Pre-eclampsia is a complication of pregnancy which is discussed hotly because of its high morbidity, mortality and increasingly risk of chronic diseases in postpartum such as diabetes, hypertension and cardiovascular diseases. However, the pathogenesis of pre-eclampsia is poorly understood and there is no effective treatment other than childbirth in current[1]. Many studies have shown that after the embryo implanted into the endometrium in physical, the ability of EVT cells to proliferate, invade and migrate gradually increased. On the contrary, when the pre-eclampsia occurs, the invasion and migration capacity of EVT cells appear to impaired apparently, which leads to insufficient remodeling of the uterine spiral artery, resulting in low placental blood perfusion and superficial placental implantation in the uterus[21]. Thus, the proliferation, invasion and migration functions of EVT cells are thought to be essential for pregnancy[22].
Similar to the tumor cells, EVT cells exhibit high proliferation, aggressiveness, and migration features[23]. In the first trimester, EVTs, together with natural killer cells and macrophages, invade the maternal endometrium under the role of matrix metalloproteinases, gradually migrating to the uterine spiral arteries, invading the blood vessels, replacing the endothelial cells[24], which physiologically reconstituted the uterine spiral arteries into a high-drain, low-resistance type to increase the blood flow for fetus[4]. HTR8/SV-neo cell lines are widely used to study the adhesion, migration and invasion functions of trophoblasts and are generated by transfecting human primary trophoblasts with SV40-containing large T antigens[25]. Therefore, it is important to use HTR8/SV-neo cell lines as an in vitro model of human extravillous trophoblasts to observe changes in proliferation, invasion and migration functions of HTR8/SV-neo cell lines and explore possible potential mechanisms.
Forskolin is a specific activator of cAMP signaling pathway and our study also found that intracellular cAMP levels increased dose-dependently with increasing forskolin concentrations. Due to the poor solubility of forskolin in water, it requires up to 3% by mass of DMSO solution to dissolve it, whereas in most intact mammalian cell experiments, it can only tolerate up to 1% concentration of DMSO solution[16]. In this experiment, the cells were treated with different concentration of forskolin, which was dissolved with DMSO solution in necessity. In order to exclude the possible influence of DMSO solution on the experimental results, a toxic solution to cells in a certain concentration, we added the largest amount of DMSO solution in control group, compared to the highest DMSO concentration in the experimental group. As a result, in the control group, in 25µl DMSO solution mixed in 3ml culture medium, HTR8/SV-neo cells were cultured. In addition, the high hydrophobicity of forskolin indicates that there is a slow rate of dissociation, which keeps a certain drug prolonging effect, and means it can be effectively achieved applying forskolin to treat cells for 48h and 10min.
By treating HTR8/SV-neo cells with different concentrations of forskolin solution, we found that the proliferation, invasion and migration ability of HTR8/SV-neo cells also increased significantly within increasing intracellular cAMP levels, but there was no significant difference between 10µM and 50µM concentration. At 100µM concentration, the ability of cells to proliferate did not change significantly compared to controls. We speculate that intracellular cAMP may improve the proliferative properties of cells within a certain concentration range, because studies have shown that elevated cAMP levels can promote cell proliferation in some physical cells or tumors, and some data suggest that cAMP may cause cell growth cycle arrest or even apoptosis[12, 26]. When it exceeds a certain level, the proliferation of cells can be decreased or stopped or even prone to apoptosis, such as in tumor.
The cAMP/PKA/CREB signaling pathway and MAPK signaling pathway are two classical pathways that regulate cell growth and metabolism, and have been extensively studied in a variety of tumor cells and trophoblasts[27]. In trophoblasts, the cAMP/PKA/CREB signaling pathway has been found to be involved in the regulation of trophoblast fusion function[28, 29]. Therefore, we further investigated the specific mechanism of the cAMP signaling pathway and the MAPK signaling pathway in regulating trophoblasts. In our study, we found that when the cAMP level was increased in cells, the phosphorylation levels of c-Raf259, MEK1/2 and ERK1/2 in the MAPK signaling pathway were significantly increased. Previous studies in lung cancer cells found that after activation of the MAPK signaling pathway, the phosphorylation level of c-Raf259 protein was reduced, while the phosphorylation level of c-Raf338 protein was significantly increased, the growth of lung cancer cells was inhibited, and the functions of angiogenesis and epithelium-mesenchyme transformation were downregulated[30]. And in our experiments, the results are as the same. Therefore, we believe that EVT cells may promote cell proliferation and invasion abilities through activation of the phosphorylation level of Ser259 locus of c-raf protein in the MAPK signaling pathway.