Exosomal miRNA Profiles Research Articles

Diagnostic Role of Cell-Free miRNAs in Identifying Placenta Accreta Spectrum during First-Trimester Screening.

Placenta accreta spectrum (PAS) is a severe complication of pregnancy associated with excessive invasion of cytotrophoblast cells at the sites of the endometrial-myometrial interface and the myometrium itself in cases of adherent (creta) and invasive (increta and percreta) forms, respectively. This leads to a high risk of massive blood loss, maternal hysterectomy, and preterm birth. Despite advancements in ultrasound protocols and found associations of alpha-fetoprotein, PAPP-A, hCG, PLGF, sFlt-1, IL-8, and IL-33 peripheral blood levels with PAS, there is a high need for an additional non-invasive test to improve the diagnostic accuracy and to select the real PAS from the suspected ones in the first-trimester screening. miRNA signatures of placental tissue, myometrium, and blood plasma from women with PAS in the third trimester of pregnancy, as well as miRNA profiles in exosomes from the blood serum of women in the first trimester with physiologically progressing pregnancy, complicated by PAS or pre-eclampsia, were obtained using deep sequencing. Two logistic regression models were constructed, both featuring statistically significant parameters related to the levels of miR-26a-5p, miR-17-5p, and miR-101-3p, quantified by real-time PCR in native blood serum. These models demonstrated 100% sensitivity in detecting PAS during the first pregnancy screening. These miRNAs were identified as specific markers for PAS, showing significant differences in their blood serum levels during the first trimester in the PAS group compared to those in physiological pregnancies, early- or late-onset pre-eclampsia groups. Furthermore, these miRNAs exhibited differential expression in the PAS placenta and/or myometrium in the third trimester and, according to data from the literature, control angiogenesis. Significant correlations were found between extracellular hsa-miR-101-3p and nuchal translucency thickness, hsa-miR-17-5p and uterine artery pulsatility index, and hsa-miR-26a-5p and hsa-miR-17-5p with PLGF. The developed test system for early non-invasive PAS diagnosis based on the blood serum level of extracellular miR-26a-5p, miR-17-5p, and miR-101-3p can serve as an auxiliary method for first-trimester screening of pregnant women, subject to validation with independent test samples.

Exosome-delivered miR-410-3p reverses epithelial-mesenchymal transition, migration and invasion of trophoblasts in spontaneous abortion.

Current studies have indicated that insufficient trophoblast epithelial-mesenchymal transition (EMT), migration and invasion are crucial for spontaneous abortion (SA) occurrence and development. Exosomal miRNAs play significant roles in embryonic development and cellular communication. Hereon, we explored the roles of serum exosomes derived from SA patients on trophoblast EMT, migration and invasion. Exosomes were isolated from normal control (NC) patients with abortion for unplanned pregnancy and SA patients, then characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting. Exosomal miRNA profiles were identified by miRNA sequencing. The effects of serum exosomes on trophoblast migration and invasion were detected by scratch wound healing and transwell assays, and other potential mechanisms were revealed by quantitative real-time PCR (RT-PCR), western blotting and dual-luciferase reporter assay. Finally, animal experiments were used to explore the effects of exosomal miR-410-3p on embryo absorption in mice. The serum exosomes from SA patients inhibited trophoblast EMT and reduced their migration and invasion ability invitro. The miRNA sequencing showed that miR-410-3p was upregulated in SA serum exosomes. The functional experiments showed that SA serum exosomes restrained trophoblast EMT, migration and invasion by releasing miR-410-3p. Mechanistically, SA serum exosomal miR-410-3p inhibited trophoblast cell EMT, migration and invasion by targeting TNF receptor-associated factor 6 (TRAF6) at the post-transcriptional level. Besides, SA serum exosomal miR-410-3p inhibited the p38 MAPK signalling pathway by targeting TRAF6 in trophoblasts. Moreover, milk exosomes loaded with miR-410-3p mimic reached the maternal-fetal interface and aggravated embryo absorption in female mice. Clinically, miR-410-3p and TRAF6 expression were abnormal and negatively correlated in the placental villi of SA patients. Our findings indicated that exosome-derived miR-410-3p plays an important role between SA serum and trophoblasts in intercellular communication, suggesting a novel mechanism by which serum exosomal miRNA regulates trophoblasts in SA patients.

