Resveratrol stimulates microRNA expression during differentiation of bovine primary myoblasts

Abstract

Background: Resveratrol (RSV), a phenolic compound, is present in many human dietary sources, such as peanuts, peanut butter, grapes skin, and grape wine. RSV has been widely known for its benefits on human health. Beef from cattle skeletal muscle is one of the main sources of protein for human consumption. Previous studies have also found that pork and chicken qualities are influenced by the feed supplementation with RSV. In addition, our previous study demonstrated the RSV effects on bovine myoblast differentiation using messenger RNA (mRNA) data. In this study, we mainly focused on the influences of RSV on microRNA (miRNA) expression.
 Method: We used 20 μM RSV to treat primary bovine myoblasts and extracted RNA for miRNA sequencing. After quality control and alignment for clean reads, we conducted quantification and analysis of differentially expressed (DE) miRNAs in the case (RSV-treated) group versus control (non-RSV treated) group. Next, we predicted the target genes for the DE miRNAs and analyzed them for the enrichments of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.
 Results: Finally, we identified 93 DE miRNAs (adjusted P-value < 0.05), of them 44 were upregulated and 49 were downregulated. Bta-miR-34c was the most significantly upregulated miRNA. In silico, prediction results indicated 1,869 target genes for the 93 DE miRNAs. GO enrichment analysis for the genes targeted by DE miRNAs revealed two significant GO terms (adjusted P-value < 0.05), in which the most significant one was stereocilium (GO:0032420). KEGG enrichment analysis showed five significant pathways, and the top significant KEGG pathway was the insulin signaling pathway (bta04910) (adjusted P-value < 0.05).
 Conclusions: This study provided an improved understanding of effects of RSV on primary bovine myoblast differentiation through the miRNA modulations. The results suggested that RSV could promote differentiation of primary bovine myoblast by stimulating the miRNA expressions. The target genes of DE miRNAs were significantly enriched in the insulin signaling pathway, thus potentially contributing to improving muscle leanness by increasing the energy metabolism.

Integrated Analysis of mRNA and MicroRNA Co-expressed Network for the Differentiation of Bovine Skeletal Muscle Cells After Polyphenol Resveratrol Treatment

“…Using 18,329 mRNA and 650 miRNA data for the sample clustering, we found that the samples with RSV treatment were clustered together in mRNA analysis (Figure 3A), while Differentially expressed (DE) mRNA and miRNA results were achieved from our previous study (31,32).…”

“…The cultured primary myoblasts from the fetal beef longissimus dorsi muscle, the transcriptome sequencing datasets after quality control and alignment, and the differential expression analysis results were achieved from our previous studies (31,32).…”

“…RNA for each cell sample was isolated for mRNA sequencing to generate 125 or 150-bp paired-end reads and for miRNA sequencing to generate 50-bp single-end reads on an Illumina Hiseq platform (Illumina, USA). We removed the unqualified reads by quality control to achieve clean reads by in-house perl scripts that were used in our previous studies (31,32). Then, the miRNA tags were mapped to the reference genome of Bos taurus (UMD_3.1.1/bosTau8) by Bowtie software (version 0.12.9) (33).…”

“…From our previous study (31,32), a total of 3,856 DE mRNAs were identified from 18,329 mRNAs based on the threshold of adjusted P-value <0.05; meanwhile, 93 DE miRNAs were also identified from 650 miRNAs based on the same thresholds (Table 1). The details of log 2 FC, P-value, adjusted P-value, and DE mRNAs with FPKM and miRNAs with TPM of each sample are listed in the Supplemental File 1.…”

“…Our previous study focused on transcriptomic changes in bovine skeletal muscle cells after RSV treatment and was conducted to identify the differentially expressed (DE) genes and microRNAs (miRNAs) (31,32); therefore, this study mainly focuses on the combined co-expressed transcriptomes, i.e., messenger RNA (mRNA) and miRNA studies for bovine muscle in response to treatment with RSV, which aims to investigate the roles of RSV in inducing miRNA for the better understanding, to identify the characteristics of DE miRNAs in the key co-expressed module in regulation of mRNAs, and to reveal new candidate regulatory miRNAs and genes underlying the beef quality traits.…”