Increased 5-hydroxymethylcytosine and Ten-eleven Translocation Protein Expression in Ultraviolet B-irradiated HaCaT Cells

Background: DNA hydroxymethylation refers to a chemical modification process in which 5-methylcytosine (5mC) is catalyzed to 5- hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins. Recent studies have revealed that aberrant TETs expression or 5hmC level may play important roles in the occurrence and development of various pathological and physiological processes including cancer and aging. This study aimed to explore the relation between aberrant DNA hydroxymethylation with skin photoaging and to investigate the levels of TETs, 5mC, and 5hmC expression 24 h after 40 mJ/cm2 and 80 mJ/cm2 doses of ultraviolet B (UVB) irradiation to HaCaT cells. Methods: To explore whether aberrant DNA hydroxymethylation is also related to skin photoaging, 40 mJ/cm2 and 80 mJ/cm2 doses of UVB were chosen to treat keratinocytes (HaCaT cells). After 24 h of UVB irradiation, 5mC and 5hmC levels were determined by immunohistochemistry (IHC) and immunofluorescence (IF), and at the same time, the expression levels of matrix metalloproteinase 1 (MMP-1) and TETs were assessed by reverse transcription-polymerase chain reaction or Western blot analysis. Results: After 40 mJ/cm2 and 80 mJ/cm2 doses of UVB exposure, both IHC and IF results showed that 5hmC levels increased significantly, while the 5mC levels did not exhibit significant changes in HaCaT cells, compared with HaCat cells without UVB exposure. Moreover, compared with HaCat cells without UVB exposure, the levels of TET1, TET2, and TET3 mRNA and protein expression were significantly upregulated (mRNA: P = 0.0022 and 0.0043 for TET1; all P < 0.0001 for TET2; all P = 0.0006 for TET3; protein: P = 0.0012 and 0.0006 for TET1; all P = 0.0022 for TET2; and all P = 0.0002 for TET3), and the levels of MMP-1 mRNA expression increased dose dependently in 40 mJ/cm2 and 80 mJ/cm2 UVB-irradiated groups. Conclusion: UVB radiation could cause increased 5hmC and TET expression, which might become a novel biomarker in UVB-related skin aging