Study Of The Role Of SPARCL1 In The Pathogenesis Of Early Spontaneous Abortion

Ojectives The purpose of this study was to investigate whether SPARCL1 is associated with early spontaneous abortion and to explore the role of SPARCL1 in migration of human trophoblasts.Methods 1.Collected villous specimens of patients undergoing artificial abortion with suction evacuation who were diagnosed with arrested intrauterine pregnancy and normal pregnancy at Renji hospital affiliated to Shanghai Jiaotong University School of Medicine during 2015-2016.Total RNA and protein were extracted from villous specimens and tested by Western blotting and real-time quantitative PCR to compare the expression of SPARCL1 between the two groups.2.The p IRES2-EGFP-SPARCL1 recombinant plasmid was constructed and the empty plasmid p IRES2-EGFEnter was used as control.HTR8/SVneo and JAR cell lines were selected as the research model.The SPARCL1 gene was over-expressed by liposome transfection.The cell migration ability of the overexpression group and the control group was compared by the scratch test.3.1ug/m L human recombinant SPARCL1 protein was added to the cell culture solution,and the cell migration ability was compared with the control group by the scratch test.4.The cell proliferative activity of the overexpression group and the control group was compared by CCK8 assay.5.Total RNA and total cellular protein were extracted from HTR8/SVneo cells and JAR cells of the overexpression group and the control group.MMP2,MMP3,ECADHERIN,N-CADHERIN,and VIMENTIN were detected by real-time quantitative PCR and Western blotting.6.Furthermore,total cellular protein was extracted from HTR8/SVneo cells with scratch test prior to the addition of cellular supertanants contained with SPARCL1,and the phosphorylation activity of ERK1/2 pathway and the expression of AP-1 of the SPARCL1 group and the control group was compared by by Western blotting.Results 1.The m RNA level and protein level of SPARCL1 were significantly different(P<0.05)between the spontaneous abortion group and the normal early pregnancy group.The expression of SPARCL1 was significantly upregulated in the villous specimens of spontaneous abortion patients.2.After adding human recombinant SPARCL1 protein and overexpressing SPARCL1,the migration distance and migration area of HTR8/SVneo cells reduced compared to control.Overexpression of SPARCL did not affect HTR8/SVneo cell proliferation activity.3.Overexpressing SPARCL1 in HTR8/SVneo cells,the expression of MMP2,MMP3,and VIMENTIN reduced compared to the control group,and the expression of ECADHERIN elevated.Overexpressing SPARCL1 in JAR cells,compared with the control group,the expression of MMP3,VIMENTIN,NCADHERIN reduced,and the expression of E-CADHERIN elevated.4.After scratch test of HTR8/SVneo and addition of supernatant containing SPARCL1,the phosphorylation activity of ERK1/2 pathway and the expression of AP-1 protein were decreased compared with the control group.5.In the spontaneous abortion group,the m RNA level of SPARCL1 and MMP2 was negatively correlated(P=0.0261),the Pearson coefficient was -0.4962,and the m RNA level of SPARCL1 was negatively correlated with that of VIMENTIN(P=0.0356),the Pearson coefficient was-0.4842.Conclusions 1.The expression of SPARCL1 in the villous specimens of spontaneous abortion group was significantly higher than that of normal early pregnancy group.2.SPARCL1 can down-regulate the ability of migration and invasion of trophoblasts.3.SPARCL1 can down-regulate the expression of MMP2,MMP3,N-CADHERIN and VIMENTIN and up-regulate the expression of E-CADHERIN which may be mediated by the down-regulation of phosphorylation activity of ERK1/2 and formation of AP-1,thus affecting the differentiation and migration of trophoblasts and hampering spiral arterial remodeling and the development of the placental bed,which are involved in the pathogenesis of spontaneous abortion.