Effects Of 14-3-3 Tau Protein On The Biological Behaviors Of Human Trophoblast

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 differentiationSection 1.1 Location of 14-3-3 tau in human placental trophoblastObjectives: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 differentiationObjectives: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 G₀/G₁ 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 trophoblastObjective: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 trophoblastSection 3.1 Effects of 14-3-3 tau on endometrial stromal cells receptivityObjectives:This study aimed to investigate the effects of 14-3-3 tau on integrinα_vβ₃ 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_vβ₃ 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_vβ₃ protein in ESC.And adding human recombinant protein of 14-3-3 tau in co-culture medium significantly inhibited the expression of integrinα_vβ₃ 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 fluidObjectives: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.