14-3-3 tau蛋白对滋养细胞生物学行为影响的研究
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摘要
滋养细胞功能异常导致病理妊娠的分子机制一直被广泛研究,我们课题组在以前的研究中已运用蛋白质组学技术分别对缺氧培养的滋养细胞和子痫前期胎盘的蛋白质表达谱进行了研究,发现一种名为14-3-3 tau的蛋白质的差异表达,这引起了我们很大的兴趣。在此基础上,本研究进一步从14-3-3 tau在胎盘滋养细胞中的定位入手,再通过RNA干扰和表达质粒转染的方法下调和上调滋养细胞内14-3-3 tau的表达研究其对滋养细胞增殖、分化能力的影响,并通过蛋白质组学和生物信息学的方法从整体角度分析14-3-3 tau在滋养细胞所起的作用。此外对滋养细胞外的14-3-3 tau的功能我们也进行了探讨。旨在通过本课题对14-3-3tau在滋养细胞中所扮演的角色做出较全面的解释。
第一部分14-3-3 tau与滋养细胞的增殖和分化
第一节14-3-3 tau在胎盘滋养细胞中的定位
目的通过分析14-3-3 tau在胎盘组织中的定位和滋养细胞体外分化模型中的表达变化情况,了解滋养细胞中14-3-3 tau可能的作用。
方法用免疫组化方法检测人早孕期绒毛,中孕期胎盘、足月妊娠胎盘及子痫前期胎盘中14-3-3 tau的定位。腺苷酸环化酶激活剂Forskolin诱导BeWo细胞合体化,免疫细胞化学检测BeWo细胞中14-3-3 tau的定位变化,RT-PCR和Western Blot法检测14-3-3 tau转录水平和蛋白水平的变化。
结果1)在各孕期的胎盘绒毛中,14-3-3 tau主要在绒毛内细胞滋养细胞表达,而合体滋养细胞未见其表达,并且随着孕周增加绒毛内细胞滋养数目减少,14-3-3 tau的表达也逐渐减少。2)在早孕期侵袭绒毛和蜕膜组织中,14-3-3 tau在固定绒毛的绒毛外滋养细胞(EVT)胞浆表达,而在侵入蜕膜的EVT胞膜表达。3)病理胎盘中,子痫前期胎盘与正常足月胎盘相比呈明显的缺氧应激改变,14-3-3 tau表达也增加。4)与对照组相比,14-3-3 tau mRNA和蛋白表达在Forskolin处理组BeWo细胞中显著下降(P＜0.05),免疫细胞化学也显示BeWo细胞合体化后14-3-3 tau呈阴性。
讨论14-3-3 tau在不同类型胎盘滋养细胞中的定位提示其与滋养细胞增殖、合体化和侵袭功能相关,并可能参与子痫前期病理过程中胎盘滋养细胞对缺氧的应激调节。
第二节调节14-3-3 tau的表达对BeWo细胞增殖和分化的影响
目的妊娠过程中,细胞滋养细胞不断增殖,并通过两种途径分化,分别形成合体滋养细胞或侵袭型滋养细胞,前期研究显示14-3-3 tau在这三类细胞的表达水平不同,但其发挥的作用尚不清楚,故本研究意在探讨14-3-3 tau是否参与滋养细胞增殖、合体化和侵袭能力的调控。
方法RNA干扰方法下调人绒癌滋养细胞株BeWo细胞内14-3-3 tau的表达,构建14-3-3 tau表达质粒上调BeWo细胞内14-3-3 tau的表达。RT-PCR和Western Blot检测增殖相关分子核增殖抗原(PCNA),合体化相关分子syncytin,侵袭相关分子E钙粘素及其拮抗蛋白Snail的表达变化。BrdU法检测BeWo细胞DNA合成能力,流式细胞仪检测BeWo细胞周期变化。ELISA方法检测滋养细胞β-hCG分泌。Transwell小室实验检测BeWo细胞侵袭能力。用U0126特异性阻断ERK信号通路观察BeWo细胞侵袭和合体化能力的变化。
结果1) RNA干扰72h,BeWo细胞14-3-3 tau表达被显著被下调;转染14-3-3 tau表达质粒24h,BeWo细胞显著表达14-3-3 tau融合蛋白,提示14-3-3tau表达被上调。2)下调14-3-3 tau后BeWo细胞PCNA的转录和蛋白表达减少,而上调14-3-3 tau后PCNA的转录和蛋白表达增加,提示14-3-3 tau促进滋养细胞PCNA的表达。3)下调14-3-3tau后BeWo细胞对BrdU摄取显著减少(P＜0.05),而上调14-3-3 tau后BeWo细胞BrdU摄取呈增加趋势,提示14-3-3tau促进滋养细胞DNA合成。4)下调14-3-3 tau后BeWo细胞G_0/G_1期比例显著增加而S期比例显著减少(P＜0.05),提示14-3-3 tau促进滋养细胞G_1期向S期进级。5)下调14-3-3 tau表达后,合体化相关分子syncytin转录显著增高(P＜0.05),β-hCG分泌显著增加(P＜0.05);上调BeWo细胞14-3-3 tau表达,syncytin转录显著降低(P＜0.05),β-hCG分泌显著减少(P＜0.05)。提示14-3-3tau抑制滋养细胞合体化。6)下调14-3-3 tau表达后,E钙粘素表达减少而Snail表达增加,侵入Transwell小室下层的细胞数目增多,提示14-3-3 tau抑制滋养细胞侵袭。7)下调14-3-3 tau的表达,ERK1/2磷酸化水平增加,而U0126特异性抑制ERK1/2磷酸化上调14-3-3 tau的表达,提示14-3-3 tau负调节ERK1/2的活化。8) U0126阻断ERK1/2后,下调14-3-3 tau不能继续促进BeWo细胞β-hCG的分泌和侵袭,提示14-3-3 tau通过ERK1/2信号通路发挥作用。
结论14-3-3 tau促进滋养细胞增殖,同时抑制滋养细胞向分泌型合体滋养细胞和侵袭型滋养细胞的分化,引起细胞周期分布、分化相关分子的表达和MAPK/ERK1/2信号通路的改变,它可能是滋养细胞分化调控的重要节点分子。
第二部分蛋白质组学和生物信息学方法探讨滋养细胞内14-3-3 tau的作用
目的用比较蛋白质组学的方法和生物信息学方法,研究下调14-3-3 tau的BeWo细胞蛋白质组表达的变化,发现14-3-3 tau在滋养细胞生物功能中涉及的蛋白并探讨其可能的调节机制。
方法RNA干扰技术下调BeWo细胞内14-3-3 tau的表达。同位素标记相对和绝对定量(iTRAQ)技术结合质谱技术鉴定滋养细胞蛋白质组的变化,并进行生物信息学分析。