Exosome-Derived microRNA: Potential Target for Diagnosis and Treatment of Sepsis.

Exosome-derived microRNAs (miRNAs) are emerging as pivotal players in the pathophysiology of sepsis, representing a new frontier in both the diagnosis and treatment of this complex condition. Sepsis, a severe systemic response to infection, involves intricate immune and nonimmune mechanisms, where exosome-mediated communication can significantly influence disease progression and outcomes. During the progress of sepsis, the miRNA profile of exosomes undergoes notable alterations, is reflecting, and may affect the progression of the disease. This review comprehensively explores the biology of exosome-derived miRNAs, which originate from both immune cells (such as macrophages and dendritic cells) and nonimmune cells (such as endothelial and epithelial cells) and play a dynamic role in modulating pathways that affect the course of sepsis, including those related to inflammation, immune response, cell survival, and apoptosis. Taking into account these dynamic changes, we further discuss the potential of exosome-derived miRNAs as biomarkers for the early detection and prognosis of sepsis and advantages over traditional biomarkers due to their stability and specificity. Furthermore, this review evaluates exosome-based therapeutic miRNA delivery systems in sepsis, which may pave the way for targeted modulation of the septic response and personalized treatment options.

MiRNA profiling of granulosa cell-derived exosomes reveals their role in promoting follicle development.

To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exoscould serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis.GC-Exoswere characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.

Plasma-derived exosomal miRNA profiles reveal potential epigenetic pathogenesis of premature ovarian failure.

The role of plasma-derived exosomal miRNA in premature ovarian failure (POF) remains unclear. This study aimed to investigate the epigenetic pathogenesis of POF through exosomal miRNA sequencing. Exosomes were isolated and characterized from six POF patients and four healthy individuals using nanoparticle tracking analysis, transmission electron microscopy and western blot analysis. Exosomal miRNA sequencing was performed to identify differentially expressed miRNAs with |fold change| greater than 1.5 and p value less than 0.05. Bioinformatics analysis in GSE39501 dataset and our sequencing data was conducted to investigate underlying mechanisms of POF. The functional role of hsa-miR-19b-3p was assessed using CCK8, western blot, flow cytometry and fluorescence staining. The regulatory effect of hsa-miR-19b-3p on BMPR2 was investigated through miRNA transfection, qPCR analysis, and luciferase reporter assay. Statistical significance was determined using t-tests and one-way ANOVA (p < 0.05). Exosomal miRNA sequencing revealed 18 dysregulated miRNAs in POF patients compared to healthy controls. Functional enrichment analysis demonstrated their involvement in cell growth, oocyte meiosis and PI3K-Akt signaling pathways. Moreover, the constructed miRNA-mRNA network unveiled potential regulatory mechanisms underlying POF, particularly implicating hsa-miR-19b-3p in the regulation of BMPR2. In vitro assays conducted on KGN cells confirmed that hsa-miR-19b-3p promoted apoptosis, as evidenced by reduced cell viability, decayed mitochondrial membrane potential and increased apoptotic rate, thereby supporting its role in POF. Notably, hsa-miR-19b-3p was found to significantly downregulate BMPR2 expression via targeting its 3'UTR, while co-expression analysis revealed strong associations between BMPR2 and POF-related processes. This study sheds light on the epigenetic pathogenesis of POF by investigating exosomal miRNA profiles. Particularly, hsa-miR-19b-3p emerged as a potential regulator of BMPR2 and demonstrated its functional significance in POF through modulation of apoptosis.

