Circ＿0001578 Promotes Gestational Diabetes Mellitus By Inducing Placental Inflammation

Objective: Gestational diabetes mellitus(GDM)refers to the abnormal glucose metabolism which is diagnosed for the first time during pregnancy.GDM is mostly found in the middle or late pregnancy period.GDM is different from the diabetes diagnosed before pregnancy,which usually returns to normal soon after delivery or termination of pregnancy.It is the most common type of abnormal glucose metabolism during pregnancy.GDM may cause serious adverse effects on the pregnant mother and fetus.The incidence rate of GDM has been increasing year by year.Therefore,it is of great significance to further study the pathogenesis of GDM.Related studies have shown that inflammatory reaction may participate in the development of gestational diabetes mellitus.Inflammatory factors interact with each other to interfere with insulin signal transduction,resulting in insulin resistance and gestational diabetes mellitus.Circular RNA(circRNA)is an important member of noncoding RNA molecules with stable closed-loop structure.With the development of high-throughput sequencing technology,circRNAs are involved in the occurrence and development of some diseases,and have the potential value of disease diagnosis and treatment targets.Placenta is an important organ for material exchange between fetus and mother.It is a combined organ formed by embryo membrane and mother endometrium during human pregnancy.The fetus develops in the uterus and depends on the placenta to obtain nutrition from the mother.Placenta synthesizes a variety of hormones,enzymes and cytokines to maintain normal pregnancy.Studies have shown that there are a series of pathophysiological changes in the placenta of GDM patients,including the differential expression of circRNAs.Although preliminary studies have shown that there is a relationship between circRNAs expression,disease pathogenesis and pregnancy outcome,the specific mechanism of circRNAs in GDM remains unclear.Based on the results of RNA sequencing test,this study intends to further verify circ＿0001578.Objective to investigate the expression of circ＿0001578 in placental villi and serum exosomes of GDM and normal pregnant women,and to further explore whether placental inflammatory response is involved in the occurrence and development of GDM,whether circ＿0001578 involved in the regulation of NF-κ B and JNKs,thus interpreting circ＿0001578 plays an important role in the development of GDM through placental inflammation.Method: 1.Circ＿0001578 was detected by qRT-PCR.The expression of circ＿0001578 in placental chorion of GDM patients was detected by transmission electron microscope and Western blot.The expression of circ＿0001578 in placenta derived exosomes and circ＿0001578 of peripheral blood exosomes in the second and third trimester of pregnancy were evaluated by ROC curve.The diagnostic value of circ＿0001578 for GDM.2.Down regulation of circ＿0001578 in human placental trophoblast cells HTR-8/Svneo by si-RNA interference.The expression of circ＿0001578 was detected by qRT-PCR.The proliferation and migration ability of HTR-8/Svneo cells were detected by CCK-8 test,plate clone formation test,transwell migration test and scratch test.The apoptosis of HTR-8/Svneo cells was detected by flow cytometry.3.Down regulation of circ＿0001578 in human placental trophoblast cells HTR-8/Svneo by si-RNA interference.The expressions of IL-1,IL-6,IL-8,TNF-α and CRP were detected by ELISA;4.The expression of circ＿0001578 in HTR-8/Svneo cells was down regulated by Si-RNA interference.Western blot was used to detect the expression of NF-κB and JNKs;5.HTR-8/Svneo cells were co transfected with si-NF-κB and si-circ＿0001578,the expression of IL-1,IL-6,IL-8,TNF-α and CRP were detected by ELISA.HTR-8/Svneo cells were co transfected with SP600125 and si-circ＿0001578,the expressions of IL-1,IL-6,IL-8,TNF-α and CRP were detected by ELISA.Results: 1.Compared with normal pregnant women,the expression of circ＿0001578in placental villi of GDM pregnant women was significantly down regulated(P <0.05).In the second and third trimester of pregnancy,the expression of circ＿0001578in peripheral blood of GDM group was down regulated(P<0.05),while in the first trimester of pregnancy,there was no significant difference between GDM group and normal pregnant women(P=0.21).ROC curve was used to evaluate the diagnostic value of circ＿0001578 in the second and third trimester of pregnancy.AUC under the curve was 0.738(P<0.05)and 0.478(P=0.091).The diagnostic value of circ＿0001578in the second trimester is better than that in the third trimester.2.si-circ＿0001578 and si-NC were transfected into HTR-8/Svneo cells respectively.CCK-8 showed that the proliferation activity of HTR-8/Svneo cells in si-circ＿0001578 group was significantly decreased(P<0.05 48 h,P<0.01 72h).Colony formation assay showed that the number of colony forming cells in transfection group decreased significantly(P<0.01).Transwell assay showed that the migration ability of transfected cells was decreased(P < 0.05).Scratch test showed that the migration ability of transfected cells was decreased(P<0.05).Annexin-V staining showed that the apoptosis of transfected cells increased significantly(P<0.01).3.Si-circ＿0001578 and si-NC were transfected into HTR-8/Svneo cells respectively.Compared with negative control group,the expression levels of IL-1,IL-6,IL-8,TNF-α and CRP in transfection group were increased(P<0.05).4.Western blot showed that the expressions of NF-κB and JNKs were significantly up-regulated in the si-circ＿0001578 group(P<0.05).5.Targeting si-NF-κB and si-circ＿0001578 co transfected in HTR-8/Svneo cells,compared with si-circ＿0001578 group,the expressions of IL-1,IL-6,IL-8,TNF-α and CRP were down regulated by ELISA.HTR-8/Svneo cells were co-treated with SP600125 and si-circ＿0001578,compared with si-circ＿0001578 group,the expression of IL-1,IL-6,IL-8,TNF-α and CRP were down regulated by ELISA.Conclusion: Circ＿0001578 is abnormally expressed in placental villi and peripheral blood placental derived exosomes of GDM pregnant women,which affects the proliferation,migration and apoptosis of placental trophoblast cells,mediates the inflammatory response of placental trophoblast cells through NF-κB and JNKs pathways,induces the release of inflammatory cytokines,and thus participates in the occurrence and development of GDM.The expression of circ＿0001578 in serum exosomes during the second trimester of pregnancy can be used as an effective GDM prediction index.