结果通过iTRAQ-MS鉴定出BeWo细胞中271种蛋白的相对定量信息,与阴性对照组相比,14-3-3 tau siRNA组中有26种蛋白表达发生变化,其中13种蛋白上调,13种蛋白下调,包括增殖相关蛋白(Proliferation-associated protein2G4、Proliferating cell nuclear antigen、plastin 3)表达降低,细胞代谢相关Alpha-2-HS-glycoprotein表达增加,缺氧调控相关蛋白Hypoxia up-regulatedprotein 1和应激蛋白Stress-induced-phosphoprotein 1的表达减少;蛋白转运相关的Sorting nexin-2表达增加,蛋白3级结构形成相关的T-complex protein 1 subunitbeta上调,铁离子转运相关蛋白Transferrin receptor protein 1上调等。这些细胞周期、谷胱甘肽代谢、PPAR信号通路、铁转运等多种机体通路,其中几种蛋白是首次在滋养细胞中被发现。
结论运用蛋白质组学技术和生物信息学方法分析14-3-3 tau对滋养细胞蛋白组影响,为14-3-3 tau在滋养细胞内的功能研究做出了较全面的解释,14-3-3 tau不仅调节滋养细胞的增殖和分化,还可能参与细胞代谢、缺氧损伤、钙化等,研究14-3-3 tau有助于了解滋养细胞功能异常导致的妊娠期疾病可能的分子机制。
第三部分14-3-3 tau在滋养细胞外的表达
第一节14-3-3 tau对子宫内膜基质细胞容受性的影响
目的探讨14-3-3 tau是否影响滋养细胞对周围细胞发挥作用及其可能的机制。
方法RNA干扰技术下调绒癌滋养细胞株BeWo的14-3-3 tau蛋白表达。建立下调14-3-3 tau的BeWo细胞和人子宫内膜基质细胞ESC的共培养体系,Western Blot方法检测共培养体系中培养液14-3-3 tau的表达以及ESC细胞中容受性分子整合素α_vβ_3的表达变化。
结果与阴性对照siRNA组相比,特异性的siRNA可显著下调BeWo细胞内和胞外培养液中14-3-3 tau蛋白的表达(P＜0.05)。共培养体系中,与阴性对照相比,下调14-3-3 tau的BeWo细胞共培养的ESC细胞内整合素α_vβ_3的表达显著增高,提示ESC细胞容受性增加。与单纯培养的ESC相比,培养液中加入14-3-3 tau纯化蛋白培养24h的ESC表达的整合素α_vβ_3显著下调(P＜0.05),提示ESC细胞容受性下降。
结论14-3-3通过调节滋养细胞分化促进合体细胞激素、细胞因子等的释放增加,以及直接作用子宫内膜基质细胞两种途径引起子宫内膜的容受性增加。
第二节14-3-3 tau在孕妇血清和羊水中的表达
目的检测孕妇血清中14-3-3 tau的表达。
方法Western Blot方法检测正常非孕妇女、早孕期妇女、中期妊娠孕妇、晚期妊娠孕妇以及子痫前期孕妇静脉血及胎儿脐带血血清中14-3-3 tau的表达。Western Blot方法检测正常中期妊娠孕妇、足月分娩孕妇、子痫孕妇羊水中14-3-3tau的表达。
结果在正常非孕妇女、早孕期妇女、中期妊娠孕妇、晚期妊娠孕妇以及子痫前期孕妇静脉血及胎儿脐带血血清均未检测到14-3-3 tau的表达。正常中期妊娠孕妇、足月分娩孕妇、子痫孕妇羊水中均检测到14-3-3 tau的表达,并随孕周增加表达增加,子痫前期羊水中14-3-3 tau表达高于足月正常妊娠羊水。
结论血清中14-3-3 tau表达低于本研究的检测水平。14-3-3 tau在羊水中的表达反映了母体、胎儿、羊水之间交换功能的改变,并可能有子痫前期的病理过程有关,但其在正常妊娠以及病理妊娠中发挥怎样的作用仍需要近一步研究。
Disturbed function of trophoblast may lead to a number of pregnancy-associated pathologies,but regulation of trophoblast differentiation and proliferation is partly understood.Recently,we found an interesting protein which named 14-3-3 tau in both hypoxia-treated human trophoblast cell line BeWo and preeclampsia placenta by proteomic analysis,it is suggested that 14-3-3 tau may play important roles in trophoblast biological process.In this research,we firstly detected the location of 14-3-3 tau in human placenta.Secondly,we applied RNA interference and Plasmid transfection techniques to explore the effects of differently expressed 14-3-3 tau on trophoblast proliferation and differentiation.Thirdly,we used proteomic and bioinformatics approaches to analysis the differential expression of proteins and the potential pathways involved in the 14-3-3 tau down-regulated trophoblast.Finally,we discussed the extracellular functions of 14-3-3 tau.We seek a better understanding of how 14-3-3 tau regulates trophoblast.
Section 1 Effects of 14-3-3 tau in trophoblast proliferation and differentiation
Section 1.1 Location of 14-3-3 tau in human placental trophoblast
