Differential Expression Analysis Of MiRNA-124 In Eczema Lesions And The Intervention Of Chinese Herbal Extracts Of Heat-clearing And Dampness

Objective:To select miR-124,which may be related to the pathogenesis of eczema,as our research subject of the present study through literature review;then investigate the expression of mi R-12 4 in eczema lesions and non-eczema tissues by QPCR;to explore the mechanism by which miR-124 and NF-kB pathway regulates each other to provide novel theoretical basis for early diagnosis and treatment of eczema.At the same time,we observed the differential expression of miR-124 after the intervention of heat-clearing and damp-drying traditional Chinese medicine extract,and explored the mechanism of traditional Chinese medicine in treating eczema,and provided the objective basis for the prevention and treatment of eczema by traditional Chinese medicine.Methods:(1)37 cases of eczema patients were collected for their eczema lesions and healthy skin tissue;total RNA was extracted uing Trizo assays;miR-124 expression in eczema and healthy tissues was determined using QPCR.(2)To confirm the previous re sults in inflammatory factor-stimulated Hacat cells and to screen out inflammatory factors that are significantly associated with this outcome;(3)After transfection of miR-124 mimic or miR-124 inhibitor into Hacat cells,miR-124 expression was verified by real-time quantitative PCR;the effect of miR-124 on p6 5 expression was then evaluated.(4)To monitor the expression levels of miR-124,p6 5 and the related downstream genes of p6 5 in miR-124 mimics-or miR-124 inhibitor-transfected Hacat cells,TNF-α-stimulated Hacat cells,and INF-γ-stimulated Hacat cells using QPCR and immunoblottin g.(5)The expression levels of miR-124 and the downstream genes of p6 5 were confirmed by real-time fluorescence Quantitative PCR in normal tissues and eczema tissues;and the correlations of the indicated factors with miR-124 expression were analyzed.(6)The extract of heat-clearing and damp-drying traditional Chinese medicine was added to the Hacat cells,TNF-α-stimulated Hacat cells and INF-γ-stimulated Hacat cells;the expression of miR-124 and the protein levels of the indicated factors were detected by QPCR or Western blot assays.Results:The expression of miR-124 was significantly down-regulated in the eczema tissues(P<0.01).The expression of miR-124 was decreased after the inflammatory cytokines stimulated Hacat cells,and the most strongly down-regulated by TNF-α and INF-γ(p<0.0 1);miR-124 inversely regulated p 6 5 by direct targeting(P<0.01);in TNF-α-or INF-γ-stimulated cells,in response to miR-124,the expression levels of IL8,C CL 8 and CCL5 were significantly different(P<0.01);the expression of P65,IL8,CCL5 and C CL 8 in eczema tissues were significantly up-regulated(P<0.01),and negatively correlated with the expression level of miR-124;the extract of Sophora j aponicus,Kochia scoparia and Sophora flavescens had a positive effect on miR-124 expression(P<0.01).Conclusion:1.The expression of miR-124 in eczema tissue was down-regulated.MiR-124 and NF-kB could inversely regulate each other and form a negative feedback loop.The up-regulation of miR-124 could inhibit the activity of NK-kB pathway;reduce the production of downstream inflammatory factors;thereby prevent the occurrence of eczema inflammation.2.The specific down-regulated miR-124 expression was rescued by the extract of heat-clearing and damp-drying traditional Chinese medicine,indicating that the heat-clearing and damp-drying traditional Chinese medicine may exert the functions through the regulation of miR-124.3.Provided experimental basis for the further miRNA targeted therapy and molecular markers for eczema.