Involvment Of SK3in Preiniplantation Embryo Development,Endometrium Growth And Cndometrial Carcinoma Developemt

Part â…  SK3Regulate blastocyst hatching by control of intracellular calcium concentrationObjective:The present study was designed to investigate the effect of small-conductance calcium-activated K+channels3(SK3) on blastocyst hatching and underlying mechanism by detecting the expression of SK3in preimplantation embryos.Materials and methods:Human preimplantation embryos were obtained from voluntary donation of patients who had successful pregnancy with in-vitro fertilization. Mouse preimplantation embryos in different stages were collected and cultured with or without siRNA cell injection. The expression of SK3was examined by RT-PCR, quantitative real-time PCR and immunofluorescence. Membrane potential was measured by patch-clamp.[Ca2+]i was measured by fluorescent imaging.Results:In human blastocysts, the level of SK3expression showed significantly lower in failed hatching blastocysts than that in successful hatching blastocysts. In mouse, the SK3mRNA and protein were not found in zygotes, but detected from the2-cell stage onwards, with the highest levels observed in blastocysts. The SK3predominately located in the trophectoderm cell membrane of expanded blastocyst. The thapsigargin (TG) stimulated an increase in the [Ca2+]i and membrane potential hyperpolarization in trophectoderm cell in vitro, which could be significantly reduced by SK3knock-down. Although the formation of expanded blastocyst was not affected, blastocyst hatching as well as F-actin formation were significantly inhibited after SK3knock-down.Conclusions:SK3-mediated [Ca2+]i elevation and membrane potential hyperpolarization in trophectoderm cell is important for blastocyst hatching, a defect of which may contribute to infertility. Objective:To investigate the value of endometrial thickness and endometrial pattern as prognostic parameters for successful pregnancy in in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET). To study the effect of small-conductance Ca2+-activated K+channels3(SK3) on facilitating the elevation of cytosolic Ca2+in cell migration during cell proliferation to form the normal structure of tissue. And investigate the correlation between SK3expression and endometrial thickness and pattern (individually and together).Materials and methods:Endometrial ultrasonographic characteristics were detected on the day of hCG administration. Endometrial thickness and pattern (individually and together) were analyzed. The correlation between SK3expression and endometrial thickness and pattern and were examined. And the relationship between SK3expression and CP outcome were examined. The expression profile of SK3was investigated in human endometrium by RT-PCR, western blot and immunofluorescence staining. The effect of SK3on the regulation of cytosolic Ca2+and the membrane potential was measured by electrophysiologic technology and cytosolic calcium measurement. Furthermore, we explored the intracellular mechanism of SK3involved in the migration of human endometrial epithelium cell after SK3knockdown by special siRNA.Results:The SK3mRNA levels in endometrium were significantly correlated with the endometrial thickness and no-triple line endometrial pattern showed a lower SK3mRNA levels. The failed group had a low SK3protein expression in IVF treatment. SK3expressed in human endometrial epithelium and was up-regulated in proliferative phase and down-regulated in secretory phase. SK3knockdown inhibited the elevation of cytosolic calcium and the hyperpolarization induced by thapsigargin. In vitro experiment also confirmed that SK3expression was estrogen-dependent. Cell migration and F-Actin assembly were significantly suppressed by SK3knockdown, which inhibited the elevation of cytosolic calcium induced by cytosolic cAMP as well as the co-interaction of S100A4with MHCIIA.Conclusions:SK3is a potenial biomarker of human endometrial structure forming. Through a cAMP-activated calcium signaling pathway, SK3plays an important role in regulating the migration of human endometrial epithelial cell during endometrial epithelium proliferation. Part â…¢ Involvement of SK3in estrogen-activated calcium signaling-induced migration and invasion of endometrial cancer cellsObjective:To explore the mechanism of SK3involved endometrial cancer cell migration and invasion induced by estrogen.Materials and methods:The expression profile of SK3was investigated in endometrial cancer tissue and3cancer cell lines by RT-PCR, Quantitative real-time PCR and western blot. The expression of SK3affected by estradiol(E2) was analysed using Ishikawa(IK) cells by western blot. SK3special siRNA transfection was used to found the in vitro knockout cell model. Cell migration and invasion analysis were used to value effect of SK3knockout on cell function. The effect of SK3on the regulation of cytosolic Ca2+induced by estrogen was measured by cytosolic calcium measurement. Western blot assay using SK3inhibitor along with E2treatment detected the downstream of calcium signaling pathway activated by estrogen in cell motility.Results:SK3was expressed in endometrial cancer tissue and cancer cell lines. SK3has a higher expression level in cell lines with lower differentiation. In IK cell, E2treatment induced upregulation of SK3expression and cell migration. Moreover, effect of E2on SK3expression were in a dose-dependent manner. Treatment of SK3inhibitor and SK3special siRNA transfection both suppressed cell migration for cultured cells even with E2treatmentt. Further, we identified that SK3expression upregulation was mainly through classic ER and cell migration was through GPR30located in the membrane for estrogen signaling. Inositol1,4,5-triphosphate receptors (IP3Rs) are involved in compartmentalized signaling, and they could increase the calcium of cytoplasma to keep the pathway activation involved by calcium in E2depended cell migration. We found that E2treatment induced a significantly increase for Cytosolic Racl and Cytosolic RhoA activation and hence increased MLC activation, while SK3inhibitor could block the effect. Conclusions:These findings suggest that E2-induced endometrial cancer cell migration was assisted by SK3which the expression was activated of estrogen mainly via binding classic ER and the function was provoked of estrogen via binding of GPR30in the membrane through calcium signaling pathway.