Objectives:We aimed to evaluate the expression,distribution,and localization of 14-3-3 tau in human placenta tissues and trophoblast differentiation model in order to help to construct a hypothesis about its function in the placenta.
Methods:14-3-3 tau was detected in placenta tissue of three trimester pregnancies by immunohistochemistry.RT-PCR,Western Blot and immunocytochemistry analysis were employed to evaluate expression changes during Forskolin induced in vitro differentiation of human trophoblast cell line(BeWo).
Results:1) In villous tissue,14-3-3 tau was immunolocalized to villous cytotrophoblast,but not present in the syncytiotrophoblast throughout pregnancy,and decreased along with reducing of cytotrophoblast in pregnancy process.2) In first trimester extravillous tissue and decidua,14-3-3 tau was stained in the cytoplasm of extravillous cytotrophoblast cells(EVT) of anchoring villi,and stained in the membrane of EVT cells that had invaded the deciduas.3 ) In preeclampsia placenta, significant morphological differences,including cytotrophoblast proliferation increase, associated with hypoxia were detected.14-3-3 tau-positive cells were widespread in the preeclamptic placentas,but were not prominent in the control placentas。4) 14-3-3 tau mRNA and protein were suppressed in Forskolin treated BeWo cells compared to vehicle control in a time dependent manner(P＜0.05).Syncytializated BeWo cells were negative stained with 14-3-3 tau antibody.
Conclusion:These results demonstrate that different localization of 14-3-3 tau in placenta may associate with trophoblast function,including proliferation, syncytialization and invasion,and may involve in preeclampsia hypoxia.
Section 1.2 Effects of regulated 14-3-3 tau expression on trophoblast proliferation and differentiation
Objectives:Cytotrophoblast proliferate and differentiate according to one of two distinct pathways.We have found that 14-3-3 tau differently expressed in distinct phenotypes of trophoblast.We aimed to investigate the potential role of 14-3-3 tau in trophoblast on proliferation and differentiation.
Methods:Small interference RNA(siRNA) targeting 14-3-3 tau and HA tagged 14-3-3 tau plasmid were transfected into human trophoblast cell line(BeWo), respectively.Invasion of BeWo cells was examined by matrigel invasion assay,DNA synthesization was detected by BrdU assay,cell cycle distribution was detected by Flow Cytometry.β-hCG secretion was detected by ELISA.The expression of proliferating cell nuclear antigen(PCNA),syncytin,E-cadherin and snail were estimated by RT-PCR or Western Blot.The phosphorylation level of extracellular-signal related kinase(ERK) 1/2 in BeWo cells was evaluated by Western Blot.
