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Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes

  • Peder Aabel
  • Tor Paaske Utheim
  • Ole Kristoffer Olstad
  • Helge Rask-Andersen
  • Rodney James Dilley
  • Magnus von Unge
Research Article

Abstract

The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.

Keywords

tympanic membrane gene expression microRNA migration FOXC2 HOX genes 

Notes

Acknowledgements

We would like to thank Marja Boström for expertise in the cell preparations, Torstein Lyberg and Jon Roger Eidet for the technical assistance in the lab, Anne Marie Siebke Trøseid for running the PCR analyses, Sumana Kalayanasundaram and Hilde Loge Nilsen at EpiGen, AHUS, for assistance in the biostatistical analyses and data presentation, and Fredrik Maxwell Hermansen for assistance in preparing the figures.

Funding information

The study was funded by Akershus University Hospital, The South-Eastern Norway Regional Health Authority and the University of Oslo.

Compliance with Ethical Standards

The study was performed according to the Declaration of Helsinki and was approved by the South-Eastern Norway Regional Committee for Medical Research Ethics (2010/1345).

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

10162_2018_660_MOESM1_ESM.pdf (407 kb)
Table S1 (PDF 406 kb)
10162_2018_660_MOESM2_ESM.pdf (167 kb)
Table S2 g:Profiler analysis with all significant gene ontology enrichment categories for the genes of high expression. (PDF 167 kb)
10162_2018_660_MOESM3_ESM.pdf (138 kb)
Table S3 g:Profiler analysis with all significant gene ontology enrichment categories for the genes of low expression. (PDF 137 kb)

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Copyright information

© Association for Research in Otolaryngology 2018

Authors and Affiliations

  • Peder Aabel
    • 1
    • 2
    • 3
  • Tor Paaske Utheim
    • 1
    • 4
  • Ole Kristoffer Olstad
    • 1
  • Helge Rask-Andersen
    • 5
  • Rodney James Dilley
    • 6
    • 7
  • Magnus von Unge
    • 2
    • 3
    • 8
  1. 1.Department of Medical BiochemistryOslo University HospitalOsloNorway
  2. 2.Ear, Nose and Throat Department, Division of SurgeryAkershus University HospitalLørenskogNorway
  3. 3.Division of Surgery, Institute of Clinical MedicineUniversity of OsloOsloNorway
  4. 4.Institute of Oral Biology, Faculty of DentistryUniversity of OsloOsloNorway
  5. 5.Department of OtolaryngologyUppsala University HospitalUppsalaSweden
  6. 6.Ear Science Institute AustraliaPerthAustralia
  7. 7.Ear Sciences Centre and Centre for Cell Therapy and Regenerative MedicineUniversity of Western AustraliaNedlandsAustralia
  8. 8.Centre for Clinical ResearchUniversity of UppsalaVästeråsSweden

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