Fgf21 Knockout Mouse MiRNA Differentially Expressed And Target Genes Prediction In Dorsal Skin Tissue

Fibroblast growth factor 21?Fgf21?is a regulatory factor of the FGF family and has been postulated to play an important role in hair follicles'development and growth cycle.To evaluate such Fgf21 role,we had established Fgf21 knockout mouse model,and proved that Fgf21 had effect on regulating hair follicles'growth and diameters.MicroRNAs?mi RNAs?are a large family of endogenous and non-coding RNAs and regulate gene expression through sequence specific base pairing with target mRNAs.Mature miRNAs silence complex target mRNA degradation or deter a translation of the target mRNA by depending on the degree of complementary guidance.Over the past decade,accumulating evidence has shown that miRNAs are involved in hair follicles'growth and development.Although Fgf21 is associated with mi RNA in biochemistry and genetics,the complex relations of Fgf21,miRNA and regulating mechanism in the role of hair follicles'growth are indistinct.1 Fgf21^-/-mice model breeding and phenotypic analysisFgf21^-/-mice were bred by using breeding methods of different heterozygous intercrossing,heterozygous,homozygous orthogonal and homozygous intercrossing.After gene identification,we finally obatained 45 Fgf21^-/-mice.Through the phenotypic analysis,compared with WT,we found that the litter size of Fgf21^-/-mice significantly reduced?p<0.05?,but physiological indexes of the birth weight,gender rate,body weight?0-8 week?and body weight of male and female?4-8 week?were no significant difference?p>0.05?,Combined Fgf21 expression profiling analysis with physiological anatomy,we found that,compared with WT,the organizations or guts of Fgf21^-/-mice were normal,especially the pancreatic tissue of Fgf21 high expression.Then by paraffin section and the number statistics of hair shafts after H&E staining,we found that the number of hair shafts of Fgf21^-/-mice significantly less than the WT mice?p<0.05?.2 Fgf21^-/-mice skin tissue for miRNA sequencingUsing Fgf21^-/-mice and WT mice skin as material,we obtained a total of 1249different length miRNAs.Through the analysis of the statistic data from miRNA differential expression,we found that 134 miRNAs,which contained 83up-regulation and 51 down-regulation miRNAs,had significant difference?p<0.05?.Then combined with the literature references,we screened 10 miRNAs from miRNA sequencing results,associated with hair follicle growth and development regulation.These miRNAs respectively were miR-377-3p,miR-335-5p,miR-214-5p,mi R-148a-3p,mi R-410-3p,miR-411-5p,mi R-376b-3p,miR-376c-3p,mi R-136 and mi R-221-3p.Using the fluorescent quantitative PCR method for validation,the results were consistent with the miRNA sequencing.3 The differentially expressed mi RNAs target genes prediction and bioinformatics analysisUsing TargetScan and miRanda software to predict target genes for significant differences 134 miRNAs?p<0.05?,we finally took its intersection to receive105,886,298 target genes.Then we launched biological information analysis of GO and KEGG target genes enrichment for the significant differences mi RNAs.Finally,we received 20,557 target genes by GO functional annotation and 7881 target genes by KEGG annotation.By analyzing differentially expressed genes using GO enrichment and classification analysis,we found there are 1023 gene function attributes of significant difference miRNAs?p<0.05?.These gene function attributes were mainly involved in protein binding,nucleus,integral component of membrane,cytoplasm and biological-process in all the functional attributes;Using KEGG pathway gene expression analysis and the differences of functional analysis,we found that a total of 202 signaling pathways were significant difference?p<0.05?and164 signaling pathways were extremely difference?p<0.01?.Further,we found that the PI3K-AKT signaling pathway,P53 signaling pathway,Notch signaling pathway,TGF-beta signaling pathway and TNF signaling pathway were extremely significant difference?p<0.01?,associated with hair follicles'growth and development regulation.Eventually,the microRNA-GO/KEGG-network diagram,which contained 10mi RNAs as the core focus of regulating and controlling the predicted target genes,was successfully drawn by using Cytoscape software.Above results provide the experimental datas for confirming that abnormal expression of miRNA may participate in the Fgf21 in hair follicles'development regulation and teasing out the relationship of Fgf21,abnormal expression of micro RNAs and target genes,associated with hair follicles'regulation mechanism and development process.