Differential expression profiles of long non­coding RNAs during the mouse pronuclear stage under normal gravity and simulated microgravity.

ABSTRACT

Pronuclear migration, which is the initial stage of embryonic development and the marker of zygote formation, is a crucial process during mammalian preimplantation embryonic development. Recent studies have revealed that long non­coding RNAs (lncRNAs) serve an important role in early embryonic development. However, the functional regulation of lncRNAs in this process has yet to be elucidated, largely due to the difficulty of assessing gene expression alterations during the very short time in which pronuclear migration occurs. It has previously been reported that migration of the pronucleus of a zygote can be obstructed by simulated microgravity. To investigate pronuclear migration in mice, a rotary cell culture system was employed, which generates simulated microgravity, in order to interfere with murine pronuclear migration. Subsequently, lncRNA sequencing was performed to investigate the mechanism underlying this process. In the present study, a comprehensive analysis of lncRNA profile during the mouse pronuclear stage was conducted, in which 3,307 lncRNAs were identified based on single­cell RNA sequencing data. Furthermore, 52 lncRNAs were identified that were significantly differentially expressed. Subsequently, 10 lncRNAs were selected for validation by reverse transcription­quantitative polymerase chain reaction, in which the same relative expression pattern was observed. The results revealed that 12 lncRNAs (lnc006745, lnc007956, lnc013100, lnc013782, lnc017097, lnc019869, lnc025838, lnc027046, lnc005454, lnc007956, lnc019410 and lnc019607), with tubulin ß 4B class IVb or actinin α 4 as target genes, may be associated with the expression of microtubule and microfilament proteins. Binding association was confirmed using a dual­luciferase reporter assay. Finally, Gene Ontology analysis revealed that the target genes of the differentially expressed lncRNAs participated in cellular processes associated with protein transport, binding, catalytic activity, membrane­bounded organelle, protein complex and the cortical cytoskeleton. These findings suggested that these lncRNAs may be associated with migration of the mouse pronucleus.