Differentially Expressed MicroRNA In Plasma Of Vitiligo And Functional Study

Objective: To screen differentially expressed mi RNA in plasma of vitiligo,further to detect mi RNA related with the immune pathogenesis of vitiligo and verify the differentially expressed mi RNA.The target genes of differentially expressed mi RNA were predicted and screened,and the expression relationship between differentially expressed mi RNA and target genes was analyzed.Normal human epidermal melanocytes were isolated and cultured,and the effects of overexpression or inhibition of differentially expressed mi RNA on the biological functions of cells were investigated.And the expression of target genes at m RNA and protein levels was investigated by functional experiments at the cell level.The differential expressed mi RNA and the regulatory mechanisms of target gene were analyzed by double-luciferase reporter assay.Methods: The expression of mi RNA in plasma was detected by mi RNA microarray,and the expression of mi RNA related to immune pathogenesis in plasma was detected by mi RNA PCR Array.Besides,differentially expressed mi RNA in plasma was verified by q RT-PCR.The target genes of differentially expressed mi RNAs were predicted based on Target Scan,and genes related with vitiligo were screened by weighted gene co-expression network analysis,and the target genes of differentially expressed mi RNA were selected.Primary melanocytes were isolated and cultured.The mimics and inhibitor of differentially expressed micro RNA were synthesized,and transfected to the melanocytes.CCK-8 assay,flow cytometry were adopt to detect the cell activity,cell proliferation and cell apoptosis after transfection in order to investigate the changes of the biological behavior of primary culture melanocyte.In addition,the q RT-PCR and western blot were also adopt to detect the expression of target gene of differentially expressed mi RNA after transfection with mi RNA mimics and inhibitor at m RNA and protein level.Besides,the dual luciferase reporter assay was used to explore the regulation mechanism between differentially expressed mi RNA and its target gene.Results: In plasma of vitiligo,compared with control group,mi R-223-3p?mi R-6089?mi R-6800-5p?mi R-2861?mi R-328-5p?mi R-5703?mi R-574-5p?mi R-6125?mi R-630?mi R-8069 were screened by mi RNA microarray.And mi R-223-3p?mi R-15b-5p?let-7e-5p?let-7g-5p?mi R-20a-5p were detected by mi RNA PCR Array related with immune pathogenesis.The mi R-223-3p was detected both in mi RNA microarray and mi RNA PCR Array.And mi R-223-3p was verified by q RT-PCR in plasma of vitiligo.Compared with the control group,the expression of mi R-223-3p in the plasma of vitiligo was significantly upregulated.The target genes of mi R-223-3p were predicted and screened,based on bioinformatics,FOXO3 was preliminarily screened.The expression of FOXO3 in the public dataset of vitiligo,GSE75819,GSE53146 and GSE90880,were all down-regulated.Functional experiments at cell level showed that overexpression of mi R-223-3p in primary culture melanocyte significantly inhibited the cell activity and cell proliferation,as well as promoted cell apoptosis.However,inhibition of mi R-223-3p expression showed the opposite effect to the overexpression.On the other hand,the expression of FOXO3 at m RNA and protein levels was significantly decreased after overexpression of mi R-223-3p.The results of the dual luciferase reporter assay showed that mi R-223-3p directly binded to the 3 'UTR of FOXO3 gene,which was involved in regulating the proliferation and apoptosis of primary culture melanocyte.Conclusion: The mi R-223-3p is highly expressed in vitiligo plasma.Mi R-223-3p directly binds to the 3'UTR of FOXO3 gene.Mi R-223-3p negatively regulates FOXO3 expression and participates in the regulation of the proliferation and apoptosis of primary culture melanocyte,which is expected to provide a new therapeutic target and research direction for vitiligo.