Urinary exosomal miRNA signature of IgA nephropathy: a case–control study

IgA nephropathy is the most common primary glomerulonephritis worldwide and can progress to end-stage kidney disease (ESKD). The current “gold standard” for diagnosis is kidney biopsy, which is invasive and associated with morbidity. miRNAs are small, non-coding endogenous RNA that may serve as non-invasive biomarkers, and that are found in urinary exosomes. Thus far, there is a paucity of studies of the miRNA profile for the diagnosis of IgA nephropathy. Hence, we aimed to study the urinary exosomal miRNA signature of Indian patients with IgA nephropathy. Fifty biopsy-proven IgA nephropathy patients, 50 healthy controls and 25 patients with ESKD (IgA nephropathy) were recruited over 2 years (2020–2022). Urinary exosomes were isolated from which miRNA was extracted . Analysis of urinary exosomal miRNA was done using the digital multiplexed nCounter® human v3 miRNA Expression Assay which contains 799 unique miRNA barcodes. Candidate miRNAs were identified using Lasso regression and consensus clustering. The mean age of IgA nephropathy patients was 36.32 ± 3.067 years, mean creatinine was 2.26 ± 0.318 mg/dl and mean proteinuria was 2.69 ± 0.64 g/day. Compared to healthy controls, the majority (N = 150) of miRNAs were significantly downregulated. Five candidate miRNAs (hsa.miR.146b.3p, hsa.miR.599, hsa.miR.4532, hsa.miR.664b.5p and hsa.miR.221.5p) were able to differentiate between IgA nephropathy cases and controls (AUC > 0.90); the presence of all 5 was associated with 100% specificity and sensitivity for diagnosing IgA nephropathy cases. This study of Indian patients identified that there was a significant difference in the urinary exosomal miRNA profile between IgA nephropathy cases and healthy controls, suggesting that miRNAs may be valuable in the non-invasive diagnosis of IgA nephropathy.

Motility-related microRNAs identified in pig seminal plasma exosomes by high-throughput small RNA sequencing

Boar fertility is a key determinant of the production efficiency of the whole pig breeding industry and boar sperm motility is the seminal parameter with the greatest impact on the fecundity of a sow. Exosomes are small, extracellular vesicles found in many body fluids. Seminal plasma exosomes, which are secreted by the epididymis, prostate, seminal vesicles, and testes, contain a large number of miRNAs, the types and levels of which can reflect the physiological state of source cells. It has been shown that the expression profile of seminal plasma exosomal miRNA differs between low-motility semen and normal semen. The aim of this study was to investigate the relationship between semen motility and exosomal miRNA profiles to obtain information that would allow to predict boar fertility, as well as contribute to the understanding of the mechanisms by which exosomal miRNAs regulate semen motility. Three high-motility (semen motility >90 %) and three low-motility (semen motility <80 %) semen samples were collected from Landrace and Yorkshire boars, respectively, and seminal plasma exosomes were extracted by ultracentrifugation. Exosome characterization was performed using transmission electron microscopy, NTA, and Western blot. The expression profiles of exosomal miRNAs associated with semen motility in the two boar breeds were subsequently determined by small RNA sequencing. The results showed that 297 known miRNAs and 295 novel RNAs were co-expressed in the four groups. Notably, six miRNAs (ssc-miR-122–5p, ssc-miR-486, ssc-miR-451, ssc-miR-345–3p, ssc-miR-362, and ssc-miR-500–5p) were found to be differentially expressed in both boar breeds. Enrichment analysis of the target genes of the differentially expressed miRNAs showed that they were mainly involved in biological processes such as regulation of transcription from RNA polymerase II promoter, regulation of gene expression, and intracellular signal transduction and signaling pathways such as the PI3K-Akt, MAPK, and Ras signaling pathways. The six differentially expressed miRNAs identified in this study have significant potential as noninvasive markers of boar semen motility. Meanwhile, the results of the enrichment analysis provide novel insights into the mechanisms underlying the regulation of semen motility.

Investigation of Urinary Exosomal miRNA and lncRNA Expression Profiles and ceRNA Network in Diabetic Kidney Disease

Investigation of Urinary Exosomal miRNA and lncRNA Expression Profiles and ceRNA Network in Diabetic Kidney Disease

Small RNA sequencing of exosomal microRNAs reveals differential expression of microRNAs in preeclampsia.

