–604. CrossRefPubMedGoogle Scholar
  15. Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY (1996) Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol 134:793–799CrossRefPubMedGoogle Scholar
  16. Gonzalez G, Behringer RR (2009) Dicer is required for female reproductive tract development and fertility in the mouse. Mol Reprod Dev 76:678–688. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Hawkins SM, Andreu-Vieyra CV, Kim TH et al (2012) Dysregulation of uterine signaling pathways in progesterone receptor- Cre knockout of Dicer. Mol Endocrinol 26:1552–1566. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Hong X, Luense LJ, McGinnis LK et al (2008) Dicer1 is essential for female fertility and normal development of the female reproductive system. Endocrinology 149:6207–6212. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Hossain MM, Sohel MMH, Schellander K, Tesfaye D (2012) Characterization and importance of microRNAs in mammalian gonadal functions. Cell Tissue Res 349:679–690. CrossRefPubMedGoogle Scholar
  20. Hossini AM, Quast AS, Plötz M et al (2016) PI3K/AKT signaling pathway is essential for survival of induced pluripotent stem cells. PLoS One 11:e0154770. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hung C-M, Garcia-Haro L, Sparks CA, Guertin DA (2012) mTOR-dependent cell survival mechanisms. Cold Spring Harb Perspect Biol.
  22. Kennedy SG, Wagner AJ, Conzen SD et al (1997) The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal. Genes Dev 11:701–713. CrossRefPubMedGoogle Scholar
  23. Li M, Liu Y, Wang T et al (2011) Repertoire of porcine microRNAs in adult ovary and testis by deep sequencing. Int J Biol Sci 7:1045–1055CrossRefPubMedPubMedCentralGoogle Scholar
  24. Li J, Li J, Wei T, Li J (2016) Down-regulation of MicroRNA-137 improves high glucose-induced oxidative stress injury in human umbilical vein endothelial cells by up-regulation of AMPKα1. Cell Physiol Biochem 39:847–859. CrossRefPubMedGoogle Scholar
  25. Li M, Yang Y, Kuang Y et al (2017) miR-365 induces hepatocellular carcinoma cell apoptosis through targeting Bcl-2. Exp Ther Med 13:2279–2285. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Liu J, Tu F, Yao W et al (2016) Conserved miR-26b enhances ovarian granulosa cell apoptosis through HAS2-HA-CD44-Caspase-3 pathway by targeting HAS2. Sci Rep 6:21197. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Magenta A, Cencioni C, Fasanaro P et al (2011) miR-200c is upregulated by oxidative stress and induces endothelial cell apoptosis and senescence via ZEB1 inhibition. Cell Death Differ 18:1628–1639. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mancini A, Di Segni C, Raimondo S et al (2016) Thyroid hormones, oxidative stress, and inflammation. Mediat Inflamm 2016:6757154. CrossRefGoogle Scholar
  29. Manna PR, Stocco DM (2011) The role of specific mitogen-activated protein kinase signaling cascades in the regulation of steroidogenesis. J Signal Transduct 2011:821615. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Mebratu Y, Tesfaigzi Y (2009) How ERK1/2 activation controls cell proliferation and cell death: is subcellular localization the answer? Cell Cycle 8:1168–1175. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nagaraja AK, Andreu-Vieyra C, Franco HL et al (2008) Deletion of Dicer in somatic cells of the female reproductive tract causes sterility. Mol Endocrinol 22:2336–2352. CrossRefPubMedPubMedCentralGoogle Scholar
  32. Nakahara T, Iwase A, Nakamura T et al (2012) Sphingosine-1-phosphate inhibits H2O2-induced granulosa cell apoptosis via the PI3K/Akt signaling pathway. Fertil Steril 98:1001–1008.e1. CrossRefPubMedGoogle Scholar
  33. Ngamwongsatit P, Banada PP, Panbangred W, Bhunia AK (2008) WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line. J Microbiol Methods 73:211–215. CrossRefPubMedGoogle Scholar
  34. Ni J, Wang X, Chen S et al (2015) MicroRNA let-7c-5p protects against cerebral ischemia injury via mechanisms involving the inhibition of microglia activation. Brain Behav Immun 49:75–85. CrossRefPubMedGoogle Scholar
  35. Noferesti SS, Sohel MMH, Hoelker M et al (2015) Controlled ovarian hyperstimulation induced changes in the expression of circulatory miRNA in bovine follicular fluid and blood plasma. J Ovarian Res 8:81. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Pastorelli LM, Wells S, Fray M et al (2009) Genetic analyses reveal a requirement for Dicer1 in the mouse urogenital tract. Mamm Genome 20:140–151. CrossRefPubMedGoogle Scholar
  37. Peng Y, Croce CM (2016) The role of microRNAs in human cancer. Signal Transduct Target Ther 2016:1–9. CrossRefGoogle Scholar
  38. Ray PD, Huang B-W, Tsuji Y (2012) Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 24:981–990. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Romaine SPR, Tomaszewski M, Condorelli G, Samani NJ (2015) MicroRNAs in cardiovascular disease: an introduction for clinicians. Heart 101:921–928. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Sangokoya C, Telen MJ, Chi J-T et al (2010) microRNA miR-144 modulates oxidative stress tolerance and associates with anemia severity in sickle cell disease. Blood 116:4338–4348. CrossRefPubMedPubMedCentralGoogle Scholar
  41. Shkolnik K, Tadmor A, Ben-Dor S et al (2011) Reactive oxygen species are indispensable in ovulation. Proc Natl Acad Sci U S A 108:1462–1467. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Simpson K, Wonnacott A, Fraser DJ, Bowen T (2016) MicroRNAs in diabetic nephropathy: from biomarkers to therapy. Curr Diab Rep 16:35. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Sohel MMH (2016) Extracellular/circulating microRNAs: release mechanisms, functions and challenges. Achiev Life Sci 10:175–186. CrossRefGoogle Scholar
  44. Sohel MMH, Cinar MU (2015) Advancement in molecular genetics to understand the molecular reproduction of livestock – follicular development. Res Agric Livest Fish 1:47–60. CrossRefGoogle Scholar
  45. Sohel MMH, Hoelker M, Noferesti SS et al (2013) Exosomal and non-exosomal transport of extra-cellular microRNAs in follicular fluid: implications for bovine oocyte developmental competence. PLoS One.
