Characterization of the hacat keratinocyte cell line in which PLAGL2 is silenced by the CRISPR/Cas9 method

Keratinocytes are the main cell of the multilayered epidermis, which acts as a protective barrier against the external environment. The p63 transcription factor coordinates keratinocyte differentiation, which results in the formation and regeneration of the epidermis. RIPK4 is a protein which has an important role in keratinocyte differentiation. In our previous studies in order to identify the mechanism of RIPK4 function in this process, we screened human keratinocyte cDNA library using RIPK4 as a bait with large scale yeast-two hybrid method and found transcription factor PLAGL2 as a RIPK4 interacting partner. This interaction has been confirmed both in yeast with a small-scale yeast-two hybrid assay and in human cell lines with different protein-protein interaction methods. Interaction between RIPK4 and PLAGL2 and transcriptional regulation of PLAGL2 by p63 similar to RIPK4, revealed the hypothesis that PLAGL2, like RIPK4, has a role in keratinocytes. The studies so far have mainly focused on defining the role of PLAGL2 in carcinogenesis. There is no study in the literature showing the role of PLAGL2 in keratinocytes. In this context, our thesis aims to investigate whether PLAGL2 has a role in basic cellular functions in keratinocytes. For this purpose as a first step, PLAGL2 expression was silenced in the HaCaT keratinocyte cell line by using CRISPR/Cas9n gene editing system. Afterwards, PLAGL2 knock-out cells were analyzed in terms of basic cellular functions including morphology, growth rate, cell cycle, migration and cell death. We also investigated the WNT/β-catenin signaling pathway in these cells, which has been demonstrated to be controlled by PLAGL2 in different cell types. In conclusion, silencing of PLAGL2 did not cause any change in the cell cycle in keratinocytes, but increased cell death and cell migration significantly. Morphological examination of knock-out cells revealed increased cell size and cell migration associated filopod structures. In addition, WNT/β-catenin signaling pathway activity was down-regulated in PLAGL2 knock-out cells compared to control. Our results provide clues that PLAGL2 may have a role in skin development.