The Role Of Leptin In Proliferation Of HTR8-SVNEO Cells In Vitro And Its Mechanism

Pregnancy is a delicate and complicated process, the implantation of embryo and maintenance of pregnancy is highly depended on the normal proliferation of trophoblasts. The abnormal proliferation of trophoblasts is associated with variant gynecologic disorder, such as spontaneous abortion or choriocarcinoma. Nevertheless, the regulatory mechanism of trophoblasts proliferation is still under investigation. It has been demonstrated that leptin promotes proliferation of multiple kinds of tumor cells, as well as angiogenesis, suggested that it plays important role in the regulation of proliferation. During pregnancy, the level of leptin is significantly increased in maternal circulation, and this state of hyperleptinemia gradually returns to normal after delivery, indicated that leptin may also regulate the proliferation of trophoblasts, but the precise mechanism still remains unclear. Recent studies have showed that ID (inhibitor of differentiation) is highly expressed in variant kinds of tumor cells, incluing glioma, lung cancer and melanoma, and promotes their proliferation. Moreover, Udagawa et al.have found that leptin regulates the expression of ID mRNA in glial cells of mouse embryos, and is related to glial and neuronal development in embryos. Based on the above investigations, we supposed that leptin and its receptors may promote the proliferation of trophoblasts through the induced expression of ID.Objective:To observe the effect of leptin on HTR8-SVneo cells proliferation in vitro and the level of ID2. To explore the regulation mechanism of leptin on HTR8-SVneo cells proliferation.Methods:Cultured HTR8-SVneo cells in vitro and test the proliferation under the various concentration of leptin (0ng/mL,10ng/mL,50ng/mL,100ng/mL250ng/mL,500ng/mL) by means of MTT. Detected leptin receptor expression on the surface of HTR8-SVneo cells by flow cytometry. Then HTR8-SVneo cells were divided into3groups:control (Ong/ml), leptin (100ng/mL), leptin (500ng/mL). We employed RT-PCR for detecting the expression of leptin receptors, and real time PCR for ID mRNA profiling. Furthermore, we detected the protein level of ID2by western blot. HTR8-SVneo cells were divided into6groups under the presence of ID2-siRNA or not, to observe the proliferation of HTR8-SVneo cells:leptin (Ong/mL), leptin (Ong/mL)+ID2-siRNA, leptin (100ng/mL), leptin (100ng/mL)+ID2-siRNA, leptin (500ng/mL), leptin (500ng/mL)+ID2-siRNA. At last we employed western blot to detect the level of p-AKT at different time point (5min、10min、15min、30min、60min) after leptin (500ng/mL) stimulation, then observed the effect of AKT inhibitor on expression of ID2in HTR8-SVneo cells.Results:1. Compared with the control group, the high level of leptin (100ng/mL,250ng/mL,500ng/mL) enhances the proliferation of HTR8-SVneo cells (P<0.05).2. HTR8-SVneo cells express leptin receptors incluing OB-RL、HuB219.1、 HuB219.3.3. Leptin increases the expression of ID2and OB-RL in a dose-dependent manner (P<0.05).4. ID2-siRNA inhibits the expression of ID2and reversed the effect of leptin on the proliferation of HTR8-SVneo cells.5. Leptin promotes the phosphonation of AKT, and AKT inhibitor down-regulates the expression of ID2induced by leptin.Conclusion:HTR8-SVneo cells express the leptin recptor OB-RL, and the interation of leptin-leptin receptor exerts its function on HTR8-SVneo cells. Leptin promotes the proliferation of HTR8-SVneo cells by the pathway of leptin R-ID2. AKT signaling pathway is involved in leptin-induced expression of ID2. Our results provide the new data for clarifying the regulation mechanism of trophoblasts proliferation.