Transcriptome-Wide Sequencing Identifies Non-Coding RNAs and Their Competing Endogenous RNA Networks During the Stages of pPGCLCs-Induced Differentiation.

Abstract

Primordial germ cells (PGCs) are undifferentiated embryonic germ cells with the unique potential to develop into gametes. It is widely recognized that PGC development involves the activation of germ cell-specific genes and the repression of certain pluripotency genes. However, little is known about the noncoding RNAs that play a crucial role in regulating cellular functions during PGC development. In this study, to investigate the ncRNA regulatory network during PGC differentiation, whole transcriptome sequencing technology was employed during pPGCLC differentiation from porcine skin-derived stem cells (pSDSCs). Our findings unveiled that the TGF-β signaling pathway was indispensable for PGC cell fate commitment in pPGCLCs. Specifically, SMAD3 and ACVR2B, genes associated with the TGF-β pathway, showed marked upregulation at 20 d. We then identified their target microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), including ssc-miR-504, ssc-miR-125a, ssc-let-7c, MSTRG.8397, MSTRG.5581, MSTRG.4342, MSTRG.4186, and MSTRG.1058 and subsequently constructed the competitive endogenous RNA (ceRNA) network. To validate our analysis, we transfected miRNA inhibitors into cells. RT-qPCR analysis revealed a notable upregulation in the expression levels of MSTRG.8397, MSTRG.5581, MSTRG.4342, MSTRG.4186, MSTRG.1058, SMAD3, and ACVR2B compared to the negative control (NC) group. This is the first study to describe ncRNAs during the induction of pSDSCs to pPGCLCs. Our findings help to elucidate the molecular mechanism involved in the induction of pPGCLCs from pSDSCs.