Exosomal MiRNA As A Diagnostic Biomarker For Endometriosis And Its Pathological Mechanism

BackgroundEndometriosis(EMs)refers to the presence of endometrial glands and stroma outside the uterus cavity,and these ectopic lesions mainly appear on the ovaries and peritoneum.The main clinical symptoms of EMs include dysmenorrhea,chronic pelvic pain,dyspareunia,infertility and so on.EMs is one of the most common gynecological diseases among women of childbearing age.About 10%of women of childbearing age suffer from EMs,however the prevalence among women with infertility has increased to 35-50%.Although many hypotheses have been proposed for the cause of EMs,each hypothesis has unexplainable conditions.Therefore,the exact pathogenesis of EMs remains unclear.In addition,there is lack of effective non-invasive diagnostic markers for EMs,resulting in the delayed diagnosis and poor treatment effect.Hence,it is important to find new molecular markers and explore the mechanism of EMs,which are particularly critical for the early diagnosis and treatment strategies of EMs.miRNA(microRNA)is a kind of endogenous small non-coding RNA(about 22-24 nucleotides in length),which regulates translation by binding to the 3’ UTR of target mRNA.Previous studies have confirmed that miRNAs in peripheral blood can be used as non-invasive diagnostic markers for many diseases,but these miRNAs are easily affected by RNA enzymes.Exosome is a membrane extracellular vesicle with a diameter of 50-150nm,widely distributed in urine,blood,cerebrospinal fluid and other body fluids.Exosomes can be secreted by almost all cell types.Exosomes contain various biological molecules,such as proteins,lipids,and miRNAs,which can be taken up by adjacent or distant cells,and then regulate the function of recipient cells.Thus exosomes can serve as a bridge for biological communication between different cells.miRNA in exosomes cannot be degraded by RNA enzymes,which means that peripheral exosomal miRNAs are stable.Studies have found that exosomal miRNAs can be used as diagnostic markers for many diseases and participate in many biological processes.However,the diagnostic value and mechanism of exosomal miRNAs in EMs have not been explored.Present studies suggest that immune cells play an important role in the origin and development of EMs.Immune cells and cell-mediated dysfunction lead to poor clearance of ectopic endometrium.Immune cells in peritoneal microenvironment include neutrophils,macrophages,dendritic cells.The dysfunction of these cells results in proliferation,adhesion and invasion of ectopic endometrial cells,and angiogenesis.It has been showed that peritoneal macrophages(pMφ)in EMs are in an activated state,and can secrete cytokines(e.g.L-10 and TGF-β),which act on ectopic endometrial stromal cells(eESCs).At present,it has been confirmed that peritoneal macrophages can release exosomal miRNAs into the surrounding microenvironment and can affect proliferation and infiltration of recipient cells in other diseases.However,the role of exosomal miRNA secreted by peritoneal macrophages in EMs has not been reported.In this study,we selected exosomal miRNA as a new non-invasive diagnostic marker for EMs,and explored the role of exosomal miRNA derived from peritoneal macrophages in EMs.Our goal was to provide new ideas for the diagnosis and treatment of EMs.This study was divided into two parts.In the first part,we detected exosomal miRNA profiling from the serum of EMs,and evaluated the diagnostic value of serum exosomal in EMs.In the second part,we detected the expression of miR-22-3p and miR-320a in exosomes derived from EMs peritoneal macrophages and explored the mechanism that peritoneal macrophages regulated the biological behavior of ectopic endometrial stromal cells through exosomal miR-22-3p.PART ISerum exosomal microRNAs as potential circulating biomarkers for endometriosisObjectiveOur aim was to screen and identify the differentially expressed miRNAs between serum exosomes from EMs patients and control patients,then evaluate the diagnostic value of serum specific exosomal miRNAs in EMs and the relationship between specific exosomal miRNAs and clinicopathological features.We tried to provide a new more accurate biomarker for EMs.Methods1.Exosomes were isolated from the serum of EMs and control patients by differential ultracentrifugation,and identified by transmission electron microscopy,NT A and western blot.2.The exosomal miRNA profiling between two groups was screened by Human miRNA microarray.3.qRT-PCR was used to further verify the expression of these specific exosomal miRNAs.4.ROC curve was used to explore the diagnostic value of specific exosomal miRNAs.5.Bioinformatics was used to predict the potential target genes of specific exosomal miRNAs,which were analyzed through GO and KEGG analysis.6.Clinicopathological features were collected and analyzed to find the relationship with specific exosomal miRNAs.Results1.Identification of serum exosomesTransmission electron microscope showed that serum exosomes had a round or oval shape with central pale and sharp edges,and its diameter was between 50-150 nm.NTA demonstrated that the diameter of exosomes ranged from approximately 50 nm to 200 nm and the average diameter of exosomes was 93±4.1nm.Western blot exhibited that CD9 and CD63 were highly expressed in exosomes.2.miRNA microarray