| Background and Objectives:Placenta accrete spectrum disorders(PAS)represent severe pregnancy complications with an incompletely understood pathogenesis.Recent studies indicate a crucial role of long non-coding RNAs(LncRNAs)in the process of placental implantation.This study explores the expression profile of LncRNA KCNQ10T1 in PAS and its regulatory mechanism on trophoblast cell behaviour,as well as its potential impact on placental implantation through the miR-505-3p/hnRNPM axis.By comprehensively understanding this regulatory network,our aim is to elucidate the pathophysiological mechanisms underlying PAS and provide new theoretical foundations and methodological support for its diagnosis and treatment.Methods:1.The GSE148952 dataset from the GEO database was analyzed to identify the downregulated LncRNA KCNQ10T1 in placental implantation.Tissue samples from 42 PAS patients and 40 control subjects were collected,and qPCR was employed to analyze the differential expression of LncRNA KCNQ10T1 between PAS and normal placental tissues.PAS patients were further categorized into adhesive,invasive,and penetrative groups based on surgical findings,and the correlation between LncRNA KCNQ10T1 expression and disease severity was assessed by qPCR.In vitro experiments involved transfecting HTR-8/SVneo cells with pcDNA3.1-KCNQ1OT1,followed by assessment of cell proliferation,invasion,and migration using CCK8,EdU,Transwelland Western Blot assays to examine the expression of EMT-related proteins(E-cadherin,Vimentin,and Snail).2.The StarBase database was used to predict potential miRNA-505-3p binding sites on KCNQ10T1,which were subsequently validated through dual-luciferase reporter assays.CCK8,EdU,and Transwell assays were performed to evaluate the proliferation,invasion,and migration of miR-505-3p knockdown or overexpressing cells,along with their respective controls.Western Blot analysis was conducted to assess the impact on EMT-related proteins.Using predictions from the miRDB database and results of PAS placental tissue transcriptome sequencing,Venn diagrams were constructed to identify potential downstream target genes of miR-505-3p,validated through dual-luciferase reporter assays.Pearson correlation analysis was employed to examine the correlation between miRNA and target gene expression in PAS placental tissue.Changes in mRNA and protein levels of target genes following miRNA overexpression or knockdown were assessed by qPCR and Western Blot.Rescue experiments were performed to determine whether the knockdown of target genes could partially reverse the inhibitory effects of miRNA inhibitors on trophoblast cell proliferation,invasion,and migration.Changes in mRNA and protein levels of target genes following overexpression of LncRNA KCNQ10T1 were analyzed by qPCR and Western Blot.Subsequent rescue experiments investigated whether inhibiting target gene expression could reverse the inhibitory effects of LncRNA KCNQ10T1 overexpression on trophoblast cell biological functions.3.Female SD rats,24 hours postpartum,were divided into two groups based on surgical procedures:the first group underwent full-layer incision and suturing of one side of the uterine mesentery,with the contralateral side serving as self-control;the second group underwent full-layer incision and suturing of both sides of the uterine mesentery,with a sham operation as the control.After 14 days,the rats were euthanized at gestational day 17 to evaluate the PAS model,including fetal number,absorption rate,HE staining to observe placental and muscle cell morphology,and immunohistochemistry for evaluating the expression of Cytokeratin 8.Finally,qPCR experiments were conducted to validate the expression of lncRNA KCNQ10T1/miR-505-3p/hnRNPM in the placental tissues of rats in the implantation and non-implantation groups.Results:1.Database analysis revealed a significant downregulation of LncRNA KCNQ10T1 expression in the placental tissues of PAS patients compared to the control group.Clinical sample analysis confirmed that KCNQ10T1 expression in PAS placental tissues was significantly lower than that in normal control tissues,and further analysis demonstrated that KCNQ10T1 expression in placental implantation tissues was significantly lower than that in adhesive tissues,indicating a correlation with disease severity.In vitro experiments demonstrated that overexpression of KCNQ10T1 inhibited the proliferation,invasion,and migration of HTR-8/SVneo cells,with Western Blot results showing increased E-cadherin expression and decreased Vimentin and N-cadherin expression.2.Dual-luciferase reporter assays confirmed specific binding sites between lncRNA KCNQ10T1 and miR-505-3p.qPCR analysis confirmed a negative correlation between KCNQ10T1 and miR-505-3p expression in HTR-8/SVneo cells and PAS placental tissues.Knockdown of miR-505-3p inhibited trophoblast cell proliferation,invasion,and migration,with Western Blot results showing upregulation of E-cadherin and downregulation of Vimentin and N-cadherin expression.Conversely,overexpression of miR-505-3p promoted trophoblast cell proliferation,invasion,and migration.Transfection with miR-505-3p mimics restored the inhibitory effects of KCNQ10T1 overexpression on the HTR-8/SVneo cell proliferation,migration,invasion,and epithelial-mesenchymal transition(EMT)process.3.Dual-luciferase reporter assays revealed specific binding sites between miR-505-3p and hnRNPM,which was further validated by qPCR in HTR-8/SVneo cells and PAS placental tissues.Rescue experiments demonstrated that the knockdown of hnRNPM restored the inhibitory effects of miR-505-3p inhibitor on HTR-8/SVneo cell proliferation,invasion,migration,and the EMT process.4.qPCR and Western Blot results indicated a positive correlation between KCNQ10T1 and hnRNPM expression in PAS placental tissues and HTR-8/SVneo cells.Knockdown of hnRNPM restored the inhibitory effects of KCNQ10T1 overexpression on HTR-8/SVneo cell proliferation,migration,invasion,and EMT process.Furthermore,a rat model of placental implantation was successfully established through surgical procedures,with an assessment confirming the presence of placental implantation.qPCR experiments validated the downregulation of KCNQ10T1 and hnRNPM and the upregulation of miR-505-3p in the placental tissues of rats in the implantation group compared to the non-implantation group.Conclusion:Placental tissues of patients with placental implantation disorders exhibit downregulation of LncRNA KCNQ10T1 and hnRNPM expression,along with upregulation of miR-505-3p.LncRNA KCNQ1OT1 regulates trophoblast cell biology by binding to miR-505-3p and modulating hnRNPM expression,thereby influencing the occurrence and progression of placental implantation.This study elucidates the regulatory mechanism of the LncRNA KCNQ10T1/miR-505-3p/hnRNPM axis on trophoblast cell proliferation,invasion,and migration,providing experimental evidence for the pathogenesis of placental implantation disorders.These findings suggest that LncRNA KCNQ10T1,miR-505-3p,and hnRNPM may serve as novel targets for diagnosing and treating these disorders. |
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