Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes
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.
Keywordstympanic membrane gene expression microRNA migration FOXC2 HOX genes
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.
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.
- Beaufort N, Leduc D, Eguchi H, et al (2007) The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions. 292:L1263–L1272. https://doi.org/10.1152/ajplung.00191.2006
- Hwang J-A, Lee BB, Kim Y et al (2014) HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non-small cell lung cancer. Mol Carcinog. https://doi.org/10.1002/mc.22180
- Michel M, Torok N, Godbout MJ et al (1996) Keratin 19 as a biochemical marker of skin stem cells in vivo and in vitro: keratin 19 expressing cells are differentially localized in function of anatomic sites, and their number varies with donor age and culture stage. J Cell Sci 109(Pt 5):1017–1028PubMedGoogle Scholar
- Mimoso C, Lee D-D, Zavadil J et al (2013) Analysis and meta-analysis of transcriptional profiling in human epidermis. Methods Mol Biol. https://doi.org/10.1007/7651_2013_60
- Severino P, Bruggemann H, Andreghetto FM et al (2013) MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation. BMC Cancer 13:533. https://doi.org/10.1186/1471-2407-13-533 CrossRefPubMedPubMedCentralGoogle Scholar
- Yang BL, Zhang Y, Cao L, Yang BB (1999) Cell adhesion and proliferation mediated through the G1 domain of versican. J Cell Biochem 72:210–220. https://doi.org/10.1002/(SICI)1097-4644(19990201)72:2<210::AID-JCB5>3.0.CO;2-E CrossRefPubMedGoogle Scholar
- Yoshinaga S, Nakahori Y, Yasuoka S (1998) Fibrinogenolytic activity of a novel trypsin-like enzyme found in human airway. J Med Investig 45:77–86Google Scholar