Modulation Mechanism of Wuniuzao Dark Tea Polysaccharide on Lipid Metabolism in Hyperlipidemic Mice Induced by High-Fat Diet.

The aim of this study was to investigate the functional mechanism of Wuniuzao dark tea polysaccharide (WDTP) that protect against hyperlipidemia in mice induced by high-fat diet. WDTP was extracted by hot water, isolated and purified by DEAE-52 chromatography and characterized by high-performance liquid chromatograph (HPLC), Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscope (SEM). Different doses (200 or 800 mg/kg/day) of WDTP were orally administered to mice induced by high-fat diet to evaluate the mechanism of WDTP regulating lipid metabolism. And these results showed that average molecular weight of WDTP was nearly 63,869 Da. And WDTP intervention significantly reduced body weight, lipid accumulation, and modulated blood lipid levels. The mechanism of WDTP ameliorating lipid metabolism was associated with regulating the expression of lipid metabolism-related genes and serum exosomes miR-19b-3p, and modulating the community structure of gut microbiota in mice.

Serum exosomal hsa-circ-0004771 modulates the resistance of colorectal cancer to 5-fluorouracil via regulating miR-653/ZEB2 signaling pathway.

BACKGROUND: Drug resistance is a major obstacle causing chemotherapy failure, and enabling cancer progression. Exosome excreted by cancer cells is participated in cancer progression and chemoresistance, and can be used as an prognostic biomarker. Previous studies have revealed that serum exosomal hsa-circ-0004771 is over-expressed in colorectal cancer (CRC) sufferers and suggested it as a predictive biomarker for early diagnosis and prognosis of CRC. This work will to investigate the role and mechanism of serum exosomal hsa-circ-0004771 in mediating resistance to 5-fluorouracil (5-FU) in CRC. METHODS: Serum and tissue samples were collected from 60 patients with CRC/ benign intestinal disease, and 60 healthy control. Exosomes were isolated and identified from serum samples and cell cultured media with TEM, WB, NTA, and flow cytometry. qRT-PCR and WB were performed to evaluate mRNA expressions of exosomal has-circ-0004771 and miR-653, and ZEB2 protein expression, respectively. Cell proliferation, migration, invasion, and apoptosis abilities were assessed with BrdU and colony formation assay, wound-healing assay, and flow cytometry, respectively. RESULTS: Exosomal hsa-circ-0004771 was over-expressed in CRC serum and cell cultured media, while miR-653 was lower-expressed in CRC tissues and cells. Negative correlations existed between exosomal hsa-circ-0004771 in the patients' serum/cell culture media and miR-653 in CRC tissues/cells, and between miR-653 and ZEB2 in CRC cells. Exosomal hsa-circ-0004771 in CRC cell cultured media was positively related to ZEB2 in CRC cells. MiR-653 was associated with poor prognosis of CRC patients, and its upregulation restrained CRC cell proliferation, migration and invasion, and stimulated apoptosis. Exosomal hsa-circ-0004771 was higher-expressed in 5-FU-resistant CRC serum and cell cultured media, miR-653 was downregulated and ZEB2 was overexpressed in 5-FU-resistant CRC cells. In vitro, exosomal hsa-circ-0004771 in cell cultured media may be involved in 5-FU-resistance by modulating miR-653/ZEB2 pathway. CONCLUSIONS: miR-653 plays as a tumour suppressor in CRC progression, and serum exosomal hsa-circ-0004771 may be a predictive biomarker for 5-FU-resistance in CRC patients, potentially through miR-653/ZEB2 axis.

The Comparison of Serum Exosome Protein Profile in Diagnosis of NSCLC Patients.

