The Effect Of Dual Specificity Phosphatase5on The Proliferation And Apoptosis Of Trophoblast And Research Of Related Molecular Mechanisms

Background and ObjectiveTrophoblast cells, which stem from the endoderm, can be divided into three groupsaccording to morphosis and structure: the cytotrophoblast, the syncytiotrophoblast and theintermediate trophoblast. The proliferation, differentiation, apoptosis, and procedural invasionof trophoblast cells play an important role on the embryo implantation, placenta formation,fetal growth and pregnancy process. Studies have shown that, the occurrence of pre-eclampsia is closely related to cell apoptosis.There is excessive apoptosis of trophoblast cellsin placenta of preeclampsia, and the degree of apoptosis of placental trophoblastic cells ispositively correlated with the severity of preeclampsia.Pre-eclampsia (pre-eclampsia, PE) is an acute and critical ill complication in pregnantwomen, also known as placenta-dependence disease,which is one of the main causes ofmaternal and fetal death.PE appears after20weeks gestation, mainly manifested as suddenhigh blood pressure and proteinuria,along with organ damage; once the gestationterminated,all of the clinical symptoms will disappear.The study of the etiology andpathogenesis of preeclampsia has been a hot topic in the field of obstetrics and gynecologyowing to its rapid, complex and lethal properties. Over the past century, although thepathogenesis had not been understood clearly, several theories had been formed: the immunetheory, cell ischemia theory, genetic theory, vaso-active substance theory, the disorder ofcalcium balance theory and vascular endothelial damage theory, of which cell ischemia theoryis more accepted at present. That is, in the early pregnancy, the trophoblast cells are short ofthe invasion ability, only reach to the decidual layer, making the uterine spiral artery"physiological recasting" difficult, lead to the placental pathological physiology changes,eventually lead to the occurrence of preeclampsia.Dual specificity phosphatase family, a subfamily of protein tyrosine phosphatase superfamily, is not has not been discovered until recent years. It can not only dephosphorylatethe phosphorylation of tyrosine, but also dephosphorylate the phosphorylation ofsilk/threonine. There are two types of DUSP family: typical DUSP and atypical DUSP. Manystudies have shown that most members of the DUSP family as negative regulator ofmitogen-activated protein kinase phosphatases, involved in cell proliferation, differentiation,metabolism, gene transcription, ion channels, cell-cell communication, immune response andthe tumorigenesis.Although DUSPs has such a wide range of functions, their functions in trophoblastwere poorly understood. There has been only one research claim reported that the expressionof DUSP9has is closely related to the development of placenta and the occurrenceof preeclampsia. So, we simulated dynamic mapping of DUSPs in normal pregnancy villitissue, normal and preeclampsia pregnant placenta tissues by PCR technique.Combined withthe previous research and literature at home and abroad, and screen out the mostsignificant differential expression gene----DUSP5gene as aim gene in this research.Then proposed the establishment of DUSP5overexpression and gene silencing stabletransfected cell line, in-depth study of effect of DUSP5on proliferation and apoptosis oftrophoblast cells, and to detect what is the guide signal transduction pathways mediatedtrophoblast cell proliferation and apoptosis，in order to clarifying the pathogenesis ofpreeclampsia and laying the new molecular mechanism, and providing the strategies forclinical treatment.BeWo cell line, which comes from human chorionic carcinoma group, is more similarwith the original generation of villi nourish cells in the capacity of secrete human chorionicgonadotropin and fusion of trophoblast cells compared with other human chorionic carcinomasuch JARs and JEG-3. So, we chose BeWo cell line as the in vitro cell model in this study.Materials and methods:1. The different expression of DUSPs in normal pregnancy villi tissue, normal andpreeclampsia pregnant placenta tissues(1) Subjects and groups:20patients of preeclampia who were diagnosed and deliverd inour hospital (from May,2012to May,2013) as the preeclampia groups. At the same period,20cases of normal late pregnancy women as the control group, while20cases of earlypregnancy women as the early pregnancy group. (2) Methods：RT-PCR and qPCR technique were applied to detect the differentexpression of DUSPs in three groups.Western blot was used to verificated the different proteinexpression of DUSP5in three groups.2. The establishment of the stably transfected cell lines of DUSP5overexpression andgene silence(1) Materials: The normal BeWo cell lines, DUSP5cDNA expression lentiviralvector, DUSP5RNAi lentiviral vector and their corresponding empty viral vectors(2) Methods: Lentiviral vectors transfected the BeWo cell lines, than sorting the positivecells by flow cytometry. qPCR and Western blot technology were applided to identify thegene expression of DUSP5.3. The effect of dual specificity phosphatase5on the proliferation and apoptosis oftrophoblast and research of related molecular mechanisms.