Results:1) After transfeeting 14-3-3 tau siRNA 72h,14-3-3 tau was significantly down-regulated in BeWo cells.After transfecting HA tagged 14-3-3 tau plasmid for 24h,BeWo cells expressed a HA tagged 14-3-3 tau protein.2) Reduced 14-3-3 tau apparently inhibited PCNA expression(P＜0.05),but increased 14-3-3 tau stimulated PCNA expression(P＜0.05).3) Reduced 14-3-3 tau apparently stimulated BeWo cells to absorb BrdU,but increased 14-3-3 tau inhibited BeWo cells absorb BrdU(P＜0.05). 4) Reduced 14-3-3 tau significantly increased the percentages of cells in G_0/G_1 phases while decreased that in S phase(P＜0.05).5) Reduced 14-3-3 tau stimulated syncytin transcription andβ-hCG secretion(P＜0.05),but increased 14-3-3 tau inhibited syncytin transcription andβ-hCG secretion(P＜0.05).6) Reduced 14-3-3 tau inhibited E-cadherin expression(P＜0.05),stimulated snail expression(P＜0.05) and increased the invasive cell-numbers of BeWo.7) Reduced 14-3-3 tau activated ERK1/2,but a U0126,a MAPK/ERK inhibitor,inhibited up-regulated 14-3-3 tau of BeWo cells.8) U0126 inhibited the enhanced invasiveness andβ-hCG secretion in these cells induced by 14-3-3 tau down-regulation.
Conclusion:14-3-3 tau may stimulate trophoblast proliferation while inhibited trophoblast differentiated into syncytiotrophoblast or into invasive phenotype,through affecting the cell cycle distribution,expression of differentiation associated molecules, and the ERK1/2 signaling pathway.These results suggested that 14-3-3 tau is a potential check point that control outcomes of trophoblast differentiation.
Section 2 Proteomic and Bioinformatics approaches to explore the role of 14-3-3 tau in trophoblast
Objective:The aim of this study was to screen key proteins or potential pathways in 14-3-3 tau down-regulated trophoblast using proteomic and bioinformatics techniques.
Methods:Small interference RNA(siRNA) targeting 14-3-3 tau was transfected into human trophoblast cell line(BeWo).A proteomic analysis was performed in the RNA interfered BeWo cells using the methods of isobaric tag for relative and absolute quantitation(iTRAQ) and LC/MS.IPI identifiers were converted to gene symbols and a total of 234 genes were submitted to DAVID for Gene ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways analysis.
Results:We identified 271 proteins from BeWo cells.Twenty-six proteins were differentially abundant in 14-3-3 tau down-regulated BeWo cells compared with negative control BeWo cells,including 13 proteins increased and 13 proteins decreased in 14-3-3 tau down-regulated BeWo cells.Involved in cell proliferation, proliferation-associated protein 2G4、proliferating cell nuclear antigen and plastin 3 were decreased;The expression of cellular metabolism associated proteins Alpha-2-HS-glycoprotein was increased;Stress-induced-phosphoprotein 1 responded to stress was decreased;we also found a decreased expression of sorting nexin-2;In addition,Transferrin receptor protein 1 was up-regulated.These proteins have been implicated in regulating cellular oxidative state,cell cycle,signal transduction,protein folding and degradation,cell mobility and cytoskeleton structure formation. Biological pathway analysis revealed that 14-3-3 tau affected several pathway associated with Glutathione metabolism,Motioning metabolism,PPAR signaling pathway,Cell cycle,Glutamate metabolism,iron transport.
Conclusion:These data provide an in depth analysis of the potential molecular mechanism of 14-3-3 tau in mediating trophoblast regulation.
Section 3 Expression of 14-3-3 tau outside trophoblast
Section 3.1 Effects of 14-3-3 tau on endometrial stromal cells receptivity
Objectives:This study aimed to investigate the effects of 14-3-3 tau on integrinα_vβ_3 protein expression in endometrial stromal cells.
Methods:Small interference RNA(siRNA) targeting 14-3-3 tau mRNA was transfected into human trophoblast cell lines(BeWo cells).The expression of 14-3-3 tau protein in the BeWo cells or its culture-medium was detected by Western Blot respectively.Then the BeWo cells transfected with 14-3-3 tau siRNA were co-cultured with human endometrial stromal cells(ESC).The expression of 14-3-3 tau protein in the co-culture-medium or integrinα_vβ_3 protein in ESC was detected by Western Blot respectively.
Results:Compared with negative control of siRNA,the expression of 14-3-3 tau protein in both BeWo cells and culture-medium were significantly inhibited by 14-3-3 tau siRNA(P＜0.05).In co-culture system,BeWo cells down-regulated by 14-3-3 tau siRNA resulted in an increased expression of integrinα_vβ_3 protein in ESC.And adding human recombinant protein of 14-3-3 tau in co-culture medium significantly inhibited the expression of integrinα_vβ_3 protein in ESC(P＜0.05).
Conclusions:14-3-3 tau protein may regulate the receptivity of human endometrial stromal cells through regulating trophoblast differentiation as well as a direct effect on endometrial stromal cells.
Section 3.2 Expression of 14-3-3 tau in maternal serum and amniotic fluid
Objectives:The aim of this study was to investigate the presence of 14-3-3 tau in maternal serum or amniotic fluid.
Methods:Venous serum samples were taken from none pregnancy women,women throughout pregnancy and pregnancy women with preeclampsia;umbilical cord blood serum were taken from fetus born by women with or without preeclampsia.Amniotic fluid samples were taken from second and third trimester women and pregnancy women with preeclampis.14-3-3 tau was detected by Western Blot.
