| As the most common hyperglycemic manifestation during pregnancy,gestational diabetes mellitus(GDM)affects short-term and long-term health in mothers and offspring.Insulin resistance(IR)is recognized as a specific pathological feature of GDM.Increasing evidence has shown that circRNAs(circRNAs)participate in the regulation of IR-related diseases,such as type 2 diabetes mellitus and metabolic syndrome,while few studies have focused on GDM.Placenta plays a crucial role in the development of the IR during pregnancy,and numerous circRNAs have been reported to exhibit differential expression profiles in the placentas of GDM patients.Exploring the effect of circRNA on IR in trophoblasts during GDM will help improve the understanding of GDM-related IR and provides new therapeutic targets for GDM.Objective: We aimed to screen the aberrant circRNAs in GDM placenta that are closely related to GDM-related IR,and explore the role of selected circRNA in GDM-related IR in trophoblast cells through competitive endogenousRNA mechanism.Methods:1.Using our previous circRNA microarray data and miRNA information in the published literature,the binding sites between differentially expressed circRNAs(DECs)and miRNAs(DEMis)in GDM placentas were predicted through bioinformatics analyses.GO and KEGG analyses were conducted to assess the underlying biological processes and pathways of DECs through their interactions with DEMis.DECs with more than 2DEMi targets in their interaction network were verified in 18 placental tissues(9 patients with GDM and 9 healthy controls)using quantitative real-time polymerase chain reaction(qRT-PCR).2.The circ?0078619/miR-6795-5p/PTPN1 axis and key markers in the insulin-PI3K/Akt signaling pathway in placental tissues were evaluated through western blotting,qRTPCR,fluorescence in situ hybridization(FISH),immunofluorescence(IF)and immunohistochemistry(IHC)in 84 placental tissues(42 patients with GDM and 42 healthy controls).The circular structure of circ?0078619 was further identified by RNase R digestion and agarose gel electrophoresis.We also analyzed the correlation between the expression of circ?0078619 and clinical parameters.3.IR model was constructed using HTR-8/SVneo cells.Interactions between miR-6795-5p and circ?0078619 or PTPN1 were identified using double luciferase reporter assay.We validated the sub-cellular localization of circ?0078619 and miR-6795-5p in trophoblst cells by FISH.shRNA,miRNA mimics,miRNA inhibitor and plasmid were transfected or co-transfected in trophoblasts.We used qRT-PCR and western blotting to detect circ?0078619/miR-6795-5p /PTPN1 axis and IRS1/PI3K/Akt pathway in trophoblast cells.The role of circ?0078619 in insulin resistance in trophoblasts was determined using the glucose uptake assay.Result:1.The DEC-DEMi interaction network was constructed,and consisted of 16 circRNA nodes,18 miRNA nodes,and 40 edges.A total of 70 KEGG pathways were enriched,and the altered circRNAs may also have affected several vital pathways involved in GDM progression,including the insulin signaling pathway,the molecular target of rapamycin(m TOR)pathway,and the adenosine monophosphate-activated protein kinase(AMPK)pathway.Among DECs that were found to bind to at least 2 DEMis in their interaction networks,circ?0078619 and circ?0087961 were dysregulated in the placental tissues of patients with GDM as compared to the placental tissues of the controls,and this was consistent with the profile data obtained.Bioinformatics analysis revealed that circ?0078619 might negtively regulate the insulin signaling pathway via miR-6795-5p/PTPN1 axis.2.The expression of circ?0078619 and PTPN1 were highly expressed in GDM placentas,while miR-6795-5p was declined,accompanied with the decreased phosphorylation of IRS1 and Akt.The annular structure of circ?0078619 was confirmed.Moreover,circ?0078619 expression in the placental tissues of GDM patients,was found to be positively correlated with weight gain during pregnancy,time-glucose area under the curve in the oral glucose tolerance test,and fasting plasma glucose level at delivery.3.The expression of circ?0078619 and PTPN1 were up-regulated in IR model of trophoblasts,whereas miR-6795-5p was down-regulated.circ?0078619 and PTPN1 can both bind to miR-6795-5p,and circ?0078619 was co-located with miR-6795-5p in the cytoplasm of trophoblast cells.circ?0078619 modulated the expression of PTPN1 via miR-6795-5p.circ?0078619 silencing or miR-6795-5p overexpression partially reversed the IR-induced decrease in glucose uptake and downregulated IRS1 and Akt phosphorylation in trophoblasts;this restoration was dysregulated again upon cotransfection with an miR-6795-5p inhibitor and PTPN1,respectively.The reduced glucose uptake and phosphorylation of IRS1 and Akt were exacerbated by tranfecting miR-6795-5p inhibitor in IR-induced trophoblast cells.Conclusion: circ?0078619 could modulate IR in trophoblasts during GDM via miR-6795-5p/PTPN1 axis,providing a novel insight into the pathogenesis of GDM and a new prospective therapeutic target for GDM patients. |
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