The Potential Role Of CircRNA＿004229 In Hair/Epidermal Regulation After Med1 Ablation In Keratinocytes

ObjectiveMediator complex subunit 1（Med1）is an important transcriptional coactivator involved in the regulation of multiple signaling pathways.Previous studies have shown that Med1 plays an essential role in hair cycling and wound repair through regulating m RNA transcription.Circular RNAs（circRNA）,as a novel class of non-coding RNAs,involved in a variety of skin biological functions.Our study aims to screen and identify circRNA expression profiles of epidermal-specific Med1 knockout mice（KO）,and to preliminary explore the biological functions of circRNA＿004229.It provide new ideas for further revealing the complex role of Med1 in hair follicles and epidermis regulation,and lays a foundation for exploring the potential mechanism of circRNA in hair follicles and epidermis.Methods1.Experimental grouping and sample acquisitionIn this study,10-weeks-old Med1 transgenic mice were divided into two groups.The experimental group was Med1^flox/flox transgenic mice expressing k14-cre（KO）,and the control group was Med1^flox/floxmice none expressing k14-cre（WT）,with 3 mice in each group.The mice were sacrificed by neck removing and the back hair was shaved.The epidermal tissue on the back of the mice was taken as an experimental sample.2.circRNA expression profile analysisThe total RNA was extracted from tissue samples,and RNA quality was quantitatively controlled and integrity controlled.The linear transcript was removed and the circRNA was specifically labeled.High-throughput sequencing was conducted through the circRNA microarry.The sequencing raw data were analyzed and differentially expressed circRNA in skin was verified by qRT-PCR.3.Prediction of circRNA/micro RNA/m RNA interaction network through bioinformatics analysisArraystar software was used to predict the miRNA binding to circRNA＿004229,and the circRNA/miRNA interaction was annotated in detail.Targeted m RNAs of miRNAs were retrieved using Target Scan,miRPath DB and miRDB databases.Based on circRNA sequencing data and micro RNA target gene prediction,a visual circRNA/micro RNA/m RNA interaction network was constructed through Cytoscape 3.5.0software.4.Preliminary exploration of the functional mechanism of circRNA＿004229The expression level of circRNA＿004229 targeted miRNA（miR-149-5p,miR-207）target gene（Tnfrsf19,Perp）was verified by qRT-PCR,and the expression level and its expression in skin tissue were determined by immunohistochemical staining（IHC）.The apoptosis levels of skin cells in the Med1-Con and Med1-KO groups were evaluated by TUNEL assay.ResultsWe identified 13,304 circRNA by circRNA chip sequencing,and 109 circRNA with significant differences were screened,among which 34 were up-regulated and 75were down-regulated（FC>2.0,P<0.05）.Most of the differentially expressed circRNA were derived from exons.Validation by qRT-PCR showed that circRNA＿004229expression was significantly down-regulated.By predicting the circRNA/miRNA interaction and sequence analysis of the micro RNA response element（MRE）,5 potential targeted miRNAs for mmu＿circRNA＿004229 were predicted:miR-149-5p,miR-207,miR-7235-3p,miR-667-5p and miR-6914-5p.The prediction of miRNA target genes indicates that the potential targeted m RNAs of miR-149-5p and miR-207 are related to apoptosis,and high scores predict the two apoptosis-related genes Tnfrsf19 and Perp.Finally,the circRNA＿004229-micro RNA-149-5p/micro RNA-207-Tnfrsf19/Perp interaction network was constructed.The results of qRT-PCR and IHC experiments showed that the m RNA expression of the target genes（Tnfrsf19,Perp）of miR-149-5p and miR-207 was significantly down-regulated in the skin of KO mice,and TUNEL analysis showed that the apoptosis level of the skin cells on the back of KO mice was significantly reduced compared to WT mice.ConclusionThe expression profile of circRNA in the skin of Med1 epidermal-specific knockout mice is significantly different,suggesting that Med1 may play a role in regulating epidermal function through the circRNA regulatory network.mmu＿circRNA＿004229 act as the role of miR-149-5p/miR-207 sponge and promotes binding of miRNA to Tnfrsf19/Perp,which may inhibit the pro-apoptotic function of Tnfrsf19/Perp in keratinocytes.This study identified the circRNA expression profiles in the epidermis of Med1epidermal-specific knockout mice and explored the biological functions of circRNAs with significant differences,enlighten us the role of Med1 in the regulation of hair follicle/epidermal function in a novel way,besides,providing potential targets for further explore the biological function of circRNA in the skin.