Results:14-3-3 tau was not detected in all the venous serum and umbilical cord blood serum samples.However,in amniotic fluid samples,14-3-3 tau was easily detected and had an increasing trend throughout pregnancy,but increased more significantly in preeclampsia amniotic fluid samples than that in normal pregnancy women.
Conclusion:The concentration of 14-3-3 tau in blood serum was below the detection level in this study.In amniotic fluid,14-3-3 tau was involved in pathological process of preeclampsia and reflected the exchange of mother with her fetus,but the roles of 14-3-3 tau played in amniotic fluid was worthy of further exploration.
第一部分14-3-3 tau与滋养细胞的增殖和分化
第一节14-3-3 tau在胎盘滋养细胞中的定位
目的通过分析14-3-3 tau在胎盘组织中的定位和滋养细胞体外分化模型中的表达变化情况,了解滋养细胞中14-3-3 tau可能的作用。
方法用免疫组化方法检测人早孕期绒毛,中孕期胎盘、足月妊娠胎盘及子痫前期胎盘中14-3-3 tau的定位。腺苷酸环化酶激活剂Forskolin诱导BeWo细胞合体化,免疫细胞化学检测BeWo细胞中14-3-3 tau的定位变化,RT-PCR和Western Blot法检测14-3-3 tau转录水平和蛋白水平的变化。
结果1)在各孕期的胎盘绒毛中,14-3-3 tau主要在绒毛内细胞滋养细胞表达,而合体滋养细胞未见其表达,并且随着孕周增加绒毛内细胞滋养数目减少,14-3-3 tau的表达也逐渐减少。2)在早孕期侵袭绒毛和蜕膜组织中,14-3-3 tau在固定绒毛的绒毛外滋养细胞(EVT)胞浆表达,而在侵入蜕膜的EVT胞膜表达。3)病理胎盘中,子痫前期胎盘与正常足月胎盘相比呈明显的缺氧应激改变,14-3-3 tau表达也增加。4)与对照组相比,14-3-3 tau mRNA和蛋白表达在Forskolin处理组BeWo细胞中显著下降(P＜0.05),免疫细胞化学也显示BeWo细胞合体化后14-3-3 tau呈阴性。
讨论14-3-3 tau在不同类型胎盘滋养细胞中的定位提示其与滋养细胞增殖、合体化和侵袭功能相关,并可能参与子痫前期病理过程中胎盘滋养细胞对缺氧的应激调节。
第二节调节14-3-3 tau的表达对BeWo细胞增殖和分化的影响
目的妊娠过程中,细胞滋养细胞不断增殖,并通过两种途径分化,分别形成合体滋养细胞或侵袭型滋养细胞,前期研究显示14-3-3 tau在这三类细胞的表达水平不同,但其发挥的作用尚不清楚,故本研究意在探讨14-3-3 tau是否参与滋养细胞增殖、合体化和侵袭能力的调控。
方法RNA干扰方法下调人绒癌滋养细胞株BeWo细胞内14-3-3 tau的表达,构建14-3-3 tau表达质粒上调BeWo细胞内14-3-3 tau的表达。RT-PCR和Western Blot检测增殖相关分子核增殖抗原(PCNA),合体化相关分子syncytin,侵袭相关分子E钙粘素及其拮抗蛋白Snail的表达变化。BrdU法检测BeWo细胞DNA合成能力,流式细胞仪检测BeWo细胞周期变化。ELISA方法检测滋养细胞β-hCG分泌。Transwell小室实验检测BeWo细胞侵袭能力。用U0126特异性阻断ERK信号通路观察BeWo细胞侵袭和合体化能力的变化。
结果1) RNA干扰72h,BeWo细胞14-3-3 tau表达被显著被下调;转染14-3-3 tau表达质粒24h,BeWo细胞显著表达14-3-3 tau融合蛋白,提示14-3-3tau表达被上调。2)下调14-3-3 tau后BeWo细胞PCNA的转录和蛋白表达减少,而上调14-3-3 tau后PCNA的转录和蛋白表达增加,提示14-3-3 tau促进滋养细胞PCNA的表达。3)下调14-3-3tau后BeWo细胞对BrdU摄取显著减少(P＜0.05),而上调14-3-3 tau后BeWo细胞BrdU摄取呈增加趋势,提示14-3-3tau促进滋养细胞DNA合成。4)下调14-3-3 tau后BeWo细胞G_0/G_1期比例显著增加而S期比例显著减少(P＜0.05),提示14-3-3 tau促进滋养细胞G_1期向S期进级。5)下调14-3-3 tau表达后,合体化相关分子syncytin转录显著增高(P＜0.05),β-hCG分泌显著增加(P＜0.05);上调BeWo细胞14-3-3 tau表达,syncytin转录显著降低(P＜0.05),β-hCG分泌显著减少(P＜0.05)。提示14-3-3tau抑制滋养细胞合体化。6)下调14-3-3 tau表达后,E钙粘素表达减少而Snail表达增加,侵入Transwell小室下层的细胞数目增多,提示14-3-3 tau抑制滋养细胞侵袭。7)下调14-3-3 tau的表达,ERK1/2磷酸化水平增加,而U0126特异性抑制ERK1/2磷酸化上调14-3-3 tau的表达,提示14-3-3 tau负调节ERK1/2的活化。8) U0126阻断ERK1/2后,下调14-3-3 tau不能继续促进BeWo细胞β-hCG的分泌和侵袭,提示14-3-3 tau通过ERK1/2信号通路发挥作用。
结论14-3-3 tau促进滋养细胞增殖,同时抑制滋养细胞向分泌型合体滋养细胞和侵袭型滋养细胞的分化,引起细胞周期分布、分化相关分子的表达和MAPK/ERK1/2信号通路的改变,它可能是滋养细胞分化调控的重要节点分子。
第二部分蛋白质组学和生物信息学方法探讨滋养细胞内14-3-3 tau的作用
目的用比较蛋白质组学的方法和生物信息学方法,研究下调14-3-3 tau的BeWo细胞蛋白质组表达的变化,发现14-3-3 tau在滋养细胞生物功能中涉及的蛋白并探讨其可能的调节机制。
