Knockout of mutant TP53 in the HaCaT cells enhances their migratory activity

About authors

Orekhovich Research Institute of Biomedical Chemistry, Moscow, Russia

Correspondence should be addressed: Alexander L. Rusanov
Pogodinskaya, 10, str. 8, Moscow, 119121, Russia; moc.liamg@vonasur.l.rednaxela

About paper

Funding: the study was carried out as part of the Program for fundamental research in the Russian Federation for the long-term period (2021–2030) (№ 122022800481-0).

Author contribution: Luzgina NG, Rusanov AL — study concept; Romashin DD, Kozhin PM, Luzgina NG, Rusanov AL — study design and literature review; Romashin DD, Kozhin PM — study planning and execution; Kozhin PM, Romashin DD, Luzgina NG, Rusanov AL — data analysis and interpretation; Kozhin PM, Romashin DD — manuscript writing; Kozhin PM, Romashin DD, Luzgina NG, Rusanov AL — manuscript editing.

Compliance with ethical standards: the study was carried out in accordance with the Declaration of Helsinki of the World Medical Association and all its revisions.

Received: 2022-12-12 Accepted: 2022-12-24 Published online: 2022-12-30

The HaCaT cell line represents the spontaneously immortalized non-carcinogenic human keratinocytes that are used as a model for studying the function of normal human keratinocytes. There are two TP53 alleles in the HaCaT cell genome, which comprise two gain-of-function (GOF) mutations acquired through spontaneous immortalization (mutTP53). Mutations result in the increased proliferation rate and violation of the stratification program. The study was aimed to assess the effects of the mutTP53 gene knockout on the HaCaT keratinocytes capability of proliferation and migration in the in vitro model of epidermal injury and regeneration (scratch test), and on the ability to form stratified epithelium in the organotypic epidermal model. To perform the scratch-test, cells were cultured until monolayer was formed, then the standardized injury was created. The organotypic model was obtained by growing keratinocytes in the polycarbonate membrane inserts with the pore size of 0.4 μm at the interface between the phases (air-liquid). It has been shown that the mutant TP53 gene knockout results in the increased migration capability of the HaCaT keratinocytes: in the HaCaT with the mutTP53 knockout, the defect closure occurred faster than in the appropriate group of the WT HaCaT (p < 0.05), on day three the defect size was 12% ± 3% and 66% ± 5% of the initial size. There is evidence that mutant TP53 in the HaCaT cells is a negative regulator of the laminin 5 expression (LAMC2 expression was 9.96 ± 1.92 times higher in the cells with the mutTP53 knockout, p < 0.05), however, this does not promote normalization of the program of epithelial differentiation and stratification followed by formation of the stratum corneum in the organotypic model.

Keywords: CRISPR/Cas9, HaCaT, р53, keratinocyte differentiation, knockout, migration