A Preliminary Study On The Inhibitory Effect Of Transcription Factor PBX1 On UVB-induced Melanogenesis In Melanocytes And Its Mechanisms

Background:Melanocytes are found in hair follicles,the epidermis and the eyes and are responsible for producing melanin,which is the basis of pigmentation in hair and skin.Ultraviolet radiation(UVR),as the main external factor,can regulate the production of melanin through the production of reactive oxygen species(ROS).Among them,UVB plays a major role in the production of pigmentation by stimulating the initiation of melanin synthesis and leading to the accumulation of pigment on the skin surface.The preb-cell leukemia homeobox 1(PBX1)gene encodes the transcription factor PBX1,which is widely expressed in a variety of tissues and plays a role in various developmental processes.PBX1 not only plays an important role in embryonic development,but also plays a regulatory role in many organs.In this study,by constructing PIG1 cells with stable overexpression of PBX1 and using UVB to construct a melanin generation model,the effects of PBX1 on the biological behavior and function of PIG1 cells were investigated,and the effects of PBX1 on the function of PIG1 cells,melanin generation and protection were preliminarily explored in the UVB induced melanin generation model.Aim:To study the effects of transcription factor PBX1 on the biological behavior and function of PIG1 cells,and to explore the role and mechanism of PBX1 in the production of melanin,ROS and DNA damage in PIG1 cells induced by UVB.To ensure the survival,genomic stability and normal function of epidermal melanocytes is very important for inhibiting the occurrence of pigment disorders and tumors.Therefore,this study attempts to comprehensively understand the role of PBX1 in melanocytes,better understand the biology of melanocytes and the role and influence of PBX1 on melanocytes,so as to provide theoretical basis for the treatment of pigment disorders and melanoma.Method:1.Lentivirus was used to construct stable overexpressed cell lines.The cell growth curve was plotted by cell counting method and the proliferation ability was tested by clonal formation experiment.Cell migration ability was detected by cell scratch assay.Apoptosis was detected by flow cytometry,and apoptosis-related proteins(PARP1,PAR,Cleaved Casepase 3,AIF,and Cyt C)were detected by Western Blot.2.The content of melanin in cells was detected by alkali lysis.The change of tyrosinase activity was detected by spectrophotometry.The expression of melaninrelated proteins was detected by Western Blot.The ROS production was detected by flow cytometry and DCFH-DA dye staining.3.The survival rate was measured by MTT method.Cell status was observed at living cell workstation.The content of melanin was detected by alkali lysis method and the activity of tyrosinase was detected by spectrophotometry.The model of melanin production by UVB irradiation was established by selecting appropriate dose of UVB irradiation.4.After UVB irradiation of cells:The content of melanin was determined by alkali lysis.The change of tyrosinase activity was detected by spectrophotometry.Western Blot was used to detect proteins related to melanin production.The ROS changes were detected by flow cytometry and DCFH-DA dye staining.DNA damage was detected by alkaline comet assay.Western Blot was used to detect proteins associated with DNA damage repair.Result:1.The effect of PBX1 on PIG1 cell biologyThe proliferation and clonogenesis of PIG1 cells were enhanced after PBX1 overexpression.Enhanced migration ability;The apoptosis rate decreased.2.Effects of PBX1 on melanin and ROS production in PIG1 cellsAfter PBX1 overexpression,melanin production was decreased in PIG1 cells.And decreased tyrosinase activity;Decreased expression of proteins related to melanin production;ROS generation decreases.3.UVB induced melanin generation modelThere was no significant difference in cell activity at 90 mJ/cm2 when treated with different UVB radiation doses.The most melanin production;Tyrosinase activity was the strongest,so 90 mJ/cm2 was selected as the experimental dose.4.PBX1 reduced the production of melanin and ROS in PIG1 cells induced by UVB irradiationPBX1 overexpression was treated with 90 mJ/cm2 UVB irradiation:(1)The content of melanin in UVB irradiation group was significantly increased;Tyrosinase activity increased;Increased expression of proteins related to melanin production;ROS generation increases.(2)The content of melanin in PBX1 group was lower than that in Control group and Vector group exposed to UVB.Tyrosinase activity decreased;Decreased expression of proteins associated with melanin production;ROS generation decreases.(3)At the same time,increased DNA damage caused by UVB irradiation and increased expression of DNA damage repair related proteins were detected,but PBX1 could reduce the increase of related indexes caused by UVB irradiation.Conclusion:1.Overexpression of PBX1 can promote the proliferation and migration of PIG1 cells,and reduce the apoptosis of PIG1 cells.2.UVB irradiation resulted in increased melanin production and ROS accumulation in PIG1 cells,accompanied by increased DNA damage.3.Overexpression of PBX1 reduced melanin production and decreased increased melanin production,ROS accumulation,and DNA damage caused by UVB exposure.