方法RNA干扰技术下调BeWo细胞内14-3-3 tau的表达。同位素标记相对和绝对定量(iTRAQ)技术结合质谱技术鉴定滋养细胞蛋白质组的变化,并进行生物信息学分析。
结果通过iTRAQ-MS鉴定出BeWo细胞中271种蛋白的相对定量信息,与阴性对照组相比,14-3-3 tau siRNA组中有26种蛋白表达发生变化,其中13种蛋白上调,13种蛋白下调,包括增殖相关蛋白(Proliferation-associated protein2G4、Proliferating cell nuclear antigen、plastin 3)表达降低,细胞代谢相关Alpha-2-HS-glycoprotein表达增加,缺氧调控相关蛋白Hypoxia up-regulatedprotein 1和应激蛋白Stress-induced-phosphoprotein 1的表达减少;蛋白转运相关的Sorting nexin-2表达增加,蛋白3级结构形成相关的T-complex protein 1 subunitbeta上调,铁离子转运相关蛋白Transferrin receptor protein 1上调等。这些细胞周期、谷胱甘肽代谢、PPAR信号通路、铁转运等多种机体通路,其中几种蛋白是首次在滋养细胞中被发现。
结论运用蛋白质组学技术和生物信息学方法分析14-3-3 tau对滋养细胞蛋白组影响,为14-3-3 tau在滋养细胞内的功能研究做出了较全面的解释,14-3-3 tau不仅调节滋养细胞的增殖和分化,还可能参与细胞代谢、缺氧损伤、钙化等,研究14-3-3 tau有助于了解滋养细胞功能异常导致的妊娠期疾病可能的分子机制。
第三部分14-3-3 tau在滋养细胞外的表达
第一节14-3-3 tau对子宫内膜基质细胞容受性的影响
目的探讨14-3-3 tau是否影响滋养细胞对周围细胞发挥作用及其可能的机制。
方法RNA干扰技术下调绒癌滋养细胞株BeWo的14-3-3 tau蛋白表达。建立下调14-3-3 tau的BeWo细胞和人子宫内膜基质细胞ESC的共培养体系,Western Blot方法检测共培养体系中培养液14-3-3 tau的表达以及ESC细胞中容受性分子整合素α_vβ_3的表达变化。
结果与阴性对照siRNA组相比,特异性的siRNA可显著下调BeWo细胞内和胞外培养液中14-3-3 tau蛋白的表达(P＜0.05)。共培养体系中,与阴性对照相比,下调14-3-3 tau的BeWo细胞共培养的ESC细胞内整合素α_vβ_3的表达显著增高,提示ESC细胞容受性增加。与单纯培养的ESC相比,培养液中加入14-3-3 tau纯化蛋白培养24h的ESC表达的整合素α_vβ_3显著下调(P＜0.05),提示ESC细胞容受性下降。
结论14-3-3通过调节滋养细胞分化促进合体细胞激素、细胞因子等的释放增加,以及直接作用子宫内膜基质细胞两种途径引起子宫内膜的容受性增加。
第二节14-3-3 tau在孕妇血清和羊水中的表达
目的检测孕妇血清中14-3-3 tau的表达。
方法Western Blot方法检测正常非孕妇女、早孕期妇女、中期妊娠孕妇、晚期妊娠孕妇以及子痫前期孕妇静脉血及胎儿脐带血血清中14-3-3 tau的表达。Western Blot方法检测正常中期妊娠孕妇、足月分娩孕妇、子痫孕妇羊水中14-3-3tau的表达。
结果在正常非孕妇女、早孕期妇女、中期妊娠孕妇、晚期妊娠孕妇以及子痫前期孕妇静脉血及胎儿脐带血血清均未检测到14-3-3 tau的表达。正常中期妊娠孕妇、足月分娩孕妇、子痫孕妇羊水中均检测到14-3-3 tau的表达,并随孕周增加表达增加,子痫前期羊水中14-3-3 tau表达高于足月正常妊娠羊水。
结论血清中14-3-3 tau表达低于本研究的检测水平。14-3-3 tau在羊水中的表达反映了母体、胎儿、羊水之间交换功能的改变,并可能有子痫前期的病理过程有关,但其在正常妊娠以及病理妊娠中发挥怎样的作用仍需要近一步研究。
Disturbed function of trophoblast may lead to a number of pregnancy-associated pathologies,but regulation of trophoblast differentiation and proliferation is partly understood.Recently,we found an interesting protein which named 14-3-3 tau in both hypoxia-treated human trophoblast cell line BeWo and preeclampsia placenta by proteomic analysis,it is suggested that 14-3-3 tau may play important roles in trophoblast biological process.In this research,we firstly detected the location of 14-3-3 tau in human placenta.Secondly,we applied RNA interference and Plasmid transfection techniques to explore the effects of differently expressed 14-3-3 tau on trophoblast proliferation and differentiation.Thirdly,we used proteomic and bioinformatics approaches to analysis the differential expression of proteins and the potential pathways involved in the 14-3-3 tau down-regulated trophoblast.Finally,we discussed the extracellular functions of 14-3-3 tau.We seek a better understanding of how 14-3-3 tau regulates trophoblast.
