Proteomic And RNAi Approaches To Explore The Relationship Between Hypoxic Trophoblast And Preeclampsia

Preeclampsia is common and results in much of the maternal morbidity and mortality related to pregnancy. Despite decades of intensive research, the pathogenesis of preeclampsia is still unsolved. Lately, the relationship between hypoxic trophoblasts and preeclampsia has been noticed. Little was known about the underlying mechanism of hypoxia injury to trophoblasts. In this research, we first investigated the effect of hypoxia on the apoptosis of cultured first trimester human cytotrophoblasts. Secondly, we used proteomic approaches to analysis hypoxia-induced responses in the syncytialization of human cytotrophoblasts, and differential expression of proteins in the placenta of preeclampsia patients, and then search the similarity and difference between two protein profiles. Finally, we applied RNA interference technique to explore the function of differently expressed proteins. We proposed to study the effect of hypoxia on cellular behaviors and proteomic profiles, to investigate the function of differently expressed proteins in trophoblasts, further we will find the relationship between hypoxic trophoblasts and preeclampsia.Section 1 Altered Bcl-2 and Bax expression is associated with cultured first trimester human cytotrophoblasts apoptosis induced by hypoxiaObjective: Preeclampsia is associated with placental hypoxia at early gestation. We thereforeinvestigated the effect of hypoxia on the apoptosis of cultured first trimester humancytotrophoblasts and the expression of apoptosis relevant proteins, Bcl-2 and Bax.Study design: First trimester human cytotrophoblasts were isolated and cultured under eithernormoxic or hypoxic conditions. Cellular apoptosis was monitored by genome DNA ladder,TUNEL and Annexin V binding, and the expression of Bcl-2 and Bax was determined byWestern blot analysis.Results: Apoptosis increased significantly in cytotrophoblasts cultured under hypoxicconditions in contrast with those cultured under normoxic conditions, meanwhile expressionof Bcl-2 reduced, and that of Bax increased.Conclusion: Hypoxia induced apoptosis in cultured first trimester cytotrophoblasts withaltered Bcl-2 and Bax expression. Further study is needed to explore the role of cytotrophoblasts apoptosis in hypoxia-induced maternal and fetal diseases.Section 2 Proteomic approaches to explore the relationship between hypoxic trophoblasts and preeclampsiaSection 2.1 Proteomic analysis of hypoxia-induced responses in the syncytialization of human placental cell Line BeWoObjective: Syncytiotrophoblast formation is affected by a number of pathological conditions, and suppressed syncytiotrophoblast formation due to hypoxia may play a role in the pathogenesis of preeclampsia. Therefore we investigated the effect of hypoxia on BeWo cells syncytialization to identify important proteins that may be involved in the hypoxic response.Study design: A proteomic analysis was performed in the syncytialization of BeWo cells under normaxia and hypoxia using two dimensional gel electrophoresis (2-DE) and MALDI-TOF-TOF-MS. Western blot was used to confirm the change of protein expression obtained in 2-DE gels.Results: Hypoxia suppressed forskolin-induced BeWo cells syncytialization. Twenty proteins were significantly up- or down- regulated under hypoxia, compared with cells under normal condition. In response to hypoxia, three antioxidants, peroxiredoxin 1, peroxiredoxin 2 and 1-Cys peroxiredoxin, were down-regulated; two proteins involved in glycolysis pathway (malate dehydrogenase and enolase) were up-regulated; the expression of two members of the annexin family (annexinA2 and annexin A5) increased; we also found a decreased expression of 14-3-3τ protein in hypoxia-treated cells; proteins implied in protein degradation and folding were also identified. The expression of two cytoskeleton components (keratin 1 and β-actin) was found to be down-regulated. In addition, galectin-3 was up-regulated. These proteins have been implicated in regulating cellular oxidative state, glycolysis, signal transduction, protein folding and degradation, cell mobility and cytoskeletal structure formation. Western blot analysis revealed that the levels of peroxiredoxin 1 and 14-3-3τ decreased, whereas the levels of annexin A5 and annexin A2 increased in BeWo cellssyncytialization under hypoxic conditions.Conclusion: These findings provided new insights into the molecular mechanism in mediating cellular responses to hypoxia in trophoblast syncytialization. Further studies are needed to explore the role of the identified up- or down- regulated proteins in response to hypoxia in the pathogenesis and therapeutic implication of preeclampsia.Section 2.2 Comparative proteomic analysis of the placental protein expression in preeclampsia patientsObjective: Altered expression of placental proteins is associated with various pathological pregnancies. Especially, preeclampsia is one of the most common