Preeclampsia (PE) is one of the most common hypertensive disorders of pregnancy. It is a dangerous condition with a high mortality rate in mothers and fetuses and is associated with a lack of early diagnosis and effective treatment. While the etiology of the disease is complex and obscure, it is now clear that the placenta is central to disease progression. Exosomal microRNAs (miRNAs) are possible mediators that regulate placenta-related physiological and pathological processes. Placental mesenchymal stem cells have considerable potential to help us understand the pathogenesis and treatment of pregnancy-related diseases. Here, we investigate the exosomal miRNA profiles of human placenta-derived mesenchymal stem cells between healthy pregnant women and those with PE. We performed small RNA sequencing to obtain miRNA profiles, and conducted enrichment analysis of the miRNA target genes to identify differentially expressed miRNAs associated with PE. Overall, we detected 1795 miRNAs; among them, 206 were differentially expressed in women with PE, including 35 upregulated and 171 downregulated miRNAs, when compared with healthy pregnant women. Moreover, we identified possible functions and pathways associated with PE, including angiogenesis, cell proliferation, migration and invasion, and the coagulation-fibrinolysis balance. Eventually, we proposed hsa-miR-675-5p, hsa-miR-3614-5p, and hsa-miR-615-5p as potential regulators of the pathogenesis of PE, and constructed a miRNA-target gene network. Our study identifies possible candidate biomarkers for the diagnosis of PE, and introduces a new direction for further understanding the pathogenesis of PE.

Simultaneous subset tracing and miRNA profiling of tumor-derived exosomes via dual-surface-protein orthogonal barcoding.

The clinical potential of miRNA-based liquid biopsy has been largely limited by the heterogeneous sources in plasma and tedious assay processes. Here, we develop a precise and robust one-pot assay called dual-surface-protein-guided orthogonal recognition of tumor-derived exosomes and in situ profiling of microRNAs (SORTER) to detect tumor-derived exosomal miRNAs and enhance the diagnostic accuracy of prostate cancer (PCa). The SORTER uses two allosteric aptamers against exosomal marker CD63 and tumor marker EpCAM to create an orthogonal labeling barcode and achieve selective sorting of tumor-specific exosome subtypes. Furthermore, the labeled barcode on tumor-derived exosomes initiated targeted membrane fusion with liposome probes to import miRNA detection reagents, enabling in situ sensitive profiling of tumor-derived exosomal miRNAs. With a signature of six miRNAs, SORTER differentiated PCa and benign prostatic hyperplasia with an accuracy of 100%. Notably, the diagnostic accuracy reached 90.6% in the classification of metastatic and nonmetastatic PCa. We envision that the SORTER will promote the clinical adaptability of miRNA-based liquid biopsy.

Plasma Exosomal miRNAs as Response Biomarkers of Immunotherapy in Extensive-Stage Small-Cell Lung Cancer

Immunotherapy combined with chemotherapy has become the first-line standard treatment for patients with extensive-stage small-cell lung cancer (ES-SCLC). The reliable biomarkers stratifying true responders of immunotherapy effectively are unknown, and it is urgent to identify novel biomarkers in clinical. Exosomal miRNAs are considered to play a role in intercellular communication among immune cells and interaction between immune cells and tumor cells. The purpose of this study was to explore the possibility of using plasma-derived exosomal miRNAs as potential biomarkers for identifying responses to immunotherapy in ES-SCLC. From March 2020 to September 2021, 24 patients with ES-SCLC who received PD-L1 inhibitors were enrolled. Tumor assessments were conducted after every two treatment cycles according to RECIST 1.1. Plasma samples of these patients were collected before administering PD-L1 inhibitors as the baseline, and after every four cycles until the occurrence of disease progression. Plasma exosomes were isolated by ultracentrifugation, then total RNA was extracted. The miRNA profile was analyzed with small RNA next-generation sequencing followed by differential expression analysis. Of the 24 patients, 15 underwent immunotherapy maintenance after completing four cycles of PD-L1 inhibitor plus chemotherapy. In order to identify biomarkers for a better response to immunotherapy, all five responders (patients achieving PR) and four non-responders (patients achieving PD) at tumor assessment within eight cycles of the maintenance phase were included for differential expression analysis. Surprisingly, hsa-miR-320c, hsa-miR-320d, and hsa-miR-320e showed a trend of increased expression in the non-responders compared with the responders at baseline and were significantly downregulated in the post-treatment plasma exosomes compared with pre-treatment samples of the responders. Exosomal miRNA profiles are discordant between responders and non-responders of anti-PD-L1 treatment. Hsa-miR-320c, hsa-miR-320d, and hsa-miR-320e were identified as potential biomarkers for predicting the efficacy of immunotherapy in patients with ES-SCLC.

