跨膜蛋白16A对胚胎着床和子宫内膜容受性的影响
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摘要
目的研究跨膜蛋白16A(TMEM16A)对子宫内膜容受性和胚胎着床的影响。方法体外培养子宫内膜Ishikawa细胞系,分为空白对照组、E_2+P_4组、抑制剂组和E_2+P_4+抑制剂组。分别加药处理后,采用实时荧光定量(qRT-PCR)和免疫印记试验(Western blot)技术检测各组TMEM16A、白血病抑制因子(LIF)和整合素αvβ3mRNA和蛋白的表达水平。采用体外细胞粘附实验检测E_2+P_4组和E_2+P_4+T16Ainh-A01组Ishikawa细胞与人绒毛膜滋养细胞HTR8-SVneo之间的粘附作用。结果 qRT-PCR和Western blot结果显示,E_2+P_4组的TMEM16A、LIF和整合素αvβ3mRNA和蛋白的表达水平最高,在加入T16Ainh-A01后3个基因的表达水平均显著降低(P<0.01),但均显著高于空白对照组(P<0.01);各组TMEM16A、LIF和整合素αvβ3表达水平均与空白对照组存在显著差异(P<0.01)。细胞粘附实验结果显示,E_2+P_4+抑制剂组中Ishikawa细胞与HTR8-SVneo细胞的粘附作用显著小于E_2+P_4组(P<0.05)。结论抑制TMEM16A的表达降低着床期LIF和整合素αvβ3的表达,可能影响子宫内膜容受性;抑制TMEM16A可降低子宫内膜细胞与滋养层细胞之间的粘附作用,可能导致胚胎着床失败。
Objective:To investigate the effect of transmembrane protein 16A on embryo implantation and endometrial receptivity.Methods:Ishikawa cells were cultured in vitro and incubated with estrogen combined progesterone(E_2+P_4 group),T16Ainh-A0 1(inhibitor group),estrogen combined progesterone added T16AinhA0 1(inhibitor+ E_2+P_4 group),and 0.1% DMSO(control group).The expressions of mRNA and the protein of TMEM16A,leukemia inhibitory factor(LIF)and Integrinαvβ3 in Ishikawa cells were detected by Q-PCR and western blot.The adhesion was detected by cell adhesion assay in vitro both in E_2+P_4+T16Ainh-A0 1 group and E_2+P_4 group in Ishikawa cell line and HTR8-SVneo cell line.Results:Q-PCR and western blot showed that the expression levels of TMEM16A,LIF and Integrinαvβ3 mRNA and protein after treated with E_2+P_4 were significantly higher than other groups(P<0.01),and the expression of the three genes were significantly reduced after adding the T16Ainh-A01(P<0.01),but they were significantly higher than the control group(P <0.01).The expression levels ofTMEM16A,LIF and integrinαvβ3 in each group were significantly different from those in the blank control group(P<0.01).The results of cell adhesion experiment showed that the adhesion of Ishikawa cells to HTR8-SVneo cells in the E_2+P_4+inhibitor group was significantly less than that of the E_2+P_4 group(P<0.05).Conclusions:Inhibiting the expression of TMEM16Acan reduce the expression of LIF and Integrinαvβ3 in the implantation period,which may affect the receptivity of endometrium.Inhibition of TMEM16A can reduce the adhesion between endometrial cells and trophoblast cells,which may lead to embryo implantation failure
Objective:To investigate the effect of transmembrane protein 16A on embryo implantation and endometrial receptivity.Methods:Ishikawa cells were cultured in vitro and incubated with estrogen combined progesterone(E_2+P_4 group),T16Ainh-A0 1(inhibitor group),estrogen combined progesterone added T16AinhA0 1(inhibitor+ E_2+P_4 group),and 0.1% DMSO(control group).The expressions of mRNA and the protein of TMEM16A,leukemia inhibitory factor(LIF)and Integrinαvβ3 in Ishikawa cells were detected by Q-PCR and western blot.The adhesion was detected by cell adhesion assay in vitro both in E_2+P_4+T16Ainh-A0 1 group and E_2+P_4 group in Ishikawa cell line and HTR8-SVneo cell line.Results:Q-PCR