Section 1 Effects of 14-3-3 tau in trophoblast proliferation and differentiation
Section 1.1 Location of 14-3-3 tau in human placental trophoblast
Objectives:We aimed to evaluate the expression,distribution,and localization of 14-3-3 tau in human placenta tissues and trophoblast differentiation model in order to help to construct a hypothesis about its function in the placenta.
Methods:14-3-3 tau was detected in placenta tissue of three trimester pregnancies by immunohistochemistry.RT-PCR,Western Blot and immunocytochemistry analysis were employed to evaluate expression changes during Forskolin induced in vitro differentiation of human trophoblast cell line(BeWo).
Results:1) In villous tissue,14-3-3 tau was immunolocalized to villous cytotrophoblast,but not present in the syncytiotrophoblast throughout pregnancy,and decreased along with reducing of cytotrophoblast in pregnancy process.2) In first trimester extravillous tissue and decidua,14-3-3 tau was stained in the cytoplasm of extravillous cytotrophoblast cells(EVT) of anchoring villi,and stained in the membrane of EVT cells that had invaded the deciduas.3 ) In preeclampsia placenta, significant morphological differences,including cytotrophoblast proliferation increase, associated with hypoxia were detected.14-3-3 tau-positive cells were widespread in the preeclamptic placentas,but were not prominent in the control placentas。4) 14-3-3 tau mRNA and protein were suppressed in Forskolin treated BeWo cells compared to vehicle control in a time dependent manner(P＜0.05).Syncytializated BeWo cells were negative stained with 14-3-3 tau antibody.
Conclusion:These results demonstrate that different localization of 14-3-3 tau in placenta may associate with trophoblast function,including proliferation, syncytialization and invasion,and may involve in preeclampsia hypoxia.
Section 1.2 Effects of regulated 14-3-3 tau expression on trophoblast proliferation and differentiation
Objectives:Cytotrophoblast proliferate and differentiate according to one of two distinct pathways.We have found that 14-3-3 tau differently expressed in distinct phenotypes of trophoblast.We aimed to investigate the potential role of 14-3-3 tau in trophoblast on proliferation and differentiation.
Methods:Small interference RNA(siRNA) targeting 14-3-3 tau and HA tagged 14-3-3 tau plasmid were transfected into human trophoblast cell line(BeWo), respectively.Invasion of BeWo cells was examined by matrigel invasion assay,DNA synthesization was detected by BrdU assay,cell cycle distribution was detected by Flow Cytometry.β-hCG secretion was detected by ELISA.The expression of proliferating cell nuclear antigen(PCNA),syncytin,E-cadherin and snail were estimated by RT-PCR or Western Blot.The phosphorylation level of extracellular-signal related kinase(ERK) 1/2 in BeWo cells was evaluated by Western Blot.
Results:1) After transfeeting 14-3-3 tau siRNA 72h,14-3-3 tau was significantly down-regulated in BeWo cells.After transfecting HA tagged 14-3-3 tau plasmid for 24h,BeWo cells expressed a HA tagged 14-3-3 tau protein.2) Reduced 14-3-3 tau apparently inhibited PCNA expression(P＜0.05),but increased 14-3-3 tau stimulated PCNA expression(P＜0.05).3) Reduced 14-3-3 tau apparently stimulated BeWo cells to absorb BrdU,but increased 14-3-3 tau inhibited BeWo cells absorb BrdU(P＜0.05). 4) Reduced 14-3-3 tau significantly increased the percentages of cells in G_0/G_1 phases while decreased that in S phase(P＜0.05).5) Reduced 14-3-3 tau stimulated syncytin transcription andβ-hCG secretion(P＜0.05),but increased 14-3-3 tau inhibited syncytin transcription andβ-hCG secretion(P＜0.05).6) Reduced 14-3-3 tau inhibited E-cadherin expression(P＜0.05),stimulated snail expression(P＜0.05) and increased the invasive cell-numbers of BeWo.7) Reduced 14-3-3 tau activated ERK1/2,but a U0126,a MAPK/ERK inhibitor,inhibited up-regulated 14-3-3 tau of BeWo cells.8) U0126 inhibited the enhanced invasiveness andβ-hCG secretion in these cells induced by 14-3-3 tau down-regulation.
