3.3 Correlation of miRNA-mRNA-protein regulatory network
348 conserved miRNAs belonging to 292 miRNA seed-based families were identified in the six small RNA libraries (M0a, M0b, M0c, M4a, M4b, and M4c). The quality control analysis of the miRNAome is shown in Table S3 , including overview of reads from raw data to cleaned sequences, summary of known and predicted miRNA, length distribution of sequences, and conservation of the identified miRNA with other species. A total of 39 differentially expressed miRNAs (DEMI) were found (P < 0.05), with 17 upregulated and 22 downregulated miRNAs (Table S2 ).
In our results, according to the DEMI, DEG and DEP datasets and miRNA-targeting information, 1809 negative miRNA-mRNA regulatory network with the expression pattern of up-down and down-up were detected (Table S4 ). Further, 12 of 20 significant enriched KEGG pathways for 1809 pairs were found to be overlapping for those of transcriptome, also involved in vessel formation, metabolism, immune response and muscle dysfunction. Additionally, similar to the DEG, the numbers of upregulated genes were greater than that of downregulated ones involved in vessel formation, catabolism, immune response, muscle dysfunction, and vice versa for anabolism (Fig. 3A ). Finally we identified 145 negative miRNA-mRNA-protein pairs (up-down-down and down-up-up) with negative correlation as the key for analysis including 21 DEMI and 60 co-DEG/DEP in total (Fig. 4A ). Moreover, five out of 11 significant enriched KEGG pathways for 145 pairs were found to be overlapping for those of proteome, such as “Citrate cycle (TCA cycle)”, “Oxidative phosphorylation”, “Tryptophan metabolism”, “Glyoxylate and dicarboxylate metabolism”, and “Galactose metabolism”, with carbohydrate metabolism of the most commonly represented subclasses. In addition, biosynthesis related to “Butirosin and neomycin biosynthesis” and “Protein export” were significantly enriched by down co-DEG/DEP (Fig. 3B ).