Effect Of Pigment Epithelium Derived Factor On Human Melanocytes

PartⅠExpression of Pigment Epithelium Derived Factor In Human Melanocytes and Malignant Melanoma Cells And TissuesObjectiveTo study the production and distribution of pigment epithelium derived factor (PEDF) in human melanocytes and malignant melanoma cells and tissues.MethodsThe expression and distribution of PEDF in healthy skins,pigmented nevus and human malignant melanoma tissues were detected by immunohistochemical method.The expression of PEDF protein in human melanocytes and malignant melanoma A375 cells were detected by Western blot analysis.The expression of PEDF mRNA values in human melanocytes and malignant melanoma A375 cells were measured by reverse transcription polymerase chain reaction(RT-PCR).ResultsPEDF could be expressed by human melanocytes.The expression of PEDF protein got lower and lower in healthy skins,pigmented nevus and human malignant melanoma tissues(P＜0.001).The expression of PEDF protein was observed in melanocytes and was almost absent in malignant melanoma A375 cells(P＜0.001).The expression of PEDF mRNA was much lower in malignant melanoma A375 cells than human melanocytes(P＜0.001).ConclusionsThe lower expression of PEDF in malignant melanoma may contribute to its pathogenesis. PartⅡEffects of Pigment Epithelium Derived Factor Inhibition by siRNA on human melanocytesObjectiveTo evaluate the effect of small interferring RNA(siRNA) targeting pigment epithelium derived factor(PEDF) gene on the proliferation of human melanocytes.MethodsAfter siRNA for PEDF gene was transfected into human melanocytes by LipofectimineTM2000,PEDF gene expression was assessed by real-time quantitive PCR,and PEDF protein expression was detected by Western blot analysis.MTT assay was used to assess the proliferation of the cells.ResultsThe expression of PEDF gene in human melanocytes could be effectively suppressed by siRNA(P＜0.001) and the proliferation rate of human melanocytes was elevateed markedly(P＜0.001).ConclusionsThe expression of PEDF gene in human melanocytes can be significantly decreased by RNAi.PEDF can inhibit exorbitant proliferation of human melanocytes.PartⅢDetection of Pigment Epithelium Derived Factor Gene Mutations In Human Malignant Melanoma Cell Line ObjectiveTo discuss the role of pigment epithelium derived factor(PEDF) gene in the pathogenesis of malignant melanoma.MethodsAll the eight exons of PEDF gene were scanned in human malignant melanoma cell line A375 by single strand conformation polymorphism analysis ofpolymerase chain reaction products(PCR-SSCP),followed by DNA sequencing.ResultsSSCP analysis of the genomic DNA from all the eight exons of PEDF gene demonstrated several polymorphisms in exons 3,4,5,6 and 7,especially in exon 5,6. Frame-shift mutation was detected in exon 5 and point mutation was detected in exon 6.ConclusionsMution of PEDF gene may contribute to the pathogenesis of human malignant melanoma.