The Mechanism Of Hsa＿circ＿0007766/miR-34b-5p Targeting CCNG1,E2F5 And RRAS2 In Unexplained Recurrent Spontaneous Abortion

Background Unexplained recurrent spontaneous abortion（URSA）,which accounts for about 50%of recurrent abortions,most of which occurs before 12 weeks of pregnancy,has serious adverse effects on the patient’s psychology and body.At present,the prevention and treatment of URSA is still a major problem.The abnormal structure and function of placental villi can lead to the occurrence of URSA.Micro RNAs（mi RNAs）and circular RNAs（circ RNAs）are relatively stable non-coding RNAs（nc RNAs）in the body,which are involved in the occurrence of various diseases and in biological processes such as cell proliferation,apoptosis and differentiation.It plays an important regulatory role and is abundantly expressed in placental villi.The most common mechanism of action of circ RNAs is to participate in the regulation of target genes by mi RNAs as competing endogenous RNAs（ce RNAs）,and play an important role in cancer and other diseases,but few studies have reported in URSA.The circ RNAs/mi RNAs regulatory network may play an important role in the pathogenesis of URSA.Objective Screen the mi RNAs and circ RNAs that may be related to the occurrence of URSA in the aborted villi,and study the related functions and mechanisms in animal and cell models,and construct the circ RNA/mi RNA ce RNA regulatory network to elucidate the pathogenesis of URSA and search for URSA new therapeutic targets.Methods（1）MiRNAs and circ RNAs that may be related to the pathogenesis of URSA were identified by the detection of aborted villus tissue in clinical specimens.The aborted villi tissues of the URSA group（5 cases）and the control group（5 cases）were collected for whole transcriptome sequencing and screened for differentially expressed mi RNAs,circ RNAs and m RNAs（|log2FC|≥0.585,P≤0.05）;According to the fold change of mi RNA expression,the consistency between groups,expression levels and literature reports,several differentially expressed mi RNAs were screened,and further verified by RT-qPCR in the villus tissue of the URSA group（22 cases）and the control group（22 cases）to find out the mi RNA which possible involvement in the pathogenesis of URSA,and according to the sequencing results,bioinformatics prediction and RT-qPCR verification,the circ RNA that could potentially bind to it were found out.（2）To study the relationship between mi RNA（mi R-34b-5p）and abortion that may be related to the pathogenesis of URSA in animal experiments.ICR mice were transfected in the uterus on the 5.5^thday of gestation,the control group（8 mice）was transfected with agomir NC,and the experimental group（8 mice）was transfected with mmu-mi R-34b-5p agomir,and the mice were sacrificed on the 11.5^thday of gestation to observe the embryos.Embryo absorption rate was calculated,uterine wet weight was weighed,and transfection efficiency was verified by RT-qPCR.（3）The effects of mi R-34b-5p on cell proliferation,apoptosis,autophagy,migration and invasion were studied in cell experiments.HTR-8/SVneo cells were transfected with mi R-34b-5p mimic or inhibitor,and the proliferation ability of mi R-34b-5p on HTR-8/SVneo cells was investigated by CCK-8 assay,Ed U assay,cell cycle detection and western blot to detect proliferation and cycle-related proteins;The effects of mi R-34b-5p on apoptosis and autophagy of HTR-8/SVneo cells were investigated by flow cytometry to detect cell apoptosis,transmission electron microscopy to observe intracellular ultrastructure,and western blot to detect apoptosis and autophagy-related proteins;The effects of mi R-34b-5p on HTR-8/SVneo cell migration and invasion were detected by scratch assay,transwell cell migration assay,transwell cell invasion assay and western blot to detect cell migration and invasion-related proteins;The MET/PI3K/AKT/m TOR signaling pathway-related proteins were detected by western blot.（4）To study the molecular mechanism of downstream targeting regulation of mi R-34b-5p in cell experiments.RNA-seqwas performed after overexpression of mi R-34b-5p in HTR-8/SVneo cells,combined with bioinformatics prediction and RT-qPCR validation,to preliminarily screen potential target genes（CCNG1,E2F5 and RRAS2）of mi R-34b-5p,and verified by dual luciferase experiments;CCNG1,E2F5 and RRAS2 were knocked down in HTR-8/SVneo cells by si RNA technology,and their effects on the proliferation,apoptosis,migration and invasion of HTR-8/SVneo cells were studied.