MicroRNA-378a-5p promotes trophoblast cell survival, migration and invasion by targeting Nodal.

Nodal is a member of the transforming growth factor-ß superfamily that plays crucial roles during embryogenesis. Recently, we have reported that Nodal inhibits trophoblast cell proliferation, migration and invasion, but induces apoptosis in the human placenta. In this study, we examined the regulation of Nodal by microRNAs. In silico analysis of Nodal 3'UTR revealed a potential binding site for miR-378a-5p. In luciferase reporter assays, we found that miR-378a-5p suppressed the luciferase activity of a reporter plasmid containing Nodal 3'UTR but this suppressive effect was completely abolished when the predicted target site was mutated. Western blot analysis showed that miR-378a-5p decreased whereas anti-miR-378a-5p increased Nodal protein levels. These results indicate that miR-378a-5p targets Nodal 3'UTR to repress its expression. Stable transfection of the miR-378a-5p precursor, mir-378a, into HTR8/SVneo cells enhanced cell survival, proliferation, migration and invasion. Transient transfection of mature miR-378a-5p mimic, and to a lesser extent, siRNA targeting Nodal, produced similar effects. However, anti-miR-378a-5p inhibited cell migration and invasion. In addition, overexpression of Nodal reversed the invasion-promoting effect of miR-378a-5p. Furthermore, miR-378a-5p enhanced, whereas anti-miR-378a-5p suppressed, the outgrowth and spreading of extravillous trophoblast cells in first trimester placental explants. Finally, miR-378a-5p was detected in human placenta throughout different stages of gestation and in preterm pregnancies, placental miR-378a-5p levels were lower in preeclamptic patients than in healthy controls. Taken together, these findings strongly suggest that miR-378a-5p plays an important role in human placental development by regulating trophoblast cell growth, survival, migration and invasion, and that miR-378a-5p exerts these effects, at least in part, through the suppression of Nodal expression.

Epidermal growth factor promotes cyclin G2 degradation via calpain-mediated proteolysis in gynaecological cancer cells.

Cyclin G2 (CCNG2) is an atypical cyclin that functions to inhibit cell cycle progression and is often dysregulated in human cancers. We have previously shown that cyclin G2 is highly unstable and can be degraded through the ubiquitin/proteasome pathway. Furthermore, cyclin G2 contains a PEST domain, which has been suggested to act as a signal for degradation by multiple proteases. In this study, we determined if calpains, a family of calcium-dependent proteases, are also involved in cyclin G2 degradation. The addition of calpain inhibitors or silencing of calpain expression by siRNAs strongly enhanced cyclin G2 levels. On the other hand, incubation of cell lysates with purified calpains or increasing the intracellular calcium concentration resulted in a decrease in cyclin G2 levels. Interestingly, the effect of calpain was found to be dependent on the phosphorylation of cyclin G2. Using a kinase inhibitor library, we found that Epidermal Growth Factor (EGF) Receptor is involved in cyclin G2 degradation and treatment with its ligand, EGF, induced cyclin G2 degradation. In addition, the presence of the PEST domain is necessary for calpain and EGF action. When the PEST domain was completely removed, calpain or EGF treatment failed to trigger degradation of cyclin G2. Taken together, these novel findings demonstrate that EGF-induced, calpain-mediated proteolysis contributes to the rapid destruction of cyclin G2 and that the PEST domain is critical for EGF/calpain actions.