MicroRNA profiling of royal jelly extracellular vesicles and their potential role in cell viability and reversing cell apoptosis.

ABSTRACT

MiRNAs are small non-coding RNA molecules that play important regulatory roles in diverse biological processes. Royal jelly, a milky-white substance produced by nurse honeybees (Apis mellifera), is the primary food of queen bees and plays a crucial role in their development. However, little is known about the microRNA (miRNAs) content of royal jelly and their potential functions. In this study, we isolated extracellular vesicles from the royal jelly of 36 samples through sequential centrifugation and targeted nanofiltration and performed high-throughput sequencing to identify and quantify the miRNA content of honeybee royal jelly extracellular vesicles (RJEVs). We found a total of 29 known mature miRNAs and 17 novel miRNAs. Through bioinformatic analysis, we identified several potential target genes of the miRNAs present in royal jelly, including those involved in developmental processes and cell differentiation. To investigate the potential roles of RJEVs in cell viability, RJEVs were supplemented to apoptotic porcine kidney fibroblasts induced by ethanol 6% exposure for 30 min. TUNEL assay showed a significant reduction in the apoptosis percentage after RJEV supplementation when compared with the non-supplemented control group. Moreover, the wound healing assay performed on the apoptotic cells showed a rapid healing capacity of RJEV-supplemented cells compared to the control group. We observed a significant reduction in the expression of the miRNA target genes such as FAM131B, ZEB1, COL5A1, TRIB2, YBX3, MAP2, CTNNA1, and ADAMTS9 suggesting that RJEVs may regulate the target gene expression associated with cellular motility and cell viability. Moreover, RJEVs reduced the expression of apoptotic genes (CASP3, TP53, BAX, and BAK), while significantly increasing the expression of anti-apoptotic genes (BCL2 and BCL-XL). Our findings provide the first comprehensive analysis of the miRNA content of RJEVs and suggest a potential role for these vesicles in the regulation of gene expression and cell survival as well as augmenting cell resurrection or anastasis.