The Role Of Histone Deacetylase SIRT1 In Recurrent Spontaneous Abortion By Regulating YB-1

Objective:Recurrent spontaneous abortion(RSA)occurs in 1%-5%of women of childbearing age and is on the rise year by year.The etiology of RSA is complex and there is no clear effective treatment at present.Repeated pregnancy failure brings serious physical and mental burden to patients and their families.Embryonic-derived trophoblast cells are the only cells in direct contact with the mother.In early gestation,proliferation,migration and invasion of trophoblast cells are the key links of embryo implantation and placenta formation.Trophoblast cells could invade the decidua of the mother,reconstruct placental blood vessels,and maintain the blood supply and growth of the fetus.The invasion process of trophoblast cells is finely regulated in time and space,and any factors that cause damage to the function of trophoblast cells may lead to abortion.In previous studies,it was found that silent type information regulator 2 homolog 1(SIRT1)was expressed in both fetal villi and trophoblast cells.And the expression in villus tissue of RSA patients was significantly increased.This raises the following scientific questions.What is the significance of abnormally elevated SIRT1?Is SIRT1 involved in regulating the biological function of trophoblast cells?It needs to be confirmed by further research.The aim of this study was to explore the regulatory effect and molecular mechanism of SIRT1 on the biological function of trophoblast cells,and to provide theoretical and experimental basis for the early precise measurement and treatment of RSA.Methods:1.The expression of SIRT1 in trophoblast cell lines was detected by immunohistochemistry,Quantitative Real-time PCR(qRT-PCR)and Western Blotting.qRT-PCR and Western Blotting were used to detect the expression of SIRT1 in villus tissues of normal early pregnancy and RSA patients.2.SIRT1 overexpression cell model was constructed.Stable overexpression SIRT1 cell lines were constructed.The SIRT1 knockout cell model was constructed.3.The effects of SIRT1 on proliferation of trophoblast cells were evaluated by real time unlabeled cell analysis(RTCA)assay and EdU assay.The effects of SIRT1 on cycle distribution of trophoblast cells were detected by flow cytometry.The effects of SIRT1 on the migration and invasion ability of trophoblast were detected by Transwell assay and Western Blotting assay.The effects of SIRT1 inhibitor EX-527 on proliferation,migration and invasion of trophoblast cells were verified by CCK-8 assay,plate clonal formation assay and Transwell assay.4.The abortion prone pregnant mice CBA/J × DBA/2(AP)and normal pregnant mice CBA/J× BALB/c(NP)models were established.Mice were injected intraperitoneally with EX-527,and the mice were killed on the 13th day of gestation and the embryos were removed.The size and morphology of mouse embryos were observed and compared,and the embryo loss rate was calculated.5.Co-immunoprecipitation(Co-IP)and liquid chromatography-tandem mass spectrometry(LC-MS/MS)were used to screen for YB-1 protein that might bind to SIRT1.The colocalization of SIRT1 and YB-1 in trophoblast cells was detected by immunofluorescence and laser scanning confocal microscope.Co-IP combined with Western Blotting experiments verified the binding between SIRT1 and YB-1.6.Acetyl Lysine Antibody was used to determine whether YB-1 could undergo acetylation modification by immunoprecipitation combined with Western Blotting,and whether exogenous EX-527 could change the YB-1 acetylation level.qRT-PCR and Western Blotting experiments were conducted to investigate the effects of SIRT1 and EX-527 on YB-1 expression.7.Rescue experiment verified the regulation of SIRT1 on trophoblast biological function through YB-1.8.The effect of SIRT1 on bioactive factors secreted by trophoblast was examined in trophoblast cells with stable overexpression of SIRT1.Results:1.SIRT1 could be expressed in trophoblast cells.SIRT1 expression was up-regulated in villi of RSA patients compared with normal early pregnancy.2.SIRT1 overexpression or knockdown cell models were constructed and their efficiency was verified.3.RTCA and EdU experiments showed that SIRT1 did not affect the proliferation ability of trophoblast cells.Flow cytometry showed that SIRT1 had no significant effect on trophoblast cycle.Transwell assay confirmed that SIRT1 inhibited the migration and invasion ability of trophoblast cells.SIRT1 inhibited the expression of invasion-related protein markers.In addition,the SIRT1 inhibitor EX-527 also had no significant effect on the proliferation of trophoblast cells and inhibited the migration and invasion ability of trophoblast cells.4.Compared with AP group,the embryo loss rate of AP+EX-527 group was lower.5.Co-IP combined with LC-MS/MS analysis screened the protein YB-1 that may bind to SIRT1.Immunofluorescence showed that SIRT1 and YB-1 were colocalized in the nucleus of trophoblast cells.Western Blotting experiments confirmed the mutual binding between SIRT1 and YB-1 protein.6.YB-1 was modified by acetylation,and the acetylation level of YB-1 was enhanced by exogenous EX-527.SIRT1 inhibited YB-1 protein expression through deacetylase activity,but had no effect on mRNA level.7.Rescue experiments showed that YB-1 could partially reverse the inhibition of SIRT1 on the migration and invasion ability of trophoblast cells.8.Trophoblast cells overexpressed with SIRT1 inhibited the expression of IL-10,but had no significant effect on TGF-β,IL-6,CCL5 and IL-35.Conclusion:1.Through in vitro experiments and spontaneous abortion animal model studies,this study proved that SIRT1 can inhibit the normal growth and development of embryos and induce the imbalance of maternal fetal tolerance by inhibiting the migration and invasion of trophoblast cells and the expression of cytokine IL-10.2.SIRT1 could bind to YB-1 and inhibited YB-1 expression depending on deacetylase activity.The inhibition of SIRT1 on the migration and invasion ability of trophoblast could be partially reversed by YB-1.