Construction and Comprehensive Analysis of miRNAs and Target mRNAs in Longissimus dorsi Muscle of Queshan Black and Large White Pigs

SIMPLE SUMMARY: In this study, the miRNA-mRNA combination analysis of the longissimus dorsi of adult Queshan Black and Large White pigs was carried out by RNA-seq technology, and the molecular mechanism affecting pork quality traits was revealed. The results showed that 39 miRNAs were differentially expressed between Queshan Black and Large White pigs. A functional enrichment analysis of their target genes showed that they were mainly enriched in pathways related to adipogenesis and cell proliferation and differentiation. In addition, miR-328, as a key miRNA, can affect the proliferation of 3T3-L1 cells. This study revealed the miRNA-mRNA regulatory axis affecting fat deposition and skeletal muscle mass and provided a theoretical basis for further study of the molecular regulation mechanism of meat quality traits. ABSTRACT: A miRNA-mRNA combination analysis was performed on the longissimus dorsi muscle of adult Queshan Black and Large White pigs by RNA-seq technology to reveal the molecular mechanism affecting pork quality traits. The sequencing results showed that 39 miRNAs were differentially expressed between Queshan Black and Large White pigs, which targeted 5234 mRNAs, and 15 differentially expressed miRNAs targeted 86 differentially expressed mRNAs. The qRT-PCR results showed that miRNAs showed similar expression patterns to RNA-seq. The GO analysis indicated that differentially expressed miRNAs with differential target mRNAs were primarily involved in biological processes such as phospholipase activity, MAP-kinase scaffold activity, lipase activity, and regulation of the extent of cell growth. The KEGG analysis also revealed that such mRNAs were significantly enriched in the ECM-receptor interaction, sphingolipid metabolism, apoptosis, PI3K-Akt signaling pathway, and AMPK signaling pathway. In addition, software predictions showed that 17 (13 of which were upregulated and four were downregulated) of 39 differentially expressed miRNAs targeted 118 negatively correlated expression mRNAs. The upregulated miRNAs contained 103 negatively correlated target mRNAs, whereas the downregulated miRNAs contained 15 negatively correlated target mRNAs. The GO analysis showed that such mRNAs were primarily involved in MAP-kinase scaffold activity, myoblast development, and peptidyl-lysine methylation, and the KEGG analysis showed significant enrichment in ECM-receptor interaction and focal adhesion. The functional enrichment analysis of miRNA target genes revealed that miR-328 was screened out as a key miRNA, and preliminary functional validation was performed. Moreover, the overexpressed miR-328 could affect the expression of proliferation-related genes, such as CDK2, CDK4, CCNB1, CCND1, CCNE1, and PCNA. These results indicated that miR-328 may regulate fat deposition and affect meat quality by influencing related pathways. This study revealed that the miRNA−mRNA regulatory axis affects fat deposition and skeletal muscle development, which provides a theoretical basis for further study on the molecular mechanism of meat quality.