profiling in serum exosomesThe result of miRNA microarray showed that there were 24 significantly different expressed miRNAs between EMs serum exosomes and control serum exosomes(P<0.05,|log2(Fold Change)|≥1).Among these miRNAs,there were 18 upregulated miRNAs and 6 downregulated miRNAs.3.Validation of the expression of exosomal miRNAsBased on the results of miRNA microarray and previous studies,qRT-PCR was used to further verify the expression of miR-134-5p,miR-197-5p,miR-22-3p,miR-320a,miR-494-3p,and miR-939-5p.Results revealed that the expression of miR-22-3p and miR-320a were significantly upregulated in serum exosomes from EMs compared with control group(P<0.05).The expression of other miRNAs did not differ significantly(P>0.05).4.Diagnostic value of serum exosomal miRNAsThe area under the ROC curve(AUC)of serum exosomal miR-22-3p and miR-320a for the diagnosis of EMs was 0.843(95%CI:0.747-0.940)and 0.824(95%CI:0.721-0.927)respectively.When miR-22-3p and miR-320a were combined,the AUC increased to 0.903(95%CI:0.846 to 0.981).The AUC of serum CA-125 is 0.775(95%CI:0.659-0.891).5.Prediction and bioinformatics analysis of the target genes of miR-22-3p and miR-320aThe venn diagram showed the target genes of miR-22-3p and miR-320a.The result of GO analysis of target gene showed that the main functions of target genes included MAPK activity,lamin binding,G-protein-coupled glutamate receptor binding,etc.KEGG analysis revealed that the target genes were enriched in TNF signaling pathway,prolactin signaling pathway,ErbB signaling pathway,etc.6.Correlation between the expression of serum exosomal miR-22-3p and miR-320a and the clinicopathological characteristics of EMsThe expression of exosomal miR-22-3p and miR-320a were both positively correlated with r-AFS stage(P<0.05).The expression of exosomal miR-320a was positively correlated with the severity of dysmenorrhea(P<0.05).Other clinicopathological features were not correlated with the expression of exosomal miR-22-3p and miR-320a(P>0.05).Conclusions1,Serum exosomal miR-22-3p and miR-320a can serve as potential circulating biomarkers for endometriosis and have higher diagnostic efficacy.2.The expression of serum exosomal miR-22-3p and miR-320a were both positively correlated with r-AFS stage.The expression of exosomal miR-320a was positively correlated with the severity of dysmenorrhea.PART ⅡExosomal miR-22-3p derived from peritoneal macrophages promotes proliferation,migration,and invasion of ectopic endometrial stromal cells by regulating SIRT1/NF-κB pathwayObjectiveOur aim was to detect the expression of miR-22-3p and miR-320a in exosomes derived from peritoneal macrophages of EMs(EMs exosomes)and control patients(control exosomes).Then we explored the role of exosomal miR-22-3p derived from peritoneal macrophages in EMs.Methods1.Peritoneal macrophages were isolated from peritoneal fluid of EMs and control group.Exosomes were isolated from the supernatant of peritoneal macrophages and identified by transmission electron microscopy,NTA and western blot.2.Ectopic endometrial stromal cells were isolated and co-cultured with exosomes labeled with Dil to determine whether labeled-exosomes can be taken up.3.CCK-8,EdU assay,wounding heal,Transwell assay and flow cytometry were used to detect the effect of EMs exosomes and control exosomes on the proliferation,migration,invasion and apoptosis of ectopic endometrial stromal cells.4.qRT-PCR was used to detect the expression of miR-22-3p and miR-320a in two kinds of exosomes,and the expression of miR-22-3p and pri-miR-22-3p in ectopic endometrial stromal cells after co-incubated with different exosomes.5.qRT-PCR was used to detect the expression of miR-22-3p in different endometrial stromal cells and different endometrial tissues.6.CCK-8,EdU assay,wounding heal,Transwell assay and flow cytometry were used to detect the effect of miR-22-3p on the proliferation,migration,invasion and apoptosis of ectopic endometrial stromal cells.7.TargetScan was used to predict the potential target gene of miR-22-3p.Dual-luciferase reporter assay was used to verify the candidate target gene SIRT18.Immunohistochemistry and western blot were used to detect the expression of SIRT1 in different endometrial tissues.CCK-8,wounding heal and Transwell assay were used to detect the effect of SIRT1 on the proliferation,migration and invasion of ectopic endometrial stromal cells.9.CCK-8,wounding heal and Transwell assay were used to confirm that SIRT1 can reverse the effect of miR-22-3p on the proliferation,migration and invasion of ectopic endometrial stromal cells.10.Immunohistochemistry and western blot were used to detect the expression of NF-κB related protein p-p65 in different endometrial tissues.Western blot was used to analyze the effect of SIRT1 on the expression of p-p65,CCK-8,wounding heal and Transwell assay were used to detect the effect of PDTC(a NF-κB inhibitor)on the proliferation,migration and invasion of ectopic endometrial stromal cells.11.CCK-8,wounding heal and Transwell assay were used to confirm that PDTC can reverse the effect of miR-22-3p on the proliferation,migration and invasion of ectopic endometrial stromal cells.Results1.Identification of peritoneal macrophages and exosomesCD68 was highly expressed in peritoneal macrophages from EMs group and control group through immunofluorescence and flow