Abstract
Simple SummaryRetinol binding protein 4 (RBP4), mainly secreted by the liver and adipocytes, is a transporter of vitamin A. RBP4 has been shown to be involved in several pathophysiological processes, such as polycystic ovary syndrome (PCOS), obesity, insulin resistance, and cardiovascular risk. However, the role of RBP4 in mammalian follicular granulosa cells (GCs) remains largely unknown. To characterize the molecular pathways associated with the effects of RBP4 on GCs, we used sRNA deep sequencing to detect differential microRNA (miRNA) expression in GCs overexpressing RBP4. A total of 17 miRNAs were significantly different between the experimental and control groups. Our results support the notion that several miRNAs are involved in important biological processes associated with folliculogenesis and pathogenesis. These results will be useful for further studies investigating the role of RBP4 in porcine GCs.Retinol binding protein 4 (RBP4) is a transporter of vitamin A that is secreted mainly by hepatocytes and adipocytes. It affects diverse pathophysiological processes, such as obesity, insulin resistance, and cardiovascular diseases. MicroRNAs (miRNAs) have been reported to play indispensable roles in regulating various developmental processes via the post-transcriptional repression of target genes in mammals. However, the functional link between RBP4 and changes in miRNA expression in porcine granulosa cells (GCs) remains to be investigated. To examine how increased expression of RBP4 affects miRNA expression, porcine GCs were infected with RBP4-targeted lentivirus for 72 h, and whole-genome miRNA profiling (miRNA sequencing) was performed. The sequencing data were validated using real-time quantitative polymerase chain reaction (RT-qPCR) analysis. As a result, we obtained 2783 known and 776 novel miRNAs. In the experimental group, 10 and seven miRNAs were significantly downregulated and upregulated, respectively, compared with the control group. Ontology analysis of the biological processes of these miRNAs indicated their involvement in a variety of biological functions. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses indicated that these miRNAs were involved mainly in the chemokine signaling pathway, peroxisome proliferators-activated receptors (PPAR) signaling pathway, insulin resistance pathway, nuclear factor-kappa B(NF-kappa B) signaling pathway, and steroid hormone biosynthesis. Our results indicate that RBP4 can regulate the expression of miRNAs in porcine GCs, with consequent physiological effects. In summary, this study profiling miRNA expression in RBP4-overexpressing porcine GCs provides an important reference point for future studies on the regulatory roles of miRNAs in the porcine reproductive system.
Highlights
Despite the hundreds of porcine follicles whose growth begins at estrus, only10–20 follicles are selected to release a mature oocyte [1]
In granulosa cells (GCs) cultured in vitro [34] and identified the effects of Retinol binding protein 4 (RBP4) on GCs by transcriptomic analysis following RBP4 overexpression [35]. These results show that there is a high level of RBP4 expression in follicular fluids in cystic follicles, which led us to assume the possible association of RBP4 with polycystic ovary syndrome (PCOS) and cystic follicles
Several studies have pointed out the potential role of miRNAs in GC follicular development and have suggested that aberrations in miRNA expression may play important roles in apoptosis and follicular atresia progression by regulating genes and pathways involved in ovarian diseases [53,54,55]
Summary
10–20 follicles are selected to release a mature oocyte [1]. Folliculogenesis involves many events at the cellular and molecular levels that occur in a highly orchestrated manner, culminating in ovulation [2]. RBP4 expression in adipose tissues is regulated by 17-estradiol, and the levels of RBP4 in serum are elevated in women with PCOS and obesity [19]. These data suggest that RBP4 might be involved in normal ovarian function. In GCs cultured in vitro [34] and identified the effects of RBP4 on GCs by transcriptomic analysis following RBP4 overexpression [35] Taken together, these results show that there is a high level of RBP4 expression in follicular fluids in cystic follicles, which led us to assume the possible association of RBP4 with PCOS and cystic follicles. The specific effects of different miRNAs and the mechanisms, the target genes and signaling pathways, by which they regulate GCs treated with RBP4 are poorly defined
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