| Placenta accreta spectrum(PAS)have emerged as an ongoing major iatrogenic public health challenge in the 21st century,known as the"killer placenta",which can result in up to 7%of maternal deaths and 77.8%of hysterectomies.PAS is a condition that is increasing at an astounding rate,with the prevalence escalating from 0.01%to 1.1%in recent years owing to an increase in cesarean delivery rates.Despite the devastating threat of PAS to mothers and infants,the underlying molecular biological mechanism is comprehensively inexplicit,and there is a lack of reliable biomarkers for prenatal diagnosis in clinical practice.The accumulating evidences have confirmed that molecular and phenotypic similarities between PAS and cancer.However,the specific role of these molecules in the pathogenesis of PAS has not been completely elucidated.The AGGF1 gene,also known as VG5Q,is a unique protein that contains both FHA and G-patch structural domains,which is commonly expressed in both normal and cancer cells and serves in a multitude of pathological processes,such as embryogenesis,tumorigenesis,apoptosis,autophagy and inflammation.AGGF1 was initially found to be highly expressed in vascular endothelial cells and it promoted angiogenesis.Subsequently,several studies showed that AGGF1,as a tumor suppressor or oncogene,mediated the proliferation,invasion,migration,and apoptosis of tumor cells,including uroepithelial,ovarian,and gastric cancers.The prominent characteristics of PAS are extensive neovascularization and excessive invasion of trophoblast cells,which are biologically similar to tumors.However,the protein expression level and the specific role of AGGF1 in PAS have not yet been reported.This study aimed to illuminate the expression of AGGF1 in placental tissue of PAS and its effect on the biological behavior of human trophoblast cells,and further explored the mechanism of AGGF1 in PAS,aiming to provide a theoretical basis and a new direction for the diagnosis and treatment of PAS.Part One The expression of AGGF1 in placental tissue from patients withPASObjective:To investigate AGGF1 m RNA and protein expression in placental tissue from patients with PAS,placenta previa and normal women with late pregnancy.Methods:1.From January 2020 to December 2021,54 participants were enrolled in this study,including 18 pregnant women with PAS,18 pregnant women with placenta previa and 18 healthy pregnant women who served as controls.All 18PAS patients in this study had placenta previa and were all preceded by at least one previous cesarean section,and none of the 18 patients with placenta previa had PAS.2.q RT-PCR was performed to detect AGGF1 m RNA expression in the placental tissue samples of the three groups.3.Western blot was conducted to examine the expression of AGGF1protein in the placental tissues of the three groups.4.Immunohistochemistry was used to confirm the expression and localization of AGGF1 in the placental tissues of the three groups,and CD34was used to label the vascular endothelial cells of the placental tissues.Results:1.Compared with the PP and CON groups,the PAS group showed a higher number of prior cesarean sections,more severe intraoperative bleeding,and a higher rate of cesarean hysterectomy(P<0.05).There were no significant differences in age,gravidity,parity or 1-min Apgar score between the PAS and the other two groups(P>0.05).In addition,differences in gestational age at delivery and neonatal weight existed between the PAS and CON groups(P<0.05).Nevertheless,no significant difference was found for gestational age at delivery between the PAS and PP groups(P>0.05).2.The AGGF1 m RNA expression apparently decreased in PAS samples compared with PP and CON samples(P<0.05),while there were no statistically differences between PP and CON samples(P>0.05).3.The AGGF1 protein expression significantly decreased in PAS samples compared with PP and CON samples(P<0.05),while there were no statistically differences between PP and CON samples(P>0.05).4.Immunohistochemical staining showed that AGGF1 was expressed vigorously in the nucleus and cytoplasm of placental trophoblast cells in the PP and CON groups,whereas the equivalent sites in PAS placental tissue presented faint staining.The AGGF1 protein expression in PAS was statistically weaker than that in the PP and CON samples(P<0.05),and no difference in AGGF1 protein expression existed between PP and CON samples(P>0.05).Pearson correlation analysis showed that AGGF1 protein was not correlated with placental microvascular density(P>0.05).5.Spearman correlation analysis showed that the expression level of AGGF1 in PAS was not correlated with the age(r_s=-0.336,P=0.173),gestational age at delivery(r_s=-0.196,P=0.435),gravidity(r_s=-0.134,P=0.596),parity(r_s=-0.184,P=0.464),number of previous cesarean sections(r_s=-0.263,P=0.291),intraoperative bleeding(r_s=-0.056,P=0.826),and hysterectomy(r_s=-0.406,P=0.094).However,the expression of AGGF1protein in placenta was significantly negatively correlated with the severity of PAS(r_s=-0.56,P=0.016).Summary:1.AGGF1 m RNA and protein were expressed in placental tissues of PAS,placenta previa and normal third trimester.2.The m RNA and protein expression levels of AGGF1 were only weakly expressed in the PAS samples and intensely exposed in the PP and normal controls.3.The expression of AGGF1 protein in placenta was significantly negatively correlated with the severity of PAS.Part Two The effect of AGGF1 on the biological behavior of trophoblastcellsObjective:To investigate the