| Objective:1)To study the effect of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)on the invasion,proliferation and apoptosis induction of trophoblast cells.2)To explore the potential mechanisms of TRAIL regulating trophoblast cells invasion.Methods:1)We used Transwell invasion experiment to investigate the effect of rsTRAIL on the invasion ability of HTR-8/SVneo cells at different concentrations(0 ng/ml、0.1ng/ml、1 ng/ml、10 ng/ml),and different stimulating time(24h、48h、72h).2)CCK8 colorimetry and Annexin V-EGFP/PI double dyeing method were used to test the proliferation and apoptosis of HTR-8/SVneo cells with variations of rsTRAIL concentration.3)The expression of CXCR4,EGFR,MMP2 and miR-146a in HTR-8/SVneo cells treated with rsTRAIL at different concentrations(0 ng/ml,0.1 ng/ml,1 ng/ml,and 10 ng/ml)was detected by qPCR and Western blotting.Result:1)At the same stimulation time,the quantity of HTR-8/SVneo cells passing through transwell chamber increased with the increasing concentration of rsTRAIL in a concentration-dependent manner.There were statistical differences in the number of invasion cells among different concentrations(p<0.05).2)At the rsTRAIL concentration of either Ong/ml or O.lng/ml,the number of HTR-8/SVneo cells crossing Transwell chamber in 24h stimulation group,48h stimulation group and 72h stimulation group showed no significant difference.While concentration of rsTRAIL reached 1ng/ml and 10 ng/ml,the number of invasive cells in 48h stimulation group increases more prominently compared with 24h stimulation group(P<0.05),since then the invasion cells counting did not change as the rsTRAIL stimulation time extended.3)There was significant interaction between the concentration and stimulation time of rsTRAIL on the invasion of HTR-8/SVneo cells(P<0.05).Stimulated by the concentration of 10 ng/ml rsTRAIL for 72h,HTR-8/SVneo cells passed through Transwell chamber the most easily and their promotion of invasiveness was the strongest.4)Comparing to the control group,there was no significant change in the proliferation of HTR-8/SVneo cells under the stimulation with rsTRAIL at concentrations of 0.1ng/ml,lng/ml and 10ng/ml.5)In comparison with the control group,the apoptosis rate of HTR-8/SVneo cells treated with rsTRAIL didn’t increase while at the conccentration of 0.1 ng/ml.However,the apoptosis rate improved at the concentration of lng/ml but it did not increase with the increasing rsTRAIL concentrations.6)As the rsTRAIL concentration increased,the mRNA expression level of CXCR4,EGFR and MMP2 in HTR-8/SVneo cells increased successively while the expression of miR-146a descended in turn,which revealed a dose-dependent effect.There was statistically significant difference by comparison of each two groups.(P<0.05)when we analysed the mRNA expression of CXCR4,EGFR and MMP2 as well as the miR-146a expression in those four groups,such as control,0.1ng/ml,lng/ml and 10ng/ml.7)There existed concentration dependence when rsTRAIL upregulated the expression of protein CXCR4,EGFR and MMP2 in HTR-8/SVneo cells.In the O.1ng/ml rsTRAIL group,the expression of EGFR protein increased significantly when compared with the control group(P<0.05).Additionally,the CXCR4,MMP2 protein expression also increased relatively but that showed no statistical difference.When concentration of rsTRAIL reached lng/ml and 10ng/ml,the protein expression of CXCR4,EGFR,MMP2 significantly increased and there was no significant difference(P<0.05).Conclusion:1)TRAIL regulates the fuction of trophoblast cells in enhancing their invasion,rather than the proliferation and apoptosis.TRAIL can significantly promote the invasion of trophoblast cells in concentration-and time-dependent manners.2)TRAIL may play a role in the regulation of trophoblastic invasiveness by upregulating the expression of CXCR4,EGFR,MMP2 and down-regulating the expression of miR-146a. |
PDF Full Text Request