and western blot showed that the expression levels of TMEM16A,LIF and Integrinαvβ3 mRNA and protein after treated with E_2+P_4 were significantly higher than other groups(P<0.01),and the expression of the three genes were significantly reduced after adding the T16Ainh-A01(P<0.01),but they were significantly higher than the control group(P <0.01).The expression levels ofTMEM16A,LIF and integrinαvβ3 in each group were significantly different from those in the blank control group(P<0.01).The results of cell adhesion experiment showed that the adhesion of Ishikawa cells to HTR8-SVneo cells in the E_2+P_4+inhibitor group was significantly less than that of the E_2+P_4 group(P<0.05).Conclusions:Inhibiting the expression of TMEM16Acan reduce the expression of LIF and Integrinαvβ3 in the implantation period,which may affect the receptivity of endometrium.Inhibition of TMEM16A can reduce the adhesion between endometrial cells and trophoblast cells,which may lead to embryo implantation failure
引文
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[12] Bernstein K,Vink JY,Fu XW,et al. Calcium-activated chloride channels anoctamin 1and 2promote murine uterine smooth muscle contractility[J].Am J Obstet Gynecol,2014,211:688.e1-e10.
[13] Sun M,Sui Y,Li L,et al.Anoctamin 1calcium-activated chloride channel downregulates estrogen production in mouse ovarian granulosa cells[J]. Endocrinology,2014,155:2787-2796.
[14] Dixon RE,Hennig GW,Baker SA,et al.Electrical Slow Waves in the Mouse Oviduct Are Dependent upon a Calcium Activated Chloride Conductance Encoded by Tmem16a[J].Biol.Reprod,2012,86:1-7.
[15]吴开林,谢青贞.跨膜蛋白16A在人正常月经周期子宫内膜表达的初步研究[J].武汉大学学报(医学版),2018,39:124-128.
[16]杨一凡,谢青贞.雌、孕激素对人子宫内膜腺癌Ishikawa细胞系中TMEM16A的表达调节[J].生殖医学杂志,2018,9:918-922.
[17] Franasiak JM,Holoch KJ,Schammel DP,et al.Prospective assessment of midsecretory endometrial leukemia inhibitor factor expression versusανβ3 testing in women with unexplained infertility[J]. Fertil Steril,2014,101:1724-1731.
[18] Lessey Ba,Castelbaum AJ,Buck CA,et al. Further characterization of endometrial integrins during the menstrual cycle and in pregnancy[J].Fertility sterility,1994,62:497-506.
[19] Apparao K,Murray M,Fritz M,et al.Osteopontin and its receptorαvβ3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially[J].J Clin Endocrinol Metab,2001,86:4991-5000.
[20] Somkuti SG,Yuan L,Fritz MA,et al.Epidermal growth factor and sex steroids dynamically regulate a marker of endometrial receptivity in Ishikawa cells[J]. J Clin Endocrinol Metab,1997,82:2192-2197.
[21] Nishida M,Kasahara K,Kaneko M,et al.Establishment of a new human endometrial adenocarcinoma cell line,Ishikawa cells,containing estrogen and progesterone receptors[J].Nihon Sanka Fujinka Gakkai Zasshi,1985,37:1103-1111.
[2] Aghajanova L.Leukemia inhibitory factor and human embryo implantation[J].Ann Ny Acad Sci,2010,1034:176-183.
[3] Apparao KB,Murray MJ,Fritz MA,et al.Osteopontin and its receptor alphavbeta(3)integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially[J].J Clin Endocrinol Metab,2001,86:4991-5000.