（5）The mechanism of circ RNA（hsa＿circ＿0007766）with potential binding sites for mi R-34b-5p was studied in cell experiments.Nucleocytoplasmic separation experiments,AGO2-RIP experiments and dual luciferase experiments were conducted to explore whether hsa＿circ＿0007766 could bind to mi R-34b-5p through the ce RNA mechanism;Hsa＿circ＿0007766 was knocked down in HTR-8/SVneo cells by si RNA technology,and the effect of hsa＿circ＿0007766 on proliferation,apoptosis,autophagy,migration and invasion ability of HTR-8/SVneo cell was investigated;The MET/PI3K/AKT/m TOR signaling pathway-related proteins were detected by western blot.Results（1）In the URSA group and the control group,290 differentially expressed mi RNAs and 94 differentially expressed circ RNAs were screened,and 2135 differentially expressed m RNAs significantly enriched in biological pathways such as oxidative phosphorylation,cell proliferation,adhesion,angiogenesis,and apoptosis;The relative expression of mi R-34b-5p in the abortive villus tissue of the URSA group was increased;Hsa＿circ＿0007766could potentially bind mi R-34b-5p,and the relative expression of hsa＿circ＿0007766 in the abortive villus tissue of the URSA group was decreased.（2）Intrauterine transfection of mi R-34b-5p agomir in ICR mice can significantly up-regulate the expression of placental mi R-34b-5p.Compared with the control group,the average embryo resorption number and embryo resorption rate increased,and the average uterine wetness decreased.（3）After overexpression of mi R-34b-5p in HTR-8/SVneo cells,the cell viability and proliferation ability decreased,and the protein expression levels of PCNA,Cyclin E,Cyclin D1 and CDK2 decreased;Apoptosis increased,typical apoptotic cells and autophagosomes were seen under the electron microscope,and the changes of apoptosis and autophagy-related proteins were consistent with the increase in apoptosis and autophagy;The ability of cell migration and invasion decreased,the expression of E-cadherin protein was up-regulated,and N-cadherin and MMP14 protein expression was down-regulated;Protein expression in the MET/PI3K/AKT/m TOR signaling pathway was down-regulated.In contrast,inhibition of mi R-34b-5p in HTR-8/SVneo cells promoted cell proliferation,migration and invasion,and inhibited apoptosis.（4）After HTR-8/SVneo cells overexpressed mi R-34b-5p,RNA-seqscreened a total of 304 differential genes,of which 217 genes were up-regulated and 87 genes were down-regulated（|log2FC|≥1,Q value≤0.05）,significantly enriched in related pathways such as cell proliferation,cell senescence,cell metabolism,vascular function,cancer,apoptosis,migration and invasion;MiR-34b-5p can target the expression of CCNG1,E2F5 and RRAS2.Knockdown of CCNG1,E2F5 and RRAS2 in HTR-8/SVneo cells can decrease cell proliferation,increase apoptosis,and decrease migration and invasion abilities.（5）Hsa＿circ＿0007766 was expressed in HTR-8/SVneo cytoplasm and could be significantly enriched by AGO2.At the same time,mi R-34b-5p,CCNG1,E2F5and RRAS2 were also significantly enriched by AGO2.Dual luciferase experiments showed that hsa＿circ＿0007766 can target binding to mi R-34b-5p;Knockdown of hsa＿circ＿0007766 resulted in decreased HTR-8/SVneo cell proliferation,increased apoptosis,decreased cell migration and invasion capacity,and inhibition of MET/PI3K/AKT/m TOR signaling pathway,which was basically consistent with the change of HTR-8/SVneo cell function caused by overexpression of mi R-34b-5p.Conclusions（1）MiR-34b-5p is up-regulated in the abortive villus tissue of the URSA group,and it leads to decreased HTR-8/SVneo cell proliferation ability,increased apoptosis and autophagy,decreased cell migration and invasion ability,and inhibites the MET/PI3K/AKT/m TOR signaling pathway by targeting CCNG1,E2F5 and RRAS2,may play an important role in the pathogenesis of URSA.（2）Hsa＿circ＿0007766 can act as a ce RNA to bind mi R-34b-5p to participate in the regulation of CCNG1,E2F5 and RRAS2,affect the proliferation,apoptosis,autophagy,migration and invasion of HTR-8/SVneo cells,and affect the MET/PI3K/AKT/m TOR signaling pathway,may play an important regulatory role in the pathogenesis of URSA.