RNA-Seq Analysis Of Transgenic Mice Skin Reveals Mechanism Of MicroRNA-137 In Coat Color Regulation

The study of coat color regulation mechanism not only provides a valuable system to understand basic mammalian biology, but also has high economic value to fiber products industry to study the regulatory mechanisms of coat color determination. Compared with fiber products made by chemical dyeing, natural fiber products with different colors have clear advantages because they draw less concerns about human health. MicroRNA (miRNA) modules multiple biological processes by inhibiting or decreasing the expression of their target genes by binding the position of the 3’UTR of the targets. Previously, we successfully created microRNA-137 (miR-137) transgenic mice which turns out lighten coat color phenotype including gray and brown mice phenotypes. In these two different coat color mice phenotypes, relative expression of miR-137 was significantly different. However, the mechanism of miR-137 in mouse coat color determination is still not well understood. To further understand regulatory mechanisms of miR-137 in coat color regulation, this study investigated gene expression profiles in gray miR-137 transgenic mice skin (transgray-4) as well as brown miR-137 transgenic mice skin (transbrown-4) by RNA sequencing (RNA-Seq). We compared the differences between gray and brown mice skin which altered by miR-137 in different expression level including gene and gene expression, signaling pathways and biological processes through the analysis of RNA-Seq data. We found two key targets of miR-137 by bioinformatic methods which might directly determine the transgenic mice coat color synthesis. The target relationship between miR-137 and its target gene was validated by Dual-luciferase assay. In additional, we found miR-137 could inhibit the melanogenesis in mouse skin melanocytes by repressing the expression of its target gene and the downstream genes of this target gene. Results reveal the mechanism of miR-137 in coat color regulation.1. All expressed genes were identified in two different color transgenic mice skin and totally 19,000 expressed genes were obtained by RNA-seq in gray and brown miR-137 transgenic mice skin. We found 93 genes with statistically different expression levels (fold change> 2,0.8<=Diverse probability<=1) and thus can be named differently expressed genes (DEGs) by comparing the gene expression levels between gray and brown mice.10 genes were randomly chose for qRT-PCR which was used to validate the quality of the sequencing results turned out it had the same tendency in gene relative expression with the results which provided by RNA-Seq data. Results provided important foundation to further study through RNA-Seq data in gene expression level.2. To better understand the biological processes and signaling pathways that differ between brown and gray miR-137 transgenic mice which altered by miR-137, Go Ontology (GO) annotation, GO functional classification and the distribution of GO functions of the identified DEGs were firstly performed using Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification system. From the analysis of GO function enrichment of DEGs which performed by Database for Annotation, Visualization and Integrated Discovery (DAVID), we found the DEGs between two different color mice skin were enriched in melanogenesis related GO functions. From the signaling pathway enrichment analysis which performed by Kyoto Encyclopedia of Genes and Genomes (KEGG), we found that DEGs between two different color mice skin were enriched in melanogenesis related KEGG signaling pathways. Results indicated that miR-137 might result in two different coat color mice skin by regulating melanogenesis process in melanocytes.3. For further explore all of the biological process which altered by miR-137 overexpression, Gene Set Enrichment Analysis (GSEA) method was used to analyze the enrichment of biological processes which all expressed genes involves in. In order to ensure the accuracy of the analysis, Hallmark gene sets which represent well-verified biological states or processes was selected in the analysis. In addition, GO biological process gene sets were selected in this analysis to verify the previous gene ontology analysis. The results indicated that the difference in melanogenesis is a bona fide molecular change in the skin tissues of brown miR-137 transgenic mice. Additionally, in gray miR-137 transgenic mice, miR-137 might regulate more obvious pigmentaion process by regulating Myc target genes and involving in Ribosome related processes.4. Target genes of miR-137 were predicted by bioinformatic prediction method in differently expressed genes. We found Kit and Myp7a were two miR-137 targets which might directly lead to two different lighten coat color synthesis. With the results of Chapter 2 and 3, we found miR-137 might determine brown mice coat color phenotype by affecting melanin synthesis due to regulate SCF/Kit signaling pathway and melanogenesis related down stream genes of the pathway.5. The target relationship between miR-137 and Kit was performed by Dual-luciferase assay. After transfecting miR-137 expression vector plasmid into normal mice skin melanocytes, the expression of Kit, Tyr, Tyrpl and Dct was down-regulated and the melanin content in melanocytes was also repressed.