The Changes Of Expression Of MiR-142a-3p And IL-6 In Mouse Models Of Chronic Itch And Their Functional Effects In Vitro

MicroRNAs(miRNAs),endogenous non-coding RNAs,are almost involved in the whole processes of eukaryotic cell development.Patients,with skin diseases such as allergic dermatitis,psoriasis and psoriasis,suffer long-term itch,which not only seriously affects the quality of patients’ life,but also brings them heavy economic burden.In order to explore different pruritus and its pathogenesis,in recent years,several animal models of chronic itch have been developed,such as AEW(acetone/ether/water,AEW)-induced dry skin model and DNFB(2,4-dinitrofluorobenzene,DNFB)-induced allergic dermatitis model.Studies have shown that Skin damage usually leads to continuously activation of the pruriceptors,which resulting in sensitization of neuro and then affecting the release of inflammatory factors.As skin damage is usually companied with chronic itch and the release of inflammatory factors can be regulated by miRNAs,we speculate that miRNAs might be participated in the regulation of chronic itch.In order to investigate the modulating effect of miRNAs on chronic itch,the present paper systematically evaluated the functional relationship of chronic itch,miRNAs and inflammatory factors,by using two chronic itch models of AEW-induced dry skin and DNFB-induced allergic dermatitis,as well as in in vitro assays.The behavioral data obtained from two chronic itch models of AEW-induced dry skin and DNFB-induced allergic dermatitis,revealed that mice exhibited a significant increase in spontaneous biting behavior directed to the treated back of the neck compared to control animals following a period of treatment with AEW or DNFB.Further,we detected the expression of 23 miRNAs that highly expressed in the spinal cord and dorsal root ganglion(DRG)obtained from the mice of these two chronic itch models,as well as 9 inflammation-related cytokines.The result obtained in real-time quantitative PCR showed that:(1)AEW treatment significantly up-regulated the expression of miR-124-3p and miR-142a-3p in the spinal cord;(2)AEW treatment significantly down-regulated miR-124-3p and miR-142a-3p in DRG tissues;(3)DNFB treatment down-regulated the expression of miR-142a-3p in DRG tissues.Taken together,the above results indicated that the expression of miR-142a-3p is significantly affected in the mice of chronic itch models.Furthermore,the expression of inflammatory factors is also tested and our data showed that:(1)AEW treatment significantly modulated the expression of IL-6、IL-17、IL-10、TNF-α and CCL2,especially the expression of IL-6 in DRG tissue was up-regulated to 296%;(2)DNFB treatment significantly modulated the expression of IL-6,IL-13,IL-17 and CCL3,especially the expression of IL-6 expression was up-regulated to 338% in the spinal cord,and to 630% in DRG.The present data indicated that the expression of IL-6 was significantly up-regulated after treatment with AEW or DNFB.Overall,the expression of miR-142a-3p was negatively correlated with the expression of IL-6 in the DRG of mice of chronic itch models.The above studies imply that there seem be a potential functional relationship between miR-142a-3p and IL-6.Furthermore,the microglioma BV-2 cell line is often used to investigate the functional interaction between miR-142a-3p and IL-6 in this paper.The present results showed that the expression of miR-142a-3p in BV-2 cells was time-dependently downregulated after treatment with lipopolysaccharide(LPS),while the expression of IL-6 was time-dependently upregulated after LPS treatment(0-8h).Moreover,the data obtained in enzyme-linked immunosorbent assay(ELISA)showed that IL-6 expression was significantly down-regulated after LPS treatment in the cells of miR-142a-3p overexpression that constructed by transfecting miRNA mimics(agomir-miR-142a-3p)into BV-2 cells,indicating that miR-142a-3p may regulate IL-6 gene.Subsequently,the bioinformatics analysis indicated IL-6 gene is a potential target of miR-142a-3p,and an effective interaction between miR-142a-3p and IL-6 by using double-stranded free energy analysis RNA hybrid.Thus,our results of in vitro functional assay indicate that miR-142a-3p might directly target IL-6 gene and regulate the expression.In summary,the present work suggested that the expression of miR-142a-3p was down-regulated in DRG tissues of mice of chronic itch model,while the expression of IL-6 was significantly up-regulated.Furthermore,in vitro studies indicate that miR-142a-3p can directly regulate the expression of IL-6.Therefore,we speculated that miR-142a-3p may directly modulate IL-6 gene,and play an indirectly role in the signal transduction of chronic itch.This study will be helpful to reveal the molecular mechanisms of the occurrence and development of chronic itch.In addition,it also provides a theoretical basis for the treatment of chronic itch.