Exosomal miRNA analysis provides new insights into exposure to nanoplastics and okadaic acid

As an emerging environmental pollutant, nanoplastics (NPs) have attracted wide attention in terms of their impact on the ecological environment and human health. Currently, researches on the cytotoxicity of NPs mainly focus on oxidative stress, damage to the cell membrane and organelles, induction of immune response and genotoxicity. Okadaic acid (OA) is the main component of diarrheal shellfish toxin. Based on the previous combined toxicity exploration of polystyrene (PS) NPs and (OA) to human gastric adenocarcinoma (AGS) cells, cell-derived exosomes were extracted and exosomal miRNA profiles were analyzed for the first time in this study. The results showed that the composition of miRNAs varied after the exposure of NPs and OA. Specifically, the expression of miR-1-3p in both PS-Exo and PS-OA-Exo was significantly reduced. And the expression of miR-1248 was upregulated most significantly by comparing the DE miRNAs between PS-Exo and PS-OA-Exo. MiR-1-3p and miR-1248 may be the key genes for the combined toxicity of NPs and OA. After analysis, we found that both the decreased expression of miR-1-3p and the increased expression of miR-1248 can increase the expression of FN1 and affect DNA replication, which was surprisingly consistent with the results of our previous cytotoxicity studies. Since exosomal miRNAs are selectively encapsulated by donor cell, we speculate that the changes of exosomal miRNAs may due to the synchronous changes of intracellular environment and the downregulation of intracellular FN1 may be attributed to decreased expression of miR-1-3p and increased expression of miR-1248 in donor cells. Accordingly, we come to the conclusion that the changes of miRNAs in the exosomes derived from AGS cells after environmental stimulation could reflect the biological effects of donor cells.

Exosomes and their miRNA/protein profile in keratoconus-derived corneal stromal cells

Keratoconus (KC) is a corneal thinning disorder and a leading cause of corneal transplantation worldwide. Exosomes are small, secreted extracellular vesicles (30–150 nm) that mediate cellular communication via their protein, lipid, and nucleic acid content. We aimed to characterize the exosomes secreted by primary corneal fibroblasts from subjects with or without KC. Using human keratoconus stromal fibroblast cells (HKC, n = 4) and healthy stromal fibroblasts (HCF, n = 4), we collected and isolated exosomes using serial ultracentrifugation. Using nanoparticle tracking analysis (NTA) with ZetaView®, we compared the size and concentration of isolated exosomes. Different exosomal markers were identified and quantified using a transmission electron microscope (TEM) (CD81) and Western blot (CD9 and CD63). Exosomal miRNA profiles were determined by qRT-PCR using Exiqon Human panel I miRNA assays of 368 pre-selected miRNAs. Proteomic profiles were determined using a label-free spectral counting method with mass spectrometry. Differential expression analysis for miRNAs and proteins was done using student's t-test with a significance cutoff of p-value ≤0.05. We successfully characterized exosomes isolated from HCFs using several complementary techniques. We found no significant differences in the size, quantity, or morphology between exosomes secreted by HCFs with or without KC. Expression of CD81 was confirmed by immuno-EM, and expression of CD63 and CD9 with western blots in all exosome samples. We detected the expression of 72–144 miRNAs (threshold cycle Ct < 36) in all exosome samples. In HKC-derived exosome samples, miR-328-3p, miR-532-5p, miR-345-5p, and miR-424-5p showed unique expression, while let-7c-5p and miR-665 have increased expression. Protein profiling identified 157 proteins in at least half of the exosome samples, with 38 known exosomal proteins. We identified 12 up- and 2 down-regulated proteins in HKC-derived exosomes. The proteins are involved in membrane-bounded vesicles, cytoskeletal, calcium binding, and nucleotide binding. These proteins are predicted to be regulated by NRF2, miR-205, and TGF-β1, which are involved in KC pathogenesis. We successfully characterized the HKC-derived exosomes and profiled their miRNA and protein contents, suggesting their potential role in KC development. Further studies are necessary to determine if and how these exosomes with differential protein/miRNA profiles contribute to the pathogenesis of KC.