A thorough study of the exosomal proteomic cargo may enable the identification of proteins that play an important role in cancer development. The aim of this study was to compare the protein profiles of the serum exosomes derived from non-small lung cancer (NSCLC) patients and healthy volunteers (control) using the high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method to identify potentially new diagnostic and/or prognostic protein biomarkers. Proteins exclusively identified in NSCLC and control groups were analyzed using several bioinformatic tools and platforms (FunRich, Vesiclepedia, STRING, and TIMER2.0) to find key protein hubs involved in NSCLC progression and the acquisition of metastatic potential. This analysis revealed 150 NSCLC proteins, which are significantly involved in osmoregulation, cell-cell adhesion, cell motility, and differentiation. Among them, 3 proteins: Interleukin-34 (IL-34), HLA class II histocompatibility antigen, DM alpha chain (HLA-DMA), and HLA class II histocompatibility antigen, DO beta chain (HLA-DOB) were shown to be significantly involved in the cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) infiltration processes. Additionally, detected proteins were analyzed according to the presence of lymph node metastasis, showing that differences in frequency of detection of protein FAM166B, killer cell immunoglobulin-like receptor 2DL1, and olfactory receptor 52R1 correlate with the N feature according to the TNM Classification of Malignant Tumors. These results prove their involvement in NSCLC lymph node spread and metastasis. However, this study requires further investigation.

Serum exosomal m6A demethylase FTO promotes gefitinib resistance in non-small cell lung cancer by up-regulating FLRT3, PTGIS and SIRPα expression.

This study investigates the molecular mechanism of FTO m6A demethylase in non-small cell lung cancer (NSCLC) and gefitinib resistance using GEO and TCGA databases. Differentially expressed genes (DEGs) were screened from RNA-seq data sets of serum exosomes of gefitinib-resistant NSCLC patients in the GEO database and the NSCLC data set in the GEPIA2 database. From this analysis, FTO m6A demethylase was found to be significantly upregulated in the serum exosomes of gefitinib-resistant NSCLC patients. To identify downstream genes affected by FTO m6A demethylase, weighted correlation network analysis and differential expression analysis were performed, resulting in the identification of three key downstream genes (FLRT3, PTGIS, and SIRPA). Using these genes, the authors constructed a prognostic risk assessment model. Patients with high-risk scores exhibited a significantly worse prognosis. The model could predict the prognosis of NSCLC with high accuracy measured by AUC values of 0.588, 0.608, and 0.603 at 1, 3, and 5 years respectively. Furthermore, m6A sites were found in FLRT3, PTGIS, and SIRPA genes, and FTO was significantly positively correlated with the expression of these downstream genes. Overall, FTO m6A demethylase promotes gefitinib resistance in NSCLC patients by upregulating downstream FLRT3, PTGIS, and SIRPA expression, with these three downstream genes serving as strong prognostic indicators.

Systematic Identification and Comparison of the Expressed Profiles of Exosomal MiRNAs in Pigs Infected with NADC30-like PRRSV Strain.

Exosomes are biological vesicles secreted and released by cells that act as mediators of intercellular communication and play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry and can cause reproductive disorders in sows, respiratory diseases in pigs, reduced growth performance, and other diseases leading to pig mortality. In this study, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs and isolate serum exosomes. Based on high-throughput sequencing technology, 305 miRNAs were identified in serum exosomes before and after infection, among which 33 miRNAs were significantly differentially expressed between groups (13 relatively upregulated and 20 relatively downregulated). Sequence conservation analysis of the CHsx1401 genome identified 8 conserved regions, of which a total of 16 differentially expressed (DE) miRNAs were predicted to bind to the conserved region closest to the 3' UTR of the CHsx1401 genome, including 5 DE miRNAs capable of binding to the CHsx1401 3' UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, ssc-miR-6529). Further analysis revealed that the target genes of differentially expressed miRNAs were widely involved in exosomal function-related and innate immunity-related signaling pathways, and 18 DE miRNAs (ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc.) associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes.

Sex-Inclined Piwi-Interacting RNAs in Serum Exosomes for Sex Determination in the Greater Amberjack (Seriola dumerili).