[4] Lee Y,Lee JK,Bae Y,et al.Suppression of 14-3-3γ-mediated surface expression of ANO1 inhibits cancer progression of glioblastoma cells[J].Sci Rep,2016,6:1-14.
[5] Stanich JE,Gibbons SJ,Eisenman ST,et al.Ano1 as a regulator of proliferation[J].Am J Physiol-Gastr L,2011,301:G1044.
[6] Yang YD,Cho H,Koo JY,et al. TMEM16A confers receptor-activated calcium-dependent chloride conductance[J].Nature,2008,455:1210-1215.
[7] Schroeder BC,Cheng T,Jan YN,et al.Expression Cloning of TMEM16Aas a Calcium-Activated Chloride Channel Subunit[J].Cell,2008,134:1019-1029.
[8] Caputo A,Caci E,Ferrera L,et al.TMEM16A,a membrane protein associated with calcium-dependent chloride channel activity[J].Science,2008,322:590-594.
[9] Huang F,Rock JR,Harfe BD,et al.Studies on expression and function of the TMEM16A calcium-activated chloride channel[J].P Natl Acad Sci USA,2009,106:21413-21418.
[10] Hwang SJ,Blair P JA,Britton FC,et al.Hwang SJ,Blair PJ,Britton FC,et al.Expression of anoctamin 1/TMEM16Aby interstitial cells of Cajal is fundamental for slow wave activity in gastrointestinal muscles[J].J Physiol,2009,587:4887-4904.
[11] Py V DW,Rahman M,Imtiaz MS,et al.Spontaneous transient depolarizations in lymphatic vessels of the guinea pig mesentery:pharmacology and implication for spontaneous contractility[J].Am J Physiol Heart Circ Physiol,2008,295:H1989-2000.
[12] Bernstein K,Vink JY,Fu XW,et al. Calcium-activated chloride channels anoctamin 1and 2promote murine uterine smooth muscle contractility[J].Am J Obstet Gynecol,2014,211:688.e1-e10.
[13] Sun M,Sui Y,Li L,et al.Anoctamin 1calcium-activated chloride channel downregulates estrogen production in mouse ovarian granulosa cells[J]. Endocrinology,2014,155:2787-2796.
[14] Dixon RE,Hennig GW,Baker SA,et al.Electrical Slow Waves in the Mouse Oviduct Are Dependent upon a Calcium Activated Chloride Conductance Encoded by Tmem16a[J].Biol.Reprod,2012,86:1-7.
[15]吴开林,谢青贞.跨膜蛋白16A在人正常月经周期子宫内膜表达的初步研究[J].武汉大学学报(医学版),2018,39:124-128.
[16]杨一凡,谢青贞.雌、孕激素对人子宫内膜腺癌Ishikawa细胞系中TMEM16A的表达调节[J].生殖医学杂志,2018,9:918-922.
[17] Franasiak JM,Holoch KJ,Schammel DP,et al.Prospective assessment of midsecretory endometrial leukemia inhibitor factor expression versusανβ3 testing in women with unexplained infertility[J]. Fertil Steril,2014,101:1724-1731.
[18] Lessey Ba,Castelbaum AJ,Buck CA,et al. Further characterization of endometrial integrins during the menstrual cycle and in pregnancy[J].Fertility sterility,1994,62:497-506.
[19] Apparao K,Murray M,Fritz M,et al.Osteopontin and its receptorαvβ3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially[J].J Clin Endocrinol Metab,2001,86:4991-5000.
[20] Somkuti SG,Yuan L,Fritz MA,et al.Epidermal growth factor and sex steroids dynamically regulate a marker of endometrial receptivity in Ishikawa cells[J]. J Clin Endocrinol Metab,1997,82:2192-2197.
[21] Nishida M,Kasahara K,Kaneko M,et al.Establishment of a new human endometrial adenocarcinoma cell line,Ishikawa cells,containing estrogen and progesterone receptors[J].Nihon Sanka Fujinka Gakkai Zasshi,1985,37:1103-1111.