Mechanisms underlying repeated ultraviolet A radiation-induced DNA damage in human skin fibroblasts

Abstract

Objective To evaluate the effects of repeated ultraviolet A (UVA) radiation on DNA damage, repair and replication processes in human skin fibroblasts, and to explore their mechanisms. Methods Fibroblasts were isolated from the circumcised foreskins of 3 children in the Department of Urological Surgery, Third Affiliated Hospital, Sun Yat-sen University, and subjected to a primary culture. Cultured human skin fibroblasts of 3rd-10th passages were divided into 2 groups: UVA group treated with repeated UVA radiation to establish a chronic photodamaged cell model, and control group receiving no treatment. Cell counting kit 8 (CCK-8) assay, β-galactosidase staining and flow cytometry were performed to assess cellular proliferative activity, and determine the proportion of photoaged cells and apoptosis rate respectively. Reverse transcription reaction was performed to establish a differentially expressed cDNA library, which was then subjected to high-throughput sequencing. The cDNA sequencing results were compared between the control group and UVA group, and the differentially expressed genes were analyzed in Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The data were compared between the two groups by using two independent sample t test. Results Compared with the control group, the UVA group showed significantly decreased cellular proliferative activity (72.0% ± 5.2% vs. 96.0% ± 3.7%, t = 6.51, P < 0.05) , but significantly increased proportion of photoaged cells (79.7% ± 5.2% vs. 6.4% ± 0.8%, t=24.12, P < 0.05) and apoptosis rate (29.0% ± 3.3% vs. 6.0% ± 5.9%, t= 5.89, P < 0.05) . Among the key enzymes involved in DNA mismatch repair, replication and base excision repair processes, the expression of DNA ligase 1 (Lig1) , ribonuclease (RNase) H2A and helicase Dna2 in the UVA group was 0.47 ± 0.13, 0.44 ± 0.07 and 0.49 ± 0.11 times (all P < 0.01) that in the control group respectively. After the UVA-induced chronic photodamage in the human skin fibroblasts, After the UVA-induced chronic photodamage in the human skin fibroblasts, Lig expression decrease could block DNA single-base excision repair, Lig and Lig1 expression decrease could block DNA multiple-base excision repair, Lig1 expression decrease could block DNA mismatch repair, and RNaseH2A, Dna2 and Lig1 expression decrease could block DNA replication. Conclusion Repeated UVA radiation can change the expression of key enzymes involved in DNA base excision repair, DNA mismatch repair and DNA replication processes in skin fibroblasts, and then affect DNA repair and DNA replication processes in skin fibroblasts. Key words: Fibroblasts; Ultraviolet rays; DNA damages; DNA repair; Skin aging