(1) Materials: The normal BeWo cell lines, the stably transfected cell lines of DUSP5overexpression/gene silence and the stably transfected cell lines of their correspondingempty viral vector.(2) Methods: CCK-8was applied to detect the proliferation of trophoblast, while flowcytometry was used to detect the apoptosis. Western blot was used to detect the proteinexpression of p-ERK1/2, total-ERK1/2, p-p38, total-p38, p-JNK and total-JNK.Results:1. The different expression of DUSPs in normal pregnancy villi tissue, normal andpreeclampsia pregnant placenta tissues(1) High expression of all DUSPs were observed in chorionic villi tissue compared withnormal late pregnancy placental tissues (P<0.001). Furthermore, DUSP1, DUSP2, DUSP3,DUSP4, DUSP5, DUSP6, DUSP9, DUSP10and DUSP23were dramatically decreased inpreeclampsia placental tissues (P<0.001) compared with normal pregnancy. OtherwiseDUSP8, DUSP14and DUSP22were markedly increased (P<0.001)and DUSP7and DUSP16had no significant difference.(2) The protein expression of DUSP5in the three groups was the same as the mRNAexpression.2. The establishment of the stably trabsfected cell lines of DUSP5overexpression andgene silence (1) After the flow cytometry sorting positive cells, the overexpression of DUSP5groupand the corresponding empty vector group with red fluorescence obviously, while the DUSP5interference group and the corresponding empty vector group with strong green fluorescenceunder the fluorescence microscopy. The blank control group showed no expression offluorescence.(2) In the experiment of DUSP5over-expression, the relative expression of DUSP5mRNA and protein in the DUSP5overexpression group were significantly higher than that inthe control group and empty vector group (P<0.05); in the experiment of DUSP5interference,relative expression of DUSP5mRNA and protein in DUSP5interference group wassignificantly lower than that in the other two groups(P<0.05).There had no changes betweenthe control group and the empty vector group.3. The effect of dual specificity phosphatase5on the proliferation and apoptosis oftrophoblast and research of related molecular mechanisms.(1) The results of CCK-8: compared to the empty vector group, the O.D level in DUSP5overexpression group was significantly increased (P<0.01), while the O.D level in DUSP5interference group was significantly reduced(P<0.01).(2) The results of flow cytometry: compared with the empty vector group,thepercentage of early apoptosis in DUSP5overexpression group+DDP was significantlyreduced (P<0.05),while the percentage of early apoptosis in DUSP5interference group wassignificantly increased (P<0.001).(3) The results of Western blot: the protein level of p-ERK1/2in DUSP5over-expressiongroup was significantly reduced (P<0.01), while that in DUSP5interference group wasincreased obviously (P<0.01). Whether DUSP5overexpression or gene silencing, theexpression of total-ERK1/2protein without obvious change. No significant change in proteinp-p38, total-p38, p-JNK and total-JNK (P>0.05).(4) The results of Western blot: after the DUSP5interference group, the empty vectorgroup and the control group co-cultured with U0126, the expression of p-ERK1/2wasinhibited, and if not co-cultured with U0126, the expression of p-ERK1/2protein wasexpressing as usual. The expression of total-ERK1/2protein showed no obvious change in allgroups.(5) The statistical results of flow cytometry: compared to the DUSP5interference group+DMSO，the percentage of early apoptosis in DUSP5interference group+U0126wassignificant reduced (P<0.001).There were not obvious change in the all control groups andempty vector groups.Conclusions:1. The successful rendering the dynamic pattens of the different expression of DUSPs innormal pregnancy villi tissue, normal and preeclampsia pregnant placenta tissues, suggestingthat DUSPs had closely related with the development of placent and the occurrence ofpre-eclamsia.2. The successful establishment of the stable transfected cell line of DUSP5over-expression and its gene silencing, provide a good platform for the further study of DUSP5function in trophoblast cells.3(1) Over-expression of DUSP5can promote the proliferation of trophoblast but inhibitthe apoptosis; while gene interference of DUSP5can inhibit proliferation of trophoblast butpromote apoptosis. This suggested that the DUSP5gene is closely related with theproliferation and apoptosis of trophoblast cells.(2) Over-expression of DUSP5was significantly down regulated the protein level ofp-ERK1/2, while that which were significantly increased after DUSP5gene silencing.Whether the over-expression or silence of DUSP5gene, the protein levels of p-p38,total-p38, p-JNK and total-JNK had no obvious changed. The results indicated that the effectof DUSP5on trophoblast cells may regulated by ERK1/2signal pathway.(3) After effectively inhibiting the ERK1/2signal transduction pathway, we found thatthe apoptosis of trophoblastic cells caused by DUSP5gene silencing was effectivelyreversed. Accordingly, we concluded that the regulatory effect of DUSP5on proliferation andapoptosis of trophoblast cells is through the ERK1/2signaling pathway.