513 Role of regnase1 in UVA-induced oxidative DNA damage and apoptosis in HaCaT cells

Abstract

Regnase 1, also known as MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein), is an important regulator of cell apoptosis but its role in the skin has never been studied. UVA (320-400nm) radiation is a well-established oxidizing agent that may lead to cells damage and apoptosis. We found that an apoptotic dose of UVA increases MCPIP1 level in HaCaT cells and normal human keratinocytes within 6-9 h after the treatment. The effect was blocked by the combination of ascorbic acid (100 μM) and α-tocopherol (1mM) which proved the engagement of reactive oxygen species in MCPIP1 increase on both transcript and protein levels. We diminished MCPIP1 expression in HaCaT cells by RNA interference to study if cells with silenced MCPIP1 are less resistant to UVA-induced oxidative stress, DNA oxidative damage, apoptosis and cell death. To evaluate impact of MCPIP1 silencing on UVA induced oxidative stress and DNA damage we performed DHR123 test and comet assay. Apoptosis and necrosis were examined by Annexin-V/ 7-aminoactinomycin D double staining and flow cytometry as well caspase 3/7 activation. Si-RNA mediated down regulation of MCPIP1 expression after UVA radiation resulted in higher sensitivity to UVA-induced DNA damage and apoptosis. However silencing of MCPIP1 resulted in decreased viability of HaCaT cells measured by MTT assay. This suggest that MCPIP1 inhibition influences the activity of signaling pathways important for this cell growth and metabolism. We conclude that MCPIP1 is involved in UVA-induced oxidative DNA damage and apoptosis in HaCaT cells.