Brain-Derived Exosomal miRNA Profiles upon Experimental SAE Rats and Their Comparison with Peripheral Exosomes.

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction secondary to body infection without overt central nervous system infection. Dysregulation of miRNA expression in the transcriptome can spread through RNA transfer in exosomes, providing an early signal of impending neuropathological changes in the brain. Here, we comprehensively analyzed brain-derived exosomal miRNA profiles in SAE rats (n = 3) and controls (n = 3). We further verified the differential expression and correlation of brain tissue, cerebrospinal fluid, and plasma exosomal miRNAs in SAE rats. High-throughput sequencing of brain-derived exosomal miRNAs identified 101 differentially expressed miRNAs, of which 16 were downregulated and 85 were upregulated. Four exosomal miRNAs (miR-127-3p, miR-423-3p, mR-378b, and miR-106-3p) were differentially expressed and correlated in the brain tissue, cerebrospinal fluid, and plasma, revealing the potential use of miRNAs as SAE liquid brain biopsies. Understanding exosomal miRNA profiles in SAE brain tissue and exploring the correlation with peripheral exosomal miRNA can contribute to a comprehensive understanding of miRNA changes in the SAE pathological process and provide the possibility of establishing early diagnostic assays.

Proteomic and genomic profiling of plasma exosomes from patients with ankylosing spondylitis

IntroductionRecent advances in understanding the biology of ankylosing spondylitis (AS) using innovative genomic and proteomic approaches offer the opportunity to address current challenges in AS diagnosis and management. Altered expression...

Identification and evaluation of circulating exosomal miRNAs for the diagnosis of postmenopausal osteoporosis

BackgroundPostmenopausal osteoporosis (PMOP) is a common condition that leads to a loss of bone density and an increased risk of fractures in women. Recent evidence suggests that exosomal miRNAs are involved in regulating bone development and osteogenesis. However, exosomal miRNAs as biomarkers for PMOP diagnosis have not been systematically evaluated. In this study, we aim to identify PMOP-associated circulating exosomal miRNAs and evaluate their diagnostic performance.MethodsWe performed next-generation sequencing and bioinformatics analysis of plasma exosomal miRNAs from 12 PMOP patients and 12 non-osteoporosis controls to identify PMOP-associated exosomal miRNAs, and then validated them in an independent natural community cohort with 26 PMOP patients and 21 non-osteoporosis controls. Exosomes were isolated with the size exclusion chromatography method from the plasma of elder postmenopausal women. The plasma exosomal miRNA profiles were characterized in PMOP paired with controls with next-generation sequencing. Potential plasma exosomal miRNAs were validated by qRT-PCR in the validation cohort, and their performance in diagnosing PMOP was systematically evaluated with the receiver operating characteristic curve.ResultsTwenty-seven miRNAs were identified as differentially expressed in PMOP versus controls in sequencing data, of which six exosomal miRNAs (miR-196-5p, miR-224-5p, miR320d, miR-34a-5p, miR-9-5p, and miR-98-5p) were confirmed to be differentially expressed in PMOP patients by qRT-PCR in the validation cohort. The three miRNAs combination (miR-34a-5p + miR-9-5p + miR-98-5p) demonstrated the best diagnostic performance, with an AUC = 0.734. In addition, the number of pregnancies was found to be an independent risk factor that can improve the performance of exosomal miRNAs in diagnosing PMOP.ConclusionsThese results suggested that the plasma exosomal miRNAs had the potential to serve as noninvasive diagnostic biomarkers for PMOP.

