Determination And Analysis Of MRNA-MicroRNA Expression Profiles In Four Portions Of Velvet Antler And Construction Of Regulation Network In Wax-like Portions

Velvet antlers are the only mammalian bone accessory organs capable of full renewal in yearly cycles.At the same time,velvet antlers are precious traditional Chinese medicine that has been used for thousands of years in China,Korea,and Southeast Asian countries.Different tissue portions along the longitudinal axis of velvet antlers contain different amount of crude proteins,amino acids,water-soluble polysaccharides,monosaccharides,fatty acids and other substances;these substances show a gradual decrease trend from the tip to the base;while the distribution of minerals(mainly calcium)shows the opposite trend.Traditionally,velvet antlers along the longitudinal axis are divided into four portions: wax like(WL),blood-color(BC),honeycomb-like(HL)and bone(B)slices from the tip to the base,but the classification lacks evidence at the molecular level.This study was to take transcriptional approach to assess the accuracy of the traditional classification for these four portions of velvet antler,and to link the expressed mRNAs and miRNAs of each portion with possible functions using bioinformatics analysis tools.On this basis,since WL covers the antler growth center,the genes that differ between WL and other regions were detected by using differential analysis,the mRNA-miRNA regulatory network was constructed,and the key genes were functionally verified by using qRT-PCR and CCK8,which ultimately provided a basis for studying the molecular mechanism underlying rapid growth of antlers.By constructing mRNA expression profiles of the four portions of velvet antlers,three multivariate statistical analysis methods(Spearman correlation analysis,hierarchical clustering analysis and principal component analysis)were used to determine the gene expression levels of each of the traditional four portions of velvet antlers,and the results showed that there was no significant difference in mRNA expression level being detected between portions HL and B,therefore,HL and B were combined into a new portion,HL+B.Further,by differential analysis and GO functional enrichment,it was found that there were 1543 mRNAs with relatively high expression levels in portion WL,whose functions were mainly related to cell division processes such as transcriptional translation of RNA;223 mRNAs with relatively high expression levels in portion BC,whose functions were mainly related to bone and cartilage development;and 435 mRNAs with relatively high expression in portion HL+B,whose functions were mainly related to angiogenesis and bone development.By constructing miRNA expression profiles of the four portions of velvet antlers,three multivariate statistical analysis methods(Spearman correlation analysis,hierarchical clustering analysis and principal component analysis)were used to determine the gene expression levels of the traditional four portions of velvet antlers,and the results showed that just like the results from mRNA expression profiling,there was no significant difference in miRNA expression levels being detected between portions HL and B,therefore,HL and B were combined into a new portion,portion HL+B.The velvet antler could be divided into three portions at the molecular level.Further,by differential analysis and GO functional enrichment,25 miRNAs were found to be relatively highly expressed in portion WL,the function of whose target genes was mainly related to the signaling pathways;3 miRNAs were found to be relatively highly expressed in portion BC,the function of whose target genes was mainly related to the development of the nervous system;4 miRNAs were found to be relatively highly expressed in portion HL+B,the function of whose target gene was not enriched by GO enrichment in the present study.Finally,we constructed a miRNA-mRNA regulatory network in portion WL and screened miR-155 and its target gene FOXO3 for possible involvement in cell proliferation by using GO functional analysis.qRT-PCR and CCK8 were used to demonstrate whether miR-155 could target FOXO3,downregulate its expression level and thus promote proliferation of antler mesenchymal stem cells.The results confirmed our expectation.Taken together,the traditionally used four portions of velvet antlers were reclassified into three portions at the transcriptional molecular level,and miR-155 and its target genes were found to be possibly related to the rapid growth of deer antler.