Conclusion:14-3-3 tau may stimulate trophoblast proliferation while inhibited trophoblast differentiated into syncytiotrophoblast or into invasive phenotype,through affecting the cell cycle distribution,expression of differentiation associated molecules, and the ERK1/2 signaling pathway.These results suggested that 14-3-3 tau is a potential check point that control outcomes of trophoblast differentiation.
Section 2 Proteomic and Bioinformatics approaches to explore the role of 14-3-3 tau in trophoblast
Objective:The aim of this study was to screen key proteins or potential pathways in 14-3-3 tau down-regulated trophoblast using proteomic and bioinformatics techniques.
Methods:Small interference RNA(siRNA) targeting 14-3-3 tau was transfected into human trophoblast cell line(BeWo).A proteomic analysis was performed in the RNA interfered BeWo cells using the methods of isobaric tag for relative and absolute quantitation(iTRAQ) and LC/MS.IPI identifiers were converted to gene symbols and a total of 234 genes were submitted to DAVID for Gene ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathways analysis.
Results:We identified 271 proteins from BeWo cells.Twenty-six proteins were differentially abundant in 14-3-3 tau down-regulated BeWo cells compared with negative control BeWo cells,including 13 proteins increased and 13 proteins decreased in 14-3-3 tau down-regulated BeWo cells.Involved in cell proliferation, proliferation-associated protein 2G4、proliferating cell nuclear antigen and plastin 3 were decreased;The expression of cellular metabolism associated proteins Alpha-2-HS-glycoprotein was increased;Stress-induced-phosphoprotein 1 responded to stress was decreased;we also found a decreased expression of sorting nexin-2;In addition,Transferrin receptor protein 1 was up-regulated.These proteins have been implicated in regulating cellular oxidative state,cell cycle,signal transduction,protein folding and degradation,cell mobility and cytoskeleton structure formation. Biological pathway analysis revealed that 14-3-3 tau affected several pathway associated with Glutathione metabolism,Motioning metabolism,PPAR signaling pathway,Cell cycle,Glutamate metabolism,iron transport.
Conclusion:These data provide an in depth analysis of the potential molecular mechanism of 14-3-3 tau in mediating trophoblast regulation.
Section 3 Expression of 14-3-3 tau outside trophoblast
Section 3.1 Effects of 14-3-3 tau on endometrial stromal cells receptivity
Objectives:This study aimed to investigate the effects of 14-3-3 tau on integrinα_vβ_3 protein expression in endometrial stromal cells.
Methods:Small interference RNA(siRNA) targeting 14-3-3 tau mRNA was transfected into human trophoblast cell lines(BeWo cells).The expression of 14-3-3 tau protein in the BeWo cells or its culture-medium was detected by Western Blot respectively.Then the BeWo cells transfected with 14-3-3 tau siRNA were co-cultured with human endometrial stromal cells(ESC).The expression of 14-3-3 tau protein in the co-culture-medium or integrinα_vβ_3 protein in ESC was detected by Western Blot respectively.
Results:Compared with negative control of siRNA,the expression of 14-3-3 tau protein in both BeWo cells and culture-medium were significantly inhibited by 14-3-3 tau siRNA(P＜0.05).In co-culture system,BeWo cells down-regulated by 14-3-3 tau siRNA resulted in an increased expression of integrinα_vβ_3 protein in ESC.And adding human recombinant protein of 14-3-3 tau in co-culture medium significantly inhibited the expression of integrinα_vβ_3 protein in ESC(P＜0.05).
Conclusions:14-3-3 tau protein may regulate the receptivity of human endometrial stromal cells through regulating trophoblast differentiation as well as a direct effect on endometrial stromal cells.
Section 3.2 Expression of 14-3-3 tau in maternal serum and amniotic fluid
Objectives:The aim of this study was to investigate the presence of 14-3-3 tau in maternal serum or amniotic fluid.
Methods:Venous serum samples were taken from none pregnancy women,women throughout pregnancy and pregnancy women with preeclampsia;umbilical cord blood serum were taken from fetus born by women with or without preeclampsia.Amniotic fluid samples were taken from second and third trimester women and pregnancy women with preeclampis.14-3-3 tau was detected by Western Blot.
Results:14-3-3 tau was not detected in all the venous serum and umbilical cord blood serum samples.However,in amniotic fluid samples,14-3-3 tau was easily detected and had an increasing trend throughout pregnancy,but increased more significantly in preeclampsia amniotic fluid samples than that in normal pregnancy women.
Conclusion:The concentration of 14-3-3 tau in blood serum was below the detection level in this study.In amniotic fluid,14-3-3 tau was involved in pathological process of preeclampsia and reflected the exchange of mother with her fetus,but the roles of 14-3-3 tau played in amniotic fluid was worthy of further exploration.
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