| Objective: STOX1(Stork head box1)is a transcription factor that is implicated in the high-prevalence human gestational disease,which studied in various types of gestational diseases using different molecular and cellular biological technology.However,the effect and detailed mechanism of STOX1 on unexplained recurrent spontaneous abortion(URSA)remains unknown.Molecular biology and pathology technology were used to determine the location and expression of STOX1 in the human villus tissues of normal early pregnancy and URSA.To identify the effect and the detailed mechanisms of STOX1 in regulating proliferation,migration and apoptosis of human trophoblast cells.To investigate the roles of downstream signaling pathway of STOX1 in regulating the function of trophoblast cell,to verify the molecular mechanisms underlined.To detect the expression of STOX1 in early unexplained recurrent spontaneous abortion and explore its role in the pathogenesis of early unexplained recurrent spontaneous abortion,thus provide a theoretical basis for further elucidate the pathogenesis of early URSA and efficient target genes are screened for the prevention and treatment of early URSA.Methods:(1)Detection of the mRNA levels of STOX1:to measured the expression of STOX1 by Real-time PCR.(2)The expression and location of STOX1 in early pregnancy villi were detected by immunohistochemistry and Western blotting(WB)(3)To detect the expression of PI3K/Akt signaling pathway proteins p85,pAKT and AKT in early pregnancy villi by WB.(4)Using lentiviral vector and plasmid construction,gene overexpression and(short hairpin RNA)shRNA interference technology were used up-regulation and down-regulation of STOX1 gene expression in HTR-8/SVneo cells.(5)Downregulation of STOX1,cell proliferation in HTR-8/SVneo cells were detected by MTT.(6)Downregulation of STOX1,transfected cells were dual stained with an annexin V-FITC/7-amino-actinimycinD(7-ADD)for measuring cell apoptosis,the expression of apoptosis-related factors Bcl-2,Bax and Bim proteins were detected by WB.(7)Downregulation of STOX1,cell migration assay was performed by the transwell chamber assay.(8)Downregulation of STOX1,To detect the relative expression levels of PI3K/Akt signaling pathway proteins by WB(9)Downregulation of STOX1,upregulation of PI3K/Akt signaling pathway,HTR-8/SVneo cell proliferation changes were detected by MTT.(10)Downregulation of STOX1,upregulation of PI3K/Akt,and detection of apoptosis by flow cytometry.(11)Downregulation of STOX1,upregulation of PI3K/Akt,cell migration of HTR-8/SVneo was detected by transwell.(12)Upregulation of STOX1,WB was used to detect the expression of PI3K/Akt signaling pathway proteins p85,pAKT.(13)Upregulating STOX1,downregulating PI3K/Akt,and cell proliferation was assessed using the MTT assays.(14)The dual luciferase gene assay was used to verify the targeting relationship between STOX1 and p85.Results:(1)RT-PCR results showed that the level of mRNA of STOX1 in the URSA group was significantly lower than that in the normal early pregnancy artificial abortion group(P<0.05).(2)The level of STOX1 protein is highly expressed in syncytiotrophoblasts,trophoblast cells and cytoplasmic cytoplasm,and expression of STOX1 in cytotrophoblasts is higher than other cells.The expression of STOX1 protein in URSA villi was similar to that in normal early pregnancy.Compared with the normal early pregnancy abortion group,immunohistochemistry and WB detection showed that the expression of STOX1 protein in the URSA group was significantly decreased(P<0.05).(3)Compared with the normal early pregnancy artificial abortion group,WB detection showed that the level of PI3K/Akt signaling pathway p85 and pAKT proteins was significantly decreased in the URSA group(P<0.05).(4)Downregulation of STOX1 resulted in decreased proliferation rate and migration number of HTR-8/SVneo cells,increased apoptosis rate,and the cell proliferation rate and apoptosis rate decreased and increased with time.Down-regulation of STOX1 leads to decreased proliferation and migration of trophoblast cells,increased apoptosis,decreased anti-apoptotic protein Bcl-2,and increased pro-apoptotic proteins Bax and Bim.(5)STOX1 was downregulated in the trophoblasts,while p85 and pAKT expression was significantly reduced.(6)STOX1 knockdown,combined with upregulation of PI3K/Akt,increased cell proliferation and decreased apoptosis.(7)Downregulation of STOX1,combined with down-regulation of PI3K/Akt,cell migration and proliferation were significantly reduced,and apoptosis was significantly increased.(8)Upregulation of STOX1,the expression levels of p85,pAKT increased;upregulation of STOX1,downregulation of PI3K/Akt,cell proliferation was significantly reduced.(9)STOX1 directly regulates p85,overexpression of STOX1 activated the PI3K/Akt signaling pathway,while STOX1 knockdown inhibited PI3K/Akt signaling pathway in the trophoblast.Conclusions:(1)STOX1 affects the function of trophoblast cells by regulating the expression of p85 and pAKT proteins of PI3K/Akt signaling pathway,it is an important regulator in the development of embryonic villi during early pregnancy.(2)STOX1 regulates p85 by directly in HTR-8/SVneo cells.STOX1 knockdown attenuated the PI3K/Akt signaling pathway,the cell proliferation and migration was significantly inhibited.STOX1 knockdown resulted in the activation of apoptotic proteins,including Bax and Bim,while reduced the Bcl-2 protein level in the HTR-8/SVneo cells.The relative protein expression level of Bcl-2 was remarkably decreased.In contrast,overexpression of STOX1 activates the PI3K/Akt signaling pathway in trophoblast cells and increases cell proliferation.This indicates the regulation of PI3K/Akt signaling pathway in trophoblast cell proliferation,migration and apoptosis.(3)The STOX1 expression is higher in normal villus tissues than in URSA villi.proteins p85 and pAKT of the PI3K/Akt signaling pathway were all decreased.Downregulation of STOX1 leads to decreased proliferation and migration of HTR-8/SVneo cells and increased apoptosis,while trophoblast proliferation,migration and apoptosis are closely related to abortion.Downregulation of STOX1 in trophoblasts can inhibit cell proliferation,migration and promote cell apoptosis by inhibiting PI3K/Akt signaling pathway,which may lead to early unexplained recurrent spontaneous abortion. |
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