Analysis Of Differential Expressions Of Mirnas In T?4 Knockout Mice And The Target Genes Prediction

Mouse thymosin beta 4(thymosin beta 4,T?4),as an important regulatory factor,plays an important role in hair follicles development and growth cycle.The T?4 knockout mice were constructed in this laboratory have been experimentally proved that the T?4 gene is involved in the regulation of hair growth rate.MicroRNAs(miRNAs)are a class of endogenous non-coding RNAs with regulatory functions that recognize target mRNA in a base-complementary pairing manner,and induce degradation of the silencing complex or suppress translation of the target mRNA according to the degree of complementarity.The latest research found that miRNA plays an important role in the regulation of hair follicle development.There is a certain relationship between T?4 gene and miRNA in biochemistry,but the interaction relationship between T?4,miRNA and hair follicles growth and development mechanism is not yet clear.1.Analysis of differential expression of miRNA in epidermal tissue of T?4knockout miceThrough expression profiling analysis,among WT mice,the expression ofT?4 was the highest in liver tissues,followed by skin,and the growth and development of hair follicles were inseparable from epidermal tissues.Therefore,epidermis was selected as the experimental materials for this study.A total of 1764 miRNAs with different lengths were detected by miRNA sequencing.TargetScan and miRanda software were used to predict the target genes of 169 miRNAs with significant differences(p <0.05),after the prediction,204,884,445 target genes were obtained by taking their intersection.GO and KEGG enrichment analyses were performed on the target genes of significantly different miRNAs.Finally,GO functional annotation obtained a total of 24,857 target genes,and KEGG annotation obtained 8,962 target genes.Through GO enrichment analysis,there were 18731 gene functions with significant differences(p <0.05),which mainly involved in five functional attribute processes of protein binding,nucleus,integral component of membrane,cytoplasm and biological-process.KEGG notes that 236 signal pathways with significant differences(p <0.05)were enriched,of which 186 signal pathways were significantly different(p <0.01),and PI3K-AKT signaling pathway,P53 signaling pathway,Notch signaling pathway,TGF-beta signaling pathway,and TNF signaling pathway have 5 significantly different(p <0.01)signaling pathways.The abnormally expressed miRNA in the above results may be involved in the regulation of T?4 on hair follicles development.At the same time,the above results provide experimental evidences for sorting out the interaction and relationship between T?4,differential miRNA,and target genes in hair follicles growth and development.2.Prediction and verification of differential miRNA target genesThe accuracy of miRNA sequencing was verified by real-time PCR during Through the verification of the real-time quantitative PCR of the preliminary screened miRNA,the accuracy of miRNA sequencing was initially confirmed.In a cycle of hair follicle development,the expression levels of T?4 and miR-148a-3p in the hair follicle's degeneration and quiescent periods are higher,and miR-148a-3p changes with the change of T?4.The dual luciferase reporter system detected that Smad2 and Meox2 are the target genes of miR-148a-3p.During the degeneration and resting periods of mouse hair follicles,the expression levels of T?4 and miR-148a-3p were higher,and Smad2 was significantly inhibited.At this time,the phosphorylation level of Smad2 / 3 also decreased.The growth and development of hair follicles can be regulated through the regulatory pathway of T?4-miR148a-3p-Smad2-TGF-?.