Mechanism Study Of METTL3 Promoting UVB-induced Skin Pigmentation

Objectives: Melasma,freckles and post-inflammatory hyperpigmentation are typical pigmented skin diseases,mainly manifested as hyperpigmentation of the skin.The disfiguring feature brings huge physical and psychological burdens to patients.The current clinical treatment methods cannot completely remove the lesions,and the symptoms are prone to recurring after treatment.Thus,the new research on the pathogenesis of pigmented skin diseases is increasingly urgent.The number of melanin granules produced by melanocytes in the basal layer of the skin is the key to determining skin pigmentation,and ultraviolet is an important exogenous stimulus for melanogenesis.N6-methyladenosine(m6A)modification is one of the important epigenetic regulatory mechanisms involved in the regulation of a variety of biological behaviors,but its role and mechanism in UV-induced melanogenesis remain unclear.This study aimed to explore the role and possible regulatory mechanisms of m6 A modification in UVB-induced melanogenesis.Methods: After low-dose UVB irradiation of human melanocytes,m6 A chip was used to explore the effect of UVB on the overall modification level of m6 A in melanocytes.Dot blotting and immunofluorescence experiments were used to detect the content of m6 A in cells.The GEPIA database analyzed the association of m6 A major methyltransferases with MITF,a key transcription factor for melanogenesis,in non-light and illuminated skin tissues,and further explored the potential link between m6 A modification and light-induced melanogenesis.Human melanoma cell line(MNT1)was used as the experimental model cell,and the content of melanin and the expression levels of melanogenesis-related genes were detected after different doses of UVB irradiation by masson-fontana staining,real-time quantitative PCR and western blotting.After overexpression of METTL3 and knockdown of METTL3,masson-fontana staining,real-time quantitative PCR,western blotting,dot blotting and immunofluorescence experiments were used to detect the content of melanin,the expression levels of melanogenesis-related genes,the levels of tyrosinase activity and m6 A content.Tissue hematoxylin and eosin staining,masson-fontana staining and immunofluorescence experiments were used to detect the expression level of METTL3 in clinical pigmented nevus samples.After overexpression of METTL3,the possible mechanism of METTL3 regulating melanogenesis was preliminarily explored by using transcriptome high-throughput sequencing data and SRAMP website.YAP1 and TEAD1,the main effector molecules of Hippo signaling pathway,were detected by western blotting.Results: The m6 A chip,dot blotting and immunofluorescence experiments confirmed that low-dose UVB can up-regulate the overall modification level of m6 A in human melanocytes and MNT1 cells.The GEPIA public database predicted that METTL3 was positively correlated with the expression of MITF,a key transcription factor in the regulation of melanogenesis.The melanin content and the expression of melanogenesis-related genes were up-regulated after overexpression of METTL3,while the melanin content and the expression of melanogenesis-related genes were down-regulated after knockdown of METTL3.Tissue immunofluorescence experiments found that the expression of METTL3 was significantly up-regulated in nevus with high melanin content.After overexpression of METTL3,the high-throughput transcriptome sequencing found that melanogenesis and Hippo signaling pathways were significantly up-regulated.Western blotting experiment confirmed that after overexpression of METTL3,the main effector molecules of Hippo signaling pathway,YAP1 and TEAD1,were up-regulated,and after knockdown of METTL3,the expressions of YAP1 and TEAD1 were down-regulated.The SRAMP website predicted that there were 11 potential m6 A modification sites on YAP1 m RNA.Conclusions: The study confirmed that UVB irradiation promotes overall m6 A modification levels in melanocytes.UVB-induced METTL3 can promote melanogenesis by regulating the expression of YAP1 and TEAD1,the Hippo signaling pathway effector molecules.