CrebH protects against liver injury associated with colonic inflammation via modulation of exosomal miRNA

BackgroundHepatic liver disease, including primary sclerosing cholangitis (PSC), is a serious extraintestinal manifestations of colonic inflammation. Cyclic adenosine monophosphate (cAMP)-responsive element-binding protein H (CrebH) is a transcription factor expressed mostly in the liver and small intestine. However, CrebH’s roles in the gut–liver axis remain unknown.MethodsInflammatory bowel disease (IBD) and PSC disease models were established in wild-type and CrebH−/− mice treated with dextran sulfate sodium, dinitrobenzene sulfonic acid, and diethoxycarbonyl dihydrocollidine diet, respectively. RNA sequencing were conducted to investigate differential gene expression. Exosomes were isolated from plasma and culture media. miRNA expression profiling was performed using the NanoString nCounter Mouse miRNA Panel. Effects of miR-29a-3p on adhesion molecule expression were investigated in bEnd.3 brain endothelial cells.ResultsCrebH−/− mice exhibited accelerated liver injury without substantial differences in the gut after administration of dextran sulfate sodium (DSS), and had similar features to PSC, including enlarged bile ducts, enhanced inflammation, and aberrant MAdCAM-1 expression. Furthermore, RNA-sequencing analysis showed that differentially expressed genes in the liver of CrebH−/− mice after DSS overlapped significantly with genes changed in PSC-liver. Analysis of plasma exosome miRNA isolated from WT and CrebH−/− mice indicates that CrebH can contribute to the exosomal miRNA profile. We also identified miR-29a-3p as an effective mediator for MAdCAM-1 expression. Administration of plasma exosome from CrebH−/− mice led to prominent inflammatory signals in the liver of WT mice with inflammatory bowel disease (IBD).ConclusionsCrebH deficiency led to increased susceptibility to IBD-induced liver diseases via enhanced expression of adhesion molecules and concomitant infiltration of T lymphocytes. Exosomes can contribute to the progression of IBD-induced liver injury in CrebH−/− mice. These study provide novel insights into the role of CrebH in IBD-induced liver injury.

Milk Exosomes from Gestational Diabetes Mellitus (GDM) and Healthy Parturient Exhibit Differential miRNAs Profiles and Distinct Regulatory Bioactivity on Hepatocyte Proliferation.

Exosomes, a novel type of bioactive component in human milk (HM), affect infant development, growth, and health. Recent studies indicate that HM exosomes and miRNAs relate to gestational diabetes mellitus (GDM). However, the miRNAs profiles and functionalities of HM exosomes from GDM parturient remain unclear. This study aims to compare the differential miRNAs in HM exosomes from GDM and healthy parturient, and investigate the HM exosomes bioactivities in regulating hepatocyte proliferation and insulin sensitivity. This study extracted HM exosomes from GDM (GDM-EXO) and healthy (NOR-EXO) parturient by ultracentrifugation, high-throughput sequenced and compared the exosomal miRNAs profiles, and explored the regulatory bioactivities on hepatocyte proliferation in HepG2 cells and Balb/c mice. As compared to NOR-EXO, GDM-EXO has similar morphology, size, concentration, and exosome-specific markers (CD9 and TSG101) expression. GDM-EXO and NOR-EXO specifically harbor 1299 and 8 miRNAs, respectively. Moreover, GDM-EXO had 176 upregulated and 47 downregulated miRNAs compared with NOR-EXO. Both GDM-EXO and NOR-EXO were absorbed in cultured HepG2 hepatocytes and mice liver. GDM-EXO inhibited hepatocytes proliferation by downregulating mammalian target of rapamycin (mTOR) possibly via exosomal miR-101-3p delivery. HM exosomes from GDM and healthy parturient exhibit differential miRNAs profiles and distinct regulatory bioactivity on hepatocyte proliferation.