cytometry.The morphology of exosomes under transmission electron microscope was round or oval,with a diameter between 50-150 nm.The average diameter of exosomes detected by NTA was 105±3.9 nm.Western blot showed that CD9 and CD63 were highly expressed in exosomes.2.Exosomes derived from peritoneal macrophages were absorbed by ectopic endometrial stromal cellsImmunofluorescence showed that red granules were located outside the nucleus of ectopic endometrial stromal cells stained with DAPI.3.Effect of peritoneal macrophage-derived exosomes on the biological behavior of ectopic endometrial stromal cellsVimentin was expressed in ectopic endometrial stromal cells through immunofluorescence.EMs exosomes significantly increased the proliferation,migration and invasion of ectopic endometrial stromal cells(P<0.05).There was no significant difference in the effect on apoptosis of ectopic endometrial stromal cells between two kinds of exosomes(P>0.05)4.Peritoneal macrophages transported miR-22-3p to ectopic endometrial stromal cells via exosomal pathwayCompared with control exosomes,the expression of miR-22-3p in EMs exosomes was significantly increased(P<0.05).There was no significant difference in the expression of miR-320a between different exosomes(P>0.05).After co-incubation with EMs exosomes,the expression of miR-22-3p in ectopic endometrial stromal cells was significantly increased(P<0.05),while the expression of pri-miR-22-3p had no significant difference(P>0.05).5.Expression of miR-22-3p in different endometrial stromal cells and different endometriumThe expression of miR-22-3p in ectopic endometrial stromal cells was significantly higher than that in eutopic endometrial stromal cells and normal endometrial stromal cells(P<0.05).The expression of miR-22-3p in ectopic endometrium was significantly higher than that in eutopic endometrium and normal endometrium(P<0.05).6.Effect of miR-22-3p on the biological behavior of ectopic endometrial stromal cellsTransfection of miR-22-3p mimics and inhibitors significantly increased the expression and significantly decreased the expressionof miR-22-3p respectively(P<0.05).miR-22-3p mimics significantly enhanced the proliferation,migration and invasion ability of ectopic endometrial stromal cells(P<0.05).miR-22-3p inhibitors significantly suppressed the proliferation,migration,and invasion of ectopic endometrial stromal cells(P<0.05).Both miR-22-3p mimics and inhibitors had no significant difference in the effect on apoptosis(P>0.05).7.Prediction and validation of the target gene of miR-22-3pTargetScan showed that the 3’UTR of SIRT1 has a binding site for miR-22-3p at 530-537.Dual-luciferase reporter assay showed that miR-22-3p specifically binds to SIRT1 3’UTR.Western blot and qRT-PCR confirmed that miR-22-3p mimics down-regulated the expression of SIRT1 protein and mRNA(P<0.05),and miR-22-3p inhibitors up-regulated the expression of SIRT1 protein and mRNA(P<0.05).8.Effects of SIRT1 on the biological behavior of ectopic endometrial stromal cellsImmunohistochemistry and western blot showed that the expression of SIRT1 in ectopic endometrium was significantly lower than that in eutopic endometrium and normal endometrium.SIRT1 siRNA significantly increased the proliferation,migration and invasion of ectopic endometrial stromal cells(P<0.05),while SIRT1 overexpression significantly suppressed the proliferation,migration and invasion of ectopic endometrial stromal cells(P<0.05).9.SIRT1 overexpression rescued the effect of miR-22-3p on the biological behavior of ectopic endometrial stromal cellsCompared with transfection with miR-22-3p mimics and miR-22-3p mimics+SIRT1 empty vector,transfection with miR-22-3p mimics+SIRT1 overexpression vector significantly inhibited the proliferation,migration and invasion of ectopic endometrial stromal cells(P<0.05)10.SIRT1 regulated the biological behavior of ectopic endometrial stromal cells by regulating NF-κBImmunohistochemistry and western blot showed that the expression of p-p65 in ectopic endometrium was significantly higher than that in eutopic endometrium and normal endometrium.Western blot showed that SIRT1 overexpression significantly decreased the expression of p-p65,and SIRT1 siRNA significantly increased the expression of p-p65.PDTC,a NF-κB inhibitor,significantly inhibited the proliferation,migration and invasion of ectopic endometrial stromal cells(P<0.05).11.PDTC rescued the effect of miR-22-3p on biological behavior of ectopic endometrial stromal cellsWestern blot showed that miR-22-3p mimics significantly increased the expression of p-p65 and miR-22-3p inhibitors significantly decreased the expression of p-p65.Compared with transfection with miR-22-3p mimics alone,transfection with miR-22-3p mimics and PDTC significantly inhibited the proliferation,migration and invasion of ectopic endometrial stromal cells(P<0.05).Conclusions1.Peritoneal macrophage-derived exosomes from EMs can be absorbed by ectopic endometrial stromal cells,and promoted the proliferation,migration and invasion of ectopic endometrial stromal cells.2.miR-22-3p was highly expressed in peritoneal macrophage-derived exosomes from EMs and can be transported to ectopic endometrial stromal cells through exosomal pathway.3.miR-22-3p can activate NF-κB through regulating SIRT1,which regulated the biological behavior of ectopic endometrial stromal cells.