In the present study, we have for the first time detected the expression of LIN28B in villi from women with URSA and have explored a possible role for LIN28B in URSA. LIN28B is a paternal imprinting gene23(i.e., is expressed by the paternal source) that promotes the development of the placenta and also stimulates the embryo or fetus to extract nutrients from the mother, thereby promoting the growth of the individual24-26. Our study found that the expression of LIN28B in the villous tissue of URSA patients decreased, implying that the expression of the parental imprinting gene LIN28B may inhibit the development of trophoblast cells and lead to the occurrence of miscarriage.
Recent studies have shown that changes in trophoblastic function are realized through various signal transduction pathways8,22,27-29. LIN28 is highly expressed in placenta and plays an important role in embryonic development and implantation as an RNA-binding protein8. Some studies have shown that the increase in LIN28B expression is positively correlated with the invasion, migration, and proliferation of trophoblast cells19,20,30,31. Canfield et al.8 found that invasive interstitial EVT expressed higher levels of LIN28B in placental sections during early pregnancy compared with non-invasive proximal trophoblast cells, and increased expression of LIN28B increased HTR8/SVneo cell proliferation, migration, and invasion in vitro. We demonstrated that HTR-8/SVneo and BeWo cells that overexpress LIN28B possessed enhanced migratory, and invasive capabilities, consistent with previous studies. Normal pregnancy requires adequate early EVT to invade the decidua, a situation highly similar to cancer metastasis with proper remodeling of the spiral artery32-34. Lin et al. 28found that LIN28B may inhibit apoptosis of ovarian cancer cells through the AKT2/FOXO3A/BIM axis. To further investigate the possible role of LIN28B in trophoblast cells, we also examined the apoptotic function and apoptosis-related proteins of HTR-8/SVneo and BeWo cells that overexpress LIN28B and discovered that anti-apoptotic ability was enhanced after overexpression of LIN28B, that the phosphorylation of the anti-apoptotic protein Akt increased, and that the phosphorylation of the pro-apoptotic proteins Bad and Bcl-2 decreased in HTR-8/SVneo cells. In BeWo cells we only detected an increase in phosphorylation of the anti-apoptotic protein Akt, which may have been due to either extremely low or very unstable protein phosphorylation in BeWo cells after overexpression of LIN28B. We hypothesized that LIN28B inhibited apoptosis of villous cells through the Akt/Bad/Bcl-2 signaling pathway, thereby changing the migration and invasion of villi and ensuring the smooth progress of pregnancy. The low expression of LIN28B in villous tissue of URSA patients may accelerate dysfunction and apoptosis in trophoblast cells, leading to the occurrence of abortion.
Cell fusion in mammals is a common physiologic process that is involved in fertilization, placental development, skeletal muscle and bone development, and immune defense responses35,36. During the development of the human placenta, it undergoes primary synthesis and secondary integration. When cytotrophoblast cells are fused to the syncytiotrophoblast, they are regulated by various cytokines and growth factors13,37,38. Syncytin-1 (ERVW-1) was the first molecule to be ascribed a direct ability to promote cell fusion36. Canfield et al. 8demonstrated that LIN28B plays a role in preeclampsia by reducing syncytialization and that JEG3-knockout of LIN28B in cells significantly decreased SYN-1 while LIN28B overexpression in HTR8/SVneo cells decreased TNF-α expression. Hypoxic culture significantly decreased the expression of LIN28B and SYN-1 in BeWo and EG3 cells and increased the expression of TNF-α. EMT is a biologic process in which differentiated epithelial cells lose epithelial characteristics and acquire mesenchymal migration. This phenomenon not only plays an important role in tumor invasion and migration, formation of endoderm, and primitive intestinal lumen but also participates in cell-fusion processes39,40. Lu et al.41speculated that EMT may play a role in the trophoblast cell assembly process and that Twist1 promotes human placental tissue. Seabrook et al.29showed that knockdown of LIN28A in human trophoblast-like ACH-3P cell lines induced spontaneous syncytialization in the early pregnancy. In the present study, we found that after overexpression of LIN28B the expression level of ERVW-1 decreased, the expression of E-cadherin and P-cadherin increased, and that the expression of the interstitial-related factors vimentin and N-cadherin fluctuated greatly. The number of cells fused was also lower than in the control group, and cell-fusion ability was attenuated. Our results indicated that by overexpression of LIN28B and induction of fusion, cell epithelial characteristics increased, while mesenchymal cells fluctuated. This may be because EMT is a transient and reversible process, consistent with studies by Li et al. 42where the EMT expression at different time-points during hESC differentiation was at a dynamic level. Generally, secondary syncytium exerts obvious effects after 12 weeks of gestation, when the synthesized contracted trophoblast cells play an important role in providing nutrition and gas exchange. The invasive capability of EVT manifests a significant time limit that only occurs in the early pregnancy43,44. Overexposure of cytotrophoblast cells in the early pregnancy, then, will result in the differentiation of invasive EVT cells, with a concomitant diminution in invasive ability accompanied by implantation failure and, ultimately, early abortion.
Several studies have shown that LIN28 plays a role in regulating stem cell activity, including self-renewal and differentiation. Therefore, during the early stages of embryonic development, early gene expression in the placenta is high, and cells continuously develop and differentiate30,31 . LIN28 is also a key factor in the regulation of developmental differentiation. In the present study, we determined the expression of LIN28B in villous tissue from some patients with early URSA due to the inherent cellular capabilities for apoptosis, invasion, migration, and cell fusion. The cells in Vivo exert their functions through various coordination mechanisms. This study has a certain limitation and cannot directly prove whether lin28b plays the same role in vivo as in vitro cells, but we will conduct further studies to prove the role of LIN28B in vivo cells or in URSA animal models.Our future aims are to further confirm that LIN28B is a contributing factor in early-abortion diseases and participates in potential mechanisms underlying placental differentiation, development, and function, thus providing a foundation for further molecular research in this area.