22-LB: MiRNA Profiling of Exosomes Derived from Perivascular Adipocyte Tissue in Type 2 Diabetic Mice

Background: Exosomes (EXs), a major type of extracellular vesicles, are emerging as a novel type of intercellular communicators. MicroRNAs (miRs) are one of the major executors of EXs. The previous study shows that lean and obesity adipocyte tissue EXs could elicit different effects in response to insulin sensitivity, the underlying mechanism is related to their carried miRs. Given EX cargoes and function vary on their cellular status, we speculate the miR profiling of EXs derived from the perivascular adipose tissue (PVAT) is altered in diabetic conditions. Methods: EXs were isolated from the PVAT tissue of type 2 diabetic db/db and control db/c mice. EX size and concentration were analyzed by nanoparticle tracking analysis. The miR profiling of PVAT-EXs was analyzed by mmu-miR miRome profiling kit. The miRs of interest were further validated by qRT-PCR. Results: 1) The PVAT-EXs level was higher in diabetic db/db mice than that in non-diabetic db/c mice; 2) In db/db mice, exosomal miRs profile was changed. The exosomal miRs, miR-223, miR-181a, miR-146a, miR-34a, and miR-210, were either down-regulated or up-regulated &amp;gt; 3-fold when comparing that in db/c mice. Among these candidate miRs, miR-181a, miR-223, miR-210, and miR-146a might be involved in modulating adipose tissue inflammation and/or vascular function. MiR-34a could participate in white adipose tissue browning, macrophage polarization, and glucose tolerance. Conclusion: Our data have demonstrated that the miR profiling of PVAT-derived EXs is changed in type 2 diabetic conditions, which suggests the functions of these EXs might be altered in diabetic conditions. Disclosure S. Chen: None. V. Polaki: None. J. Bihl: None. J. Wang: None.

Differentially expressed miRNA profiles of serum-derived exosomes in patients with sudden sensorineural hearing loss.

This study aimed to compare the expressed microRNA (miRNA) profiles of serum-derived exosomes of patients with sudden sensorineural hearing loss (SSNHL) and normal hearing controls to identify exosomal miRNAs that may be associated with SSNHL or serve as biomarkers for SSNHL. Peripheral venous blood of patients with SSNHL and healthy controls was collected to isolate exosomes. Nanoparticle tracking analysis, transmission electron microscopy, and Western blotting were used to identify the isolated exosomes, after which total RNA was extracted and used for miRNA transcriptome sequencing. Differentially expressed miRNAs (DE-miRNAs) were identified based on the thresholds of P < 0.05 and |log2fold change| > 1 and subjected to functional analyses. Finally, four exosomal DE-miRNAs, including PC-5p-38556_39, PC-5p-29163_54, PC-5p-31742_49, and hsa-miR-93-3p_R+1, were chosen for validation using quantitative real-time polymerase chain reaction (RT-qPCR). Exosomes were isolated from serum and identified based on particle size, morphological examination, and expression of exosome-marker proteins. A total of 18 exosomal DE-miRNAs, including three upregulated and 15 downregulated miRNAs, were found in SSNHL cases. Gene ontology (GO) functional annotation analysis revealed that target genes in the top 20 terms were mainly related to "protein binding," "metal ion binding," "ATP binding," and "intracellular signal transduction." Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these target genes were functionally enriched in the "Ras," "Hippo," "cGMP-PKG," and "AMPK signaling pathways." The expression levels of PC-5p-38556_39 and PC-5p-29163_54 were significantly downregulated and that of miR-93-3p_R+1 was highly upregulated in SSNHL. Consequently, the consistency rate between sequencing and RT-qPCR was 75% and sequencing results were highly reliable. This study identified 18 exosomal DE-miRNAs, including PC-5p-38556_39, PC-5p-29163_54, and miR-93-3p, which may be closely related to SSNHL pathogenesis or serve as biomarkers for SSNHL.