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Induction of apoptosis in ovarian cancer cells by miR-493-3p directly targeting AKT2, STK38L, HMGA2, ETS1 and E2F5

  • Michael Kleemann
  • Helga Schneider
  • Kristian Unger
  • Jeremias Bereuther
  • Simon Fischer
  • Philip Sander
  • E. Marion Schneider
  • Pamela Fischer-Posovszky
  • Christian U. Riedel
  • René Handrick
  • Kerstin Otte
Original Article
  • 144 Downloads

Abstract

Apoptosis is a form of directed programmed cell death with a tightly regulated signalling cascade for the destruction of single cells. MicroRNAs (miRNAs) play an important role as fine tuners in the regulation of apoptotic processes. MiR-493-3p mimic transfection leads to the induction of apoptosis causing the breakdown of mitochondrial membrane potential and the activation of Caspases resulting in the fragmentation of DNA in several ovarian carcinoma cell lines. Ovarian cancer shows with its pronounced heterogeneity a very high death-to-incidence ratio. A target gene analysis for miR-493-3p was performed for the investigation of underlying molecular mechanisms involved in apoptosis signalling pathways. Elevated miR-493-3p levels downregulated the mRNA and protein expression levels of Serine/Threonine Kinase 38 Like (STK38L), High Mobility Group AT-Hook 2 (HMGA2) and AKT Serine/Threonine Kinase 2 (AKT2) by direct binding as demonstrated by luciferase reporter assays. Notably, the protein expression of RAF1 Proto-Oncogene, Serine/Threonine Kinase (RAF1) was almost completely downregulated by miR-493-3p. This interaction, however, was indirect and regulated by STK38L phosphorylation. In addition, RAF1 transcription was diminished as a result of reduced transcription of ETS proto-oncogene 1 (ETS1), another direct target of miR-493-3p. Taken together, our observations have uncovered the apoptosis inducing potential of miR-493-3p through its regulation of multiple target genes participating in the extrinsic and intrinsic apoptosis pathway.

Keywords

MicroRNA Cancer Signalling pathways Targets RAF1 

Notes

Acknowledgements

This study was funded by the Postgraduate Scholarships Act of the Ministry for Science, Research and Arts of the Federal State Government of Baden-Wuerttemberg, Germany. Further acknowledgements address the International Graduate School in Molecular Medicine of Ulm University, Germany, for scientific encouragement and support to Michael Kleemann. Pamela Fischer-Posovszky receives funding from the Baden-Württemberg Stiftung, Germany. The results published here are based on data generated by the TCGA Research Network. In addition, the authors are grateful to Dr. Anne-Marie Mes-Masson (Centre Hospitalier de l´Université de Montréal, Canada) for providing us with the TOV21G and TOV112D cells as well as to Alex Shu-Wing Ng (Department of Obstetrics, Gynecology and Reproductive Biology, the Brigham and Women’s Hospital, Boston, USA) for the HOSE 2170 cells. The OVCAR3, A2780 and A2780-cis cells were kindly provided by Verena Jendrossek (Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Germany).

Compliance with ethical standards

Ethical standards

The authors declare that the experiments comply with the current laws of the Federal Republic of Germany.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

18_2018_2958_MOESM1_ESM.tif (71 kb)
Supplementary Figure 1 Determination of miR-493 expression after transfection. For determination of miR-493 expression after transfection, cells were seeded and transfected as described in Figure 2. The miRNA expression of miR-493 was normalized to the CT value of U6 snRNA and the NT-transfected control employing the Livak method [72]. Statistical significance was tested by an unpaired t test. [n = 3 replicates; mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001] (TIFF 70 kb)

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Copyright information

© Springer Nature Switzerland AG 2018

Authors and Affiliations

  • Michael Kleemann
    • 1
    • 2
  • Helga Schneider
    • 1
  • Kristian Unger
    • 3
  • Jeremias Bereuther
    • 4
  • Simon Fischer
    • 5
  • Philip Sander
    • 6
  • E. Marion Schneider
    • 6
  • Pamela Fischer-Posovszky
    • 7
  • Christian U. Riedel
    • 8
  • René Handrick
    • 1
  • Kerstin Otte
    • 1
  1. 1.Institute of Applied BiotechnologyUniversity of Applied Sciences BiberachBiberachGermany
  2. 2.Faculty of MedicineUniversity of UlmUlmGermany
  3. 3.Research Unit Radiation Cytogenetics, Helmholtz Zentrum München Helmholtz Center MunichGerman Research Center for Environmental HealthNeuherbergGermany
  4. 4.Apceth Biopharma GmbHOttobrunnGermany
  5. 5.Boehringer Ingelheim Pharma GmbH & Co. KG, Bioprocess and Analytical DevelopmentBiberachGermany
  6. 6.Division of Experimental AnesthesiologyUniversity Medical Center UlmUlmGermany
  7. 7.Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics and Adolescent MedicineUniversity Medical Center UlmUlmGermany
  8. 8.Faculty of MedicineUniversity of UlmUlmGermany

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