An Investigation of Keratin 15 Function by Small Interfering Ribonucleic Acid Technology

Abstract

The research presented in this thesis endeavours to further understanding of the cytoskeletal protein keratin 15 (K15), and the role this protein has on maintaining the stem cell niche. This study has focused on studying the role of K15 by using small interfering ribonucleic acid (siRNA) to knock-down K15 in different cell lines (HaCaT cells, MET 1, 2 and 4 cells and normal human epidermal keratinocytes). To observe the effect of K15 on stem cells, adhesion, migration, differentiation and proliferation were assessed following K15 knock-down by siRNA.
Although cell adhesion was not affected by K15 knock-down, cell spreading and morphology was affected. K15 knock-down cells spread more quickly than their control cells, and cells were larger following K15 ablation. Cell migration was studied using the scratch wound assay. Cells without K15 were less motile than K15-positive cells. Involucrin expression was observed as an indicator of differentiation. Following K15 knock-down, involucrin expression increased, indicating differentiation. Differentiation was assessed using the calcium switch assay, where higher levels of involucrin were observed in K15 siRNA transfected cells after only 6 hours in high calcium media. Cell proliferation was measured using the MTT assay, and K15 knock-down cells were shown to proliferate to a greater extent than control cells.
Tissue sections were also probed for K15 and CD34. K15 was observed in the basal layers of the epidermis and around the hair follicle in rat, mouse and human adult skin. This study also observed a CD34-positive/K15-positive cell population and a CD34-negative/K15-positive population in adult human interfollicular epidermis. For comparison between 2D cell culture and tissue sections, 3D (organotypic) cultures were utilised. Variable K15 expression was observed in the squamous cell carcnoma lines MET 1, MET 2 and MET 4. In HaCaT cells, K15 was observed to a greater extent at the base of the culture, similar to that observed in the epidermis.
K15 ablation has been shown to affect cell spreading and morphology during adhesion, cell migration, differentiation and proliferation. These results suggest that K15 does impact on the stem cell nature of keratinocytes, although the mechanisms require further investigation.