| The normal growth and development of the placenta in early stage is a key factor affecting the female reproductive performance and the quality of kids in goat production.Trophoblast cell is one of the main components of placental tissue in early stage,and plays an important regulatory role in the process of embryo implantation and invasion of the uterus.TET1,one member of the dioxygenase that demethylates,plays a universal role in mammalian early placental development,embryo implantation,fetal growth,genetic imprinting,and regulation of related signaling pathways.At the same time,many studies confirms that the Wnt signaling pathway initiated by Wnt molecules is widely involved in cell signal determination,fetal development,placental formation and other life processes.Therefore,in the process of mammalian placenta development,both have certain similarities and synchronicity in physiological functions,regulatory positions and role phases.However,there are few reports about that TET1 could regulates the Wnt signaling pathway related molecules in the differentiation and development of goat placental trophoblast cells(GTCs).This study was undertaken to establish goat primary placental trophoblast cell in vitro culture system,and to research the regulation of TET1 gene on Wnt pathway molecules in GTCs.Goat placentas of 45-60 days were collected,and the primary GTCs were isolated from placental chorionic membrane,then cultured by tissue block and cell co-culture method.GTCs were treated with Dox induction and piLenti-shTET1-GFP plasmid transfection,and then RT-qPCR and immunocytochemistry were used to detect mRNA and protein expression level of TET1 gene,as well as the entirety methylation level and hydroxymethylation level of the cells;RT-qPCR compared the relative expression level of Wnt signaling pathway related genes;MTS and scratches detected GTCs activity and migration ability.The results were as followings:1.The primary GTCs with homogeneous morphology and high purity were obtained by isolation and culture of tissue block and co-culture method and purified by differential digestion.The primary GTCs were fusiform at the early stage and irregular polygons at the later stage.Specific cytokeratin 7 protein was detected on the GTCs.2.GTCs with Dox treatment could increase the expression of TET1 gene.Compared with the control group,the relative expression level of TET1 mRNA was increased from 2.07 to 4.64 for 20 ng/?L Dox treatment,the difference was extremely significant(P<0.01);and the expression level of TET1 protein was significantly increased.The MTS results showed that cell activity was decreased from 0.67 to 0.52,down 22.13%(P<0.05),which was detected in GTCs treated with Dox for 2 days.The scratch test showed that the average migration ability of GTCs at the optimum concentration(20 ng/?L)Dox treatment decreased from 490.75 ?m to 177.17 ?m,down 63.90%.3.Dox treatment increased the overall 5-hydroxymethylcytosine level,but decreased the overall 5-methylcytosine level of GTCs significantly.The expression of DKK family(Except DKK1)and SFRP family genes in the upstream of Wnt pathway were uptrend entirely under the control of TET1 overexpression by Dox-induced;Among them,the relative mRNA expression of SFRP2,SFRP4,SFRP5,DKK3 and DKK4 gene raised from 1.19,2.68,283.03,562.53 and 1.55 to 10.39,3.61,621.44,1023.42 and 9.69,respectively,significant difference(P<0.05).In addition,the expression of MYC,GSK-3beta,PPARG,CCND2,CTNNB1 were tend to upward;Among them,MYC,GSK-3beta and PPARG gene mRNA expression raised from 1.10,1.05 and 1.09 to 6.42,4.22 and 24.80,respectively,extremely significant differences(P<0.01).4.One TET1 shRNA plasmid with the highest interference efficiency was successfully screened.Compared with the piLenti-GFP control group,the relative expression level of TET1 mRNA in the cells transfected with piLenti-shTET1-GFP plasmid was decreased from 4.82 to 2.46,the difference was extremely significant(P<0.01);and the expression level of TET1 protein was significantly decreased.After interfering with TET1 expression transfection of piLenti-shTET1-GFP plasmid in GTCs,the average cell migration increased from 127.01 ?m to 229.41 ?m,an increase of 80.62%.5.TET1 shRNA plasmid interference resultd the entirety 5mC levels of the cells was significantly increased,and the entirety 5hmC levels was significantly decreased.The expression of DKK family(Except DKK1)and SFRP family gene in the upstream of Wnt pathway were downtrend entirely under the control of interference with TET1 expression;meanwhile,the expression of GSK-3beta,PPARG,CCND2 and CTNNB1 were decline trend,and the relative expression of SFRP1 mRNA was decreased from 249.70 to 186.41,the difference was significant(P<0.05).In conclusion:The primary GTCs with homogeneous morphology and high purity were obtained by isolation and culture of tissue block and co-culture method and purified by differential digestion.Dox treatment could increase the expression of TET1 gene in GTCs,and one TET1 shRNA plasmid with the highest interference efficiency was successfully screened.High expression of TET1 decreased the migration ability and the the overall 5-methylcytosine level,but increased the overall 5-hydroxymethylcytosine level of GTCs significantly;after interfering with TET1 expression,it was reversed.TET1 was related to Wnt pathway molecules in goat trophoblast cells. |
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