Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu, China.
Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China.
J Cell Physiol. 2020 Dec;235(12):9895-9909. doi: 10.1002/jcp.29805. Epub 2020 May 27.
Long noncoding RNAs (lncRNAs) participate in the formation of primordial germ cells (PGCs); however, the identity of the key lncRNAs and the molecular mechanisms responsible for the formation of PGCs remain unknown. Here, we identify a key candidate lncRNA (lncRNA PGC transcript-1, LncPGCAT-1) via RNA sequencing of embryonic stem cells, PGCs, and Spermatogonial stem cells (SSCs). Functional experiments confirmed that LncPGCAT-1 positively regulated the formation of PGCs by elevating the expression of Cvh and C-kit while downregulating the pluripotency(Nanog) in vitro and in vivo; PAS staining of genital ridges in vivo also showed that interference with LncPGCAT-1 can significantly reduce the number of PGCs in genital ridges, while overexpression of LncPGCAT-1 had the opposite result. The result of luciferase reporter assay combined with CHIP-qPCR showed that the expression of LncPGCAT-1 was promoted by the transcription factor P53 and high levels of H3K4me2. Mechanistically, the luciferase reporter assay confirmed that mitogen-activated protein kinase 1 (MAPK1) was the target gene of LncPGCAT-1 and gga-mir-1591. In the ceRNA system, high levels of N methylation of LncPGCAT-1 enhanced the adsorption capacity of LncPGCAT-1 for gga-mir-1591. Adsorption of gga-mir-1591 activated the MAPK1/ERK signaling cascade by relieving the gga-mir-1591-dependent inhibition of MAPK1 expression. Moreover, LncPGCAT-1 interacted with interleukin enhancer binding factor 3 (ILF3) to regulate the ubiquitination of P53 and phosphorylation of JNK. Interaction with ILF3 resulted in positive self-feedback regulation of LncPGCAT-1 and activation of JNK signaling, ultimately promoting PGC formation. Altogether, the study expands our knowledge of the function and molecular mechanisms of lncRNAs in PGC development.
长链非编码 RNA（lncRNA）参与原始生殖细胞（PGC）的形成；然而，形成 PGC 的关键 lncRNA 及其分子机制仍不清楚。在这里，我们通过对胚胎干细胞、PGC 和精原干细胞（SSC）的 RNA 测序，鉴定出一个关键的候选 lncRNA（lncRNA PGC 转录物-1，LncPGCAT-1）。功能实验证实，LncPGCAT-1 通过上调 Cvh 和 C-kit 的表达，同时下调体外和体内多能性（Nanog），从而正向调控 PGC 的形成；体内生殖嵴的 PAS 染色也表明，干扰 LncPGCAT-1 可显著减少生殖嵴中的 PGC 数量，而过表达 LncPGCAT-1 则有相反的结果。荧光素酶报告基因检测结合 CHIP-qPCR 的结果表明，LncPGCAT-1 的表达受转录因子 P53 和高 H3K4me2 水平的促进。从机制上讲，荧光素酶报告基因检测证实丝裂原活化蛋白激酶 1（MAPK1）是 LncPGCAT-1 的靶基因，gga-mir-1591 是 LncPGCAT-1 的靶基因。在 ceRNA 系统中，LncPGCAT-1 的 N 甲基化水平升高增强了 LncPGCAT-1 对 gga-mir-1591 的吸附能力。gga-mir-1591 的吸附通过解除 gga-mir-1591 对 MAPK1 表达的抑制作用，激活 MAPK1/ERK 信号级联。此外，LncPGCAT-1 与白细胞介素增强结合因子 3（ILF3）相互作用，调节 P53 的泛素化和 JNK 的磷酸化。与 ILF3 的相互作用导致 LncPGCAT-1 的正反馈自我调节和 JNK 信号的激活，最终促进 PGC 的形成。总之，该研究扩展了我们对 lncRNA 在 PGC 发育中的功能和分子机制的认识。
