College of Animal Science, National Engineering Research Center for Breeding Swine Industry, Guangdong Provincial Key Lab of Agro‑Animal Genomics and Molecular Breeding, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong 510642, P.R. China.
Mol Med Rep. 2019 Jan;19(1):155-164. doi: 10.3892/mmr.2018.9675. Epub 2018 Nov 20.
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 non‑coding 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 single‑cell RNA sequencing data. Furthermore, 52 lncRNAs were identified that were significantly differentially expressed. Subsequently, 10 lncRNAs were selected for validation by reverse transcription‑quantitative 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 β 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 dual‑luciferase 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, membrane‑bounded organelle, protein complex and the cortical cytoskeleton. These findings suggested that these lncRNAs may be associated with migration of the mouse pronucleus.
原核迁移是胚胎发育的初始阶段和受精卵形成的标志，是哺乳动物植入前胚胎发育过程中的一个关键过程。最近的研究表明，长链非编码 RNA（lncRNA）在早期胚胎发育中发挥着重要作用。然而，lncRNA 在这一过程中的功能调控仍不清楚，这在很大程度上是因为在原核迁移发生的极短时间内，评估基因表达变化的难度很大。此前有报道称，模拟微重力会阻碍受精卵原核的迁移。为了研究小鼠原核迁移，本研究采用旋转细胞培养系统产生模拟微重力，以干扰小鼠原核迁移。随后，进行 lncRNA 测序以研究该过程的机制。在本研究中，对小鼠原核阶段的 lncRNA 谱进行了全面分析，基于单细胞 RNA 测序数据鉴定了 3307 个 lncRNA。此外，鉴定出 52 个差异表达的 lncRNA。随后，选择 10 个 lncRNA 进行逆转录定量聚合酶链反应验证，观察到相同的相对表达模式。结果表明，在 12 个 lncRNA（lnc006745、lnc007956、lnc013100、lnc013782、lnc017097、lnc019869、lnc025838、lnc027046、lnc005454、lnc007956、lnc019410 和 lnc019607）中，微管蛋白β 4B 类 IVb 或肌动蛋白α 4 作为靶基因，可能与微管和微丝蛋白的表达有关。使用双荧光素酶报告基因检测证实了结合关系。最后，GO 分析显示，差异表达 lncRNA 的靶基因参与与蛋白质转运、结合、催化活性、膜结合细胞器、蛋白质复合物和皮质细胞骨架相关的细胞过程。这些发现表明这些 lncRNA 可能与小鼠原核迁移有关。