conditions. However the pathogenesis of preeclampsia is still unclear. Therefore, the aim of this study was to screen key proteins involved in preeclampsia and investigate pathogenesis of this disease using proteomic techniques.Study design: The placental proteins expressed in patients suffering from preeclampsia (patient) and healthy volunteers (control) were analyzed using the two dimensional gel electrophoresis (2-DE) technique. The differentially expressed proteins in patients were identified with MALDI-TOF-TOF-MS and confirmed with Western blot.Results: Three proteins in patient were down-regulated compared with those of the control: Titin, calnexin and one unidentified protein. Ten proteins in patient were up-regulated compared with those of the control: heat shock 27kDa protein, 78 kDa glucose-regulated protein, prohibitin, annexin A1, NADH-ubiquinone oxidoreductase 24 kDa, chloride intracellular channel protein 3, smooth muscle and non-muscle myosin alkali light chain isoform 1, actin, keratin 10 and centrosome. Western blot analysis revealed that the level of heat shock 27kDa protein increased in patient placenta.Conclusion: Our study reveals that the expression of placental proteins in preeclampsic patients is different from that of healthy subjects and may provide further insights into the pathogenesis of preeclampsia.Section 3 RNA interference approaches to investigate the function of proteins related to hypoxic responses in trophoblastsSection 3.1 Small interfering RNA targeting 14-3-3τ inhibits forskolin induced BeWo cells syncytialization under normoxia and hypoxiaObjective: We previously searched by a proteomic screen for differentially expressed proteins in BeWo cells syncytialization under hypoxia versus normoxia, and found decreased level of 14-3-3τ protein in cells under hypoxia. The purpose of this study was to test whether reduced level of 14-3-3τ protein played a role in forskolin induced BeWo cells syncytialization under normoxia and hypoxia.Study design: BeWo cells were transfected with specific small interfering RNA (siRNA) targeting 14-3-3τ mRNA or control siRNA and subjected either to normoxia or to hypoxia. Control samples were not transfected. The efficacy of RNAi was assessed by knock down of 14-3-3τ mRNA and protein expression via real time PCR and Western blot. BeWo cells fusion was quantified by counting nuclei in syncytial cells and detecting hCG secretion in conditioned media.Results: When compared with normoxic cultures, hypoxia significantly down-regulated 14-3-3τ mRNA and protein expression and supressed the fusion of cells. Transfection with specific siRNA knocked down 14-3-3τ mRNA and protein expression by approximately 50%, whereas transfection with control siRNA had no noticeable effect on 14-3-3τ expression. A significantly lower proportion of BeWo cells transfected with specific siRNA demonstrated syncytiotrophoblast when compared with nontransfected cells. In contrast, transfection with control siRNA had no significent effect on BeWo cells fusion.Conclusion: 14-3-3τ protein plays a role in cell fusion processes and is involved in modulating hypoxia-suppressed syncytialization. Further study is needed to investigate its action.Section 3.2 Antiapoptotic action of hypoxia-inducible factor-la on BeWo cellsObjective: Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor involved inthe adaptive response to hypoxia and consists of HIF-1α and HIF-1β subunits. We evaluated the effect of RNA interference (RNAi) targeting HIF-1α messenger RNA (mRNA) on apoptosis of BeWo cells cultured under either normoxic or hypoxic conditions.Study design: BeWo cells were transfected with small interfering RNA (siRNA) targeting HIF-1α mRNA or control siRNA and subjected either to normoxia or to hypoxia for 24h. Control cells were not transfected. The efficacy of RNAi was assessed by knock down of HIF-1α mRNA and protein expression via real time PCR and Western blot. Cellular apoptosis was monitored by TUNEL, and the expression of Bcl-2 and Bax was determined by Western blot.Results: When compared with normoxic cultures, hypoxia significantly up-regulated HIF-1α mRNA and protein expression, and increased the percentage of apoptotic cells, meanwhile expression of Bcl-2 reduced, and that of Bax increased. Transfection with HIF-1α siRNA knocked down HIF-1α mRNA and protein expression in hypoxia-treated cells, whereas transfection with control siRNA had no noticeable effect on HIF-1α expression. A significantly greater proportion of BeWo cells transfected with HIF-1α siRNA and exposed to hypoxia demonstrated evidence of apoptosis when compared with nontransfected cells, accompanied with decreased expression of Bcl-2 protein and increased expression of Bax protein. In contrast, transfection with control siRNA had no significent effect on cellular apoptosis and expression of Bcl-2 proteins family.Conclusion: Our data indicate that HIF-1α exerts an antiapoptotic role in BeWo cells stressed by hypoxia via Bcl-2 proteins family pathway.