  46. Sohel MH, Cinar MU, Kalibar M et al (2016) Appropriate concentration of hydrogen peroxide and Sulforaphane for granulosa cells to study oxidative stress in vitro. J Biotechnol 231:S24. CrossRefGoogle Scholar
  47. Sohel MMH, Konca Y, Akyuz B et al (2017) Concentration dependent antioxidative and apoptotic effects of sulforaphane on bovine granulosa cells in vitro. Theriogenology 97:17–26. CrossRefPubMedGoogle Scholar
  48. Sohel MMH, Amin A, Prastowo S, et al (2018) Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway. Cell Tissue Res 1–13.
  49. Son Y, Cheong Y-K, Kim N-H et al (2011) Mitogen-activated protein kinases and reactive oxygen species: how can ROS activate MAPK pathways? J Signal Transduct 2011:792639. CrossRefPubMedPubMedCentralGoogle Scholar
  50. Sontakke SD, Mohammed BT, McNeilly AS, Donadeu FX (2014) Characterization of microRNAs differentially expressed during bovine follicle development. Reproduction 148:271–283. CrossRefPubMedGoogle Scholar
  51. Tesfaye D, Salilew-Wondim D, Gebremedhn S et al (2017) Potential role of microRNAs in mammalian female fertility. Reprod Fertil Dev 29:8–23. CrossRefGoogle Scholar
  52. Thulasingam S, Massilamany C, Gangaplara A et al (2011) miR-27b*, an oxidative stress-responsive microRNA modulates nuclear factor-kB pathway in RAW 264.7 cells. Mol Cell Biochem 352:181–188. CrossRefPubMedGoogle Scholar
  53. Wang J, Wang X, Wu G et al (2013) MiR-365b-3p, down-regulated in retinoblastoma, regulates cell cycle progression and apoptosis of human retinoblastoma cells by targeting PAX6. FEBS Lett 587:1779–1786. CrossRefPubMedGoogle Scholar
  54. Wang C, Li D, Zhang S et al (2016) MicroRNA-125a-5p induces mouse granulosa cell apoptosis by targeting signal transducer and activator of transcription 3. Menopause 23:100–107. CrossRefPubMedGoogle Scholar
  55. Wei Q, Fedail JS, Kong L et al (2018) Thyroid hormones alter estrous cyclicity and antioxidative status in the ovaries of rats. Anim Sci J 89:513–526. CrossRefPubMedGoogle Scholar
  56. Xu L, Sun H, Zhang M et al (2017) MicroRNA-145 protects follicular granulosa cells against oxidative stress-induced apoptosis by targeting Krüppel-like factor 4. Mol Cell Endocrinol 452:138–147. CrossRefPubMedGoogle Scholar
  57. Yan G, Zhang L, Fang T et al (2012) MicroRNA-145 suppresses mouse granulosa cell proliferation by targeting activin receptor IB. FEBS Lett 586:3263–3270. CrossRefPubMedGoogle Scholar
  58. Yang M, Huang C-Z (2015) Mitogen-activated protein kinase signaling pathway and invasion and metastasis of gastric cancer. World J Gastroenterol 21:11673. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Yao G, Yin M, Lian J et al (2010) MicroRNA-224 is involved in transforming growth factor-β-mediated mouse granulosa cell proliferation and granulosa cell function by targeting Smad4. Mol Endocrinol 24:540–551. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Yin M, Wang X, Yao G et al (2014) Transactivation of microRNA-320 by microRNA-383 regulates granulosa cell functions by targeting E2F1 and SF-1 proteins. J Biol Chem 289:18239–18257. CrossRefPubMedPubMedCentralGoogle Scholar
  61. Yuan S, Ortogero N, Wu Q et al (2014) Murine follicular development requires oocyte DICER, but not DROSHA1. Biol Reprod.
  62. Zhang M, Zhang Q, Hu Y et al (2017a) miR-181a increases FoxO1 acetylation and promotes granulosa cell apoptosis via SIRT1 downregulation. Cell Death Dis 8:e3088. CrossRefPubMedPubMedCentralGoogle Scholar
  63. Zhang Y-L, Wang R-C, Cheng K et al (2017b) Roles of Rap1 signaling in tumor cell migration and invasion. Cancer Biol Med 14:90–99. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Md Mahmodul Hasan Sohel
    • 1
    • 2
    Email author
  • Bilal Akyuz
    • 3
  • Yusuf Konca
    • 2
  • Korhan Arslan
    • 3
  • Serpil Sariozkan
    • 4
  • Mehmet Ulas Cinar
    • 2
  1. 1.Genome and Stem Cell CentreErciyes UniversityKayseriTurkey
  2. 2.Department of Animal Science, Faculty of AgricultureErciyes UniversityKayseriTurkey
  3. 3.Department of Genetics, Faculty of Veterinary ScienceErciyes UniversityKayseriTurkey
  4. 4.Department of Fertility and Artificial Insemination, Faculty of Veterinary ScienceErciyes UniversityKayseriTurkey

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