effects of AGGF1 on the proliferation,migration,invasion and apoptosis of trophoblast cells,and on P53 signaling pathway proteins.Methods:1.To explore the biological function of AGGF1 in the progression of PAS,we knocked down and overexpressed AGGF1 in HTR8/Snevo cells.The expression of AGGF1 m RNA and protein was detected by q RT–PCR and WB to detect the transfection efficiency.2.CCK8 assay,wound healing assay,Transwell invasion assay and flow cytometry assay were performed to monitor cell proliferation,migration,invasion and apoptosis.3.Western blot was used to detect the expression of P53/Bax/Bcl-2protein after knockdown and overexpression of AGGF1 gene.Results:1.The CCK8 results showed that knockdown of the AGGF1 gene significantly increased the proliferation rate of HTR-8/SVneo cells,demonstrating that AGGF1 gene downregulation promoted the proliferation ability of HTR-8/SVneo cells.However,the cell proliferation rate was decreased in the OV-AGGF1 group,suggesting that AGGF1 gene overexpression could inhibit HTR-8/SVneo cell proliferation(P<0.05).There was no significant difference in the proliferation rate between Si-NC and OV-NC groups(P>0.05).2.Flow cytometry results revealed that the apoptosis rate was reduced in the Si-AGGF1 group and increased in the OV-AGGF1 group compared to the corresponding control group(P<0.05).3.Compared with the Si-NC group,the migration distance of cells with Si-AGGF1 was longer at 24 h after scratching(P<0.05).Conversely,relative to the OV-NC group,the migration distance of cells with OV-AGGF1 was shorter at 24 h after scratching(P<0.05).4.Compared with the Si-NC group,the number of cells invading through the chamber membrane were apparently increased in the Si-AGGF1 group(P<0.05).In contrast,we observed a remarkable decrease in the quantity of cells crossing the chamber membrane in the OV-AGGF1 group(P<0.05).5.Western blot analysis showed that downregulation of AGGF1 inhibited the expression of P53,and Bax and enhanced the expression of Bcl2,while upregulation of AGGF1 had the opposite effects on these proteins(P<0.05).Summary:1.Downregulation of AGGF1 promoted cell proliferation,invasion and migration,inhibited apoptosis in vitro.2.Decreased AGGF1 regulated the expression of P53 signaling pathway-related proteins.3.Downregulation of placental AGGF1 may drive PAS development by affecting the biological behavior of trophoblast cells.Part Three mi R-1296-5p modulated the biological function of trophoblastcells by targeting AGGF1 expressionObjective:To explore the targeting relationship between mi R-1296-5p and AGGF1 and to investigate whether mi R-1296-5p suppressed AGGF1expression to regulate the biological functions of trophoblast cells.Methods:1.Bioinformatics analysis was used to predict the potential mi RNA targeting AGGF1 and the interaction between mi R-1296–5p and AGGF1 was detected by dual-luciferase reporter gene assay.2.q RT–PCR was used to investigate the expression levels of mi R-1296–5p in placental samples from PAS,PP and CON groups.3.The effects of mi R-1296-5p mimic/mi R-1296-5p mimic+AGGF1overexpression plasmid on cell proliferation,migration,invasion and apoptosis were detected by CCK8 assay,cell scratch assay,Transwell invasion assay and flow cytometry.4.Western blot was used to examined the expression levels of P53/Bax/Bcl-2 proteins in HTR8/SVneo cells treated with mi R-1296–5p mimic/mi R-1296-5p mimic+OV-AGGF1.Results:1.Bioinformatics analysis predicted that AGGF1 was a potential target gene of mi R-1296-5p.mi R-1296–5p mimic remarkably decreased the luciferase activity of the AGGF1–3?-UTR-WT report carrier,but did not affect the luciferase activity of the AGGF1–3?-UTR-UT report carrier(P<0.05).2.mi R1296–5p expression levels were substantially increased in PAS samples compared to PP and CON groups and mi R-1296-5p was negatively correlated with AGGF1 protein expression(P<0.05).3.Upregulation of mi R-1296–5p distinctly boosted trophoblast proliferation and invasion along with inhibition of apoptosis,and all these effects were abrogated when AGGF1 attained overexpression in HTR8/SVneo trophoblast cells(P<0.05).4.Following transfection of mi R-1296–5p mimic,AGGF1 protein expression was obviously decreased,and P53 protein and Bax protein were also suppressed,but Bcl-2 protein expression was enhanced,and the effects were abrogated when AGGF1 attained overexpression in HTR8/SVneo trophoblast cells(P<0.05).Summary:1.mi R-1296-5p was highly expressed in placental tissue with PAS and inhibited the expression of AGGF1.2.mi R-1296–5p may foster HTR8/SVneo cell proliferation,invasion,migration and repression of apoptosis by downregulating AGGF1 expression.3.Downregulation of placental AGGF1 may promote trophoblast over-invasion by mediating the P53 signaling pathway under the regulation of mi R-1296–5p.Conclusions:1.AGGF1 expression was reduced in patients with PAS and the expression of AGGF1 protein in placental tissue was negatively correlated with the severity of PAS.2.The decreased AGGF1 potentiated trophoblast proliferation,invasion migration and repressed apoptosis by regulating the P53 axis3.mi R-1296-5p was up-regulated in the placental tissues of PAS,and mi R-1296-5p could negatively mediate AGGF1 to promote proliferation,migration and invasion of trophoblast cells,and inhibit cell apoptosis.4.The downregulation of placental AGGF1 promoted trophoblast over-invasion by mediating the P53 signaling pathway under the regulation of mi R-1296–5p. |
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