| UVB can increase the number of melanocytes, activate the intracellular tyrosinase,which is one of the most important factors for pigmentation, and can also promote the transportation of melanosome body from melanocytes to the surrounding keratinocytes.Meanwhile, UVB-related pigmentation skin disease is a common clinical practice in dermatology, and the common clinical features include the pigmentation spots or spots of the face and neck. Microphthalmia-associated Transcription Factor (MITF) is a key molecule in the regulation of melanocyte cytochrome synthesis, which can regulate the transcriptional activity of more than 20 genes, including the regulation of melanogenesis genes, melanin body transporter gene, a receptor that mediates the neuroendocrine signal synthesized by the physiological melanogenesis, and the structural protein of the melanogenesis. microRNA (miRNA) is a class of single-stranded, non-coding small RNAs with a length of about 22 to 25 nt and can be combined with targeted messenger RNA (mRNA) to promote its degradation or inhibition of its protein translation. In the previous study, it was found that UVB irradiation could change the miRNA expression profile of immortalized human epidermal melanocytes (Pig-1), and the expression of miRNA-340 was time-dependent,that is, the expression of miRNA-340 was increased at 3h after UVB irradiation. The growth factor of dendritic growth factor RhoA, which promotes the growth of melanocytes by dendritic growth can be targeted by miRNA-340. At the same time, it was found that miRNA-340 decreased at 24h after UVB irradiation. It was found that MITF might be the target molecule of miRNA-340 by bioinformatics software. We further speculated that miRNA-340 might inhibit the melanogenesis by regulating the downstream molecules of MITF and its signaling pathways, such as Tyrosinase (TYR),Tyrosinase related protein-1 (TYRP-1).PART 1: Study on the correlation between miRNA-340 phase change and melanin synthesis after UVB irradiationObjective: To study the relationship between miRNA-340 and pigment-related molecules MITF, TYR and TYRP-1 in Pig-1 cells induced by UVB and to determinate pigment amount at different phase. In order to determine that the miRNA-340 regulates the association between MITF-induced UVB-induced pigment synthesis.Methods: Pigl cells were treated with 100mJ / cm2UVB, and the experimental group of Pigl cells were treated with real time quantitative PCR (qRT-PCR) at Oh, 3h, 6h,12h, 16h and 24h after irradiation. The expression of miRNA-340 in Pigl cells and the expression of MITF, TYR and TYRP-1 in Pigl cells after the same conditions were investigated. The expression of MITF, TYR and TYRP-1 mRNA in Pigl cells was detected by Western-Blotting. The changes of MITF, TYR and TYRP1 protein levels were observed at different time. The changes of melanin content in different phases after UVB irradiation were detected by sodium hydroxide dissolution method.Results: The level of miRNA-340 increased at 3h after irradiation, reached the peak at 6h, and decreased continuously from 12h to 24h. MITF level increased at 3h after irradiation, 6h decreased significantly, reached its peak at 12h, and decreased significantly at 24h. TYR levels in addition to 24h group, the other groups and the control group the difference was not statistically significant. TYRP-1 began to rise after 3h, 16h peak, 24h decreased. MITF was the highest at protein level at 16h after irradiation. TYR protein expression at each time point was not significant. TYRP-1 no significant change at 6h after irradiation, 16h expression increased, 24h expression level decreased significantly. The results of pigment determination showed that the pigment content began to increase after 6h after irradiation, and the pigment content increased by 1.4 times at 24h.Conclusion: In human melanocytes, miRNA-340 may bind to MITF, which can induce MITF degradation and inhibit its expression. It may mediate the pigment production process of melanocytes after UVB irradiation by TYRP-1.PART 2: Study on the mechanism of miRNA-340 regulating the synthesis of melanin after MITF-induced UVB irradiationObjective: To verify the binding relationship between miRNA-340 and MITF by Dual-luciferase reporter assay. After the infection of Pig-1 cells with miRNA-340 mimics and inhibitors, the effect of MITF on UVB irradiation was confirmed. And further clarify the scientific deduction of miRNA-340 by regulating MITF-induced UVB-related pigment synthesis, and provide new ideas and new targets for the clinical treatment of UVB-related pigmentation diseases.Methods: Dual-luciferase reporter assay was used to verify the targeted binding of miRNA-340 to MITF. The Pig-1 cells were infected with miRNA-340 mimics and inhibitor to prepare miRNA-340 overexpression and low expression of stable cell lines.qRT-PCR showed the time-dependent relationship between MITF, TYR and TYRP-1 in the miRNA-340 stable cell lines after UVB irradiation. The immunoblotting method was used to detect the expression of miRNA-340 cells and melanin. The expression of MITF, TYR and TYRP1 protein was detected by sodium hydroxide solution. The changes of melanin content of miRNA-340 overexpression and low expression of stable cell lines were observed by UVB irradiation.Results: The expression of luciferase was decreased (about 12%) compared with the negative control group after the 3 ’UTR plasmid and miRNA-340 plasmid co-transfected with wild-type MITF were detected by luciferase reporter assay.miRNA-340 mimic and miRNA-340 inhibitor were used to detect miRNA-340 levels in Pig-1 cells respectively by qRT-PCR, demonstrating that miRNA-340 overexpression and miRNA-340 low expression cell lines was successfully constructed.The expression of MITF, TYR and TYRP-1 was up-regulated at 16h after irradiation,and the expression of MITF, TYRP-1 was up-regulated by UVB irradiation. UVB irradiation of Pig-1 cells (NC), overexpression and miRNA-340 after 24 hours of stable cells found that low expression of miRNA-340 stable cells after UVB irradiation 24h pigment content was significantly higher than the NC group.Conclusion: miRNA-340 targets MITF-binding 3’UTR-specific bases to inhibit MITF expression and induce mRNA degradation, while suppressing the expression of multiple key molecules in the pigment synthesis pathway and also the melanin amount.It is suggested that miRNA-340 may become a new target for the clinical treatment of UVB-related pigmentation. |