The greater amberjack (Seriola dumerili) is a gonochoristic fish with no sexual dimorphism in appearance, making sex identification difficult. Piwi-interacting RNAs (piRNAs) function in transposon silencing and gametogenesis and are involved in various physiological processes, including sex development and differentiation. Exosomal piRNAs can be indicators for the determination of sex and physiological status. In this study, four piRNAs were differentially expressed in both serum exosomes and gonads between male and female greater amberjack. Three piRNAs (piR-dre-32793, piR-dre-5797, and piR-dre-73318) were significantly up-regulated and piR-dre-332 was significantly down-regulated in serum exosomes and gonads of male fish, compared to female fish, consistent with the serum exosomal results. According to the relative expression of four marker piRNAs derived from the serum exosomes of greater amberjack, the highest relative expression of piR-dre-32793, piR-dre-5797, and piR-dre-73318 in seven female fish and that of piR-dre-332 in seven male fish can be used as the standard for sex determination. The method of sex identification can ascertain the sex of greater amberjack by blood collection from the living body, without sacrificing fish. The four piRNAs did not show sex-inclined expression in the hypothalamus, pituitary, heart, liver, intestine, and muscle tissue. A piRNA-target interaction network involving 32 piRNA-mRNA pairs was generated. Sex-related target genes were enriched in sex-related pathways, including oocyte meiosis, transforming growth factor-beta signaling pathway, progesterone-mediated oocyte maturation, and gonadotropin releasing hormone signaling pathway. These results provide a basis for sex determination in greater amberjack and improve our understanding of the mechanisms underlying sex development and differentiation in the species.

Stability of exosomes in the postmortem serum and preliminary study on exosomal miRNA expression profiling in serum from myocardial infarction cadavers.

Exosome-encapsulated miRNAs could potentially be sensitive biomarkers of human diseases. Since a lipid bilayer membrane surrounds exosomes, the exosomal miRNA may stably exist in body fluids with diseases as well as biological fluids. Therefore, exosomal miRNA may be helpful for autopsy diagnosis. Assuming cadaver blood would be most useful, we initially examined serum exosome stability with regard to storage temperatures and periods. Characteristic analyses of the exosome revealed that exosomes and the content, miRNA, were stably preserved until at least three days when stored at below 20 °C. Subsequently, exosomal miRNA expression profiling was performed on the serum of acute myocardial infarction (AMI, 4 cases) autopsy bodies and on hemorrhagic shock bodies used as the control (CT, 3 cases). Results showed that significant twofold up- and downregulations of expression of 18 and 16 miRNAs were detectable in AMI as compared to the CT, respectively. miR-126-3p, which has been reported to be increased in serum of AMI patients and a mouse model, was one of the significantly upregulated miRNAs. Furthermore, dysregulation of exosomal miRNAs, such as miR-145-5p, miR-143-3p, and miR-222-3p, which are involved in cardioprotection, may be associated with AMI pathogenesis. These findings provide a novel perspective on the potential role of exosomal miRNA in determining the cause of death.

Serum-derived exosomes induce proinflammatory cytokines production in Cynoglossus semilaevis via miR-133-3p.

Exosomes are small membrane-enclosed vesicles secreted by various types of cells. In mammals, a wide range of physiological and pathological functions have been confirmed and attributed to EVs carrying a variety of molecular cargoes, including miRNAs. However, studies on the biological functions and related molecular mechanisms of serum exosomes isolated from teleost fish are limited. Indeed, the molecular mechanisms underlying the effects of serum exosomes on immune responses and inflammatory processes are unknown. Chinese tongue sole (Cynoglossus semilaevis) is an economically important species used widely in industrial aquaculture. Vibrio harveyi, a common bacterial pathogen that infects C. semilaevis and some other fish, causes excessive inflammatory reactions, which are characterized by skin ulceration. Here, we isolated serum-derived exosomes from C. semilaevis and investigated their effects on inflammatory processes following V. harveyi infection. We found that compared with uninfected fish, exosome abundance in infected fish blood