Molecular Immune Mechanism Related To Vitiligo Based On Comparative Genomics

Objective:To identify gene expression profiles of progressive halo nevus(HN),stable vitiligo(VL),nevocytic nevus(NN)and primary cutaneous melanoma(MM)from transcriptome level.Exploring the differently expressed genes in HN,through comparative analysis,in order to elucidate the molecular immunological features and characteristic molecules of VL.Selecting part of the differentially expressed genes(DEGs)screened by transcriptome sequencing(RNA-seq)for verification and comparative analysis to elaborate the gene expression characteristics in the occurrence and progression of VL,and to provide experimental basis and theoretical basis for further research on the immune mechanism of VL.Method:①The skin tissues were biopsied from 5 patients with progressive HN,5 patients with stable VL,5 patients with NN and 5patients with primary cutaneous MM.The diagnosis was clinically or pathologically confirmed.After RNA extraction and library construction,RNA-seq of the above samples was conducted on the second-generation sequencing technology platform to obtain the gene expression levels of different samples.DEGs were detected in HN vs.NN,HN vs.VL and HN vs.MM in the comparison analysis.Immune cell types and biological pathways were explored by bioinformatics analysis,according to the DEGs involved in HN,which are different from other sample groups.②Quantitative real-time PCR(RT-qPCR)was performed to verify the transcriptional expression of some DEGs that were closely related to immunity screened by sequencing.The protein expressions of CD8,KLRC1 and CD 137 genes in the tissues of patients with progressive HN,stable VL and NN were detected by immunohistochemical staining.Lastly,the proportion of CD137 positive in CD8+T cells in blood and tissue of the above patients and normal people was analyzed by flow cytometry.Results:①Analysis of the gene expression level showed that HN,NN,VL and MM samples had different expression profiles.HN,NN and VL samples had some overlap,suggesting a a certain degree of similarity of the gene expression profiles between HN,NN and VL samples.MM samples were significantly deviate from the other samples,indicating that MM gene expression was significantly different.②A total of 441 DEGs were found between HN and NN,among which 346 genes were up-regulated and 95 genes were down-regulated,respectively.Compared with stable VL,174 genes were differentially expressed,among which 162 genes were up-regulated and 12 genes were down-regulated.Compaing HN with MM,there were 1507 DEGs,including 687 up-regulated DEGs and 820 down-regulated DEGs.③Compared with NN and VL,biological pathway enrichment analysis illustrated that the up-regulated DEGs in HN were notablely enriched in immune response and immune system process,immune deficiency and immune rejection,biological stimulation(virus,bacteria),proliferation and activation of immune cells.④Immune cell composition analysis showed that expression levels of iTreg,NK,Th1,CD8+T and gamma-delta T cells in progressive HN were higher than those in other disease groups.⑤The results of RT-qPCR indicated that the results of transcriptional expression of the DEGs screened by RNA-seq were mostly consistent with the results of sequencing.Immunohistochemical scanning results showed that the expression of CD8,KLRC1 and CD 137 in progressive HN was higher than that in NN and stable VL.Flow cytometry results manifested that the proportion of CD 137 positive in CD8+T cells derived from blood and tissues of patients with progressive HN was higher than that of normal people,patients with NN and stable VL.Conclusion:We found that progressive HN and stable VL samples shared the most similar expression profile by transcriptome sequencing analysis.Compared with NN and stable VL tissues,the DEGs of progressive HN were mainly enriched in the biological pathways related to the pathogenesis of VL,which explains to a certain extent the inducing factors of VL and other diseases easily associated with VL,such as viral infection,graft-versus-host disease and other immune diseases.Both innate immunity and adaptive immunity were involved in the development of HN and VL.RT-qPCR and immunohistochemistry confirmed that the expression of KLRC1 and CD 137 in progressive HN was higher than that in other tissues.KLRC1 and CD 137 were expected to be potential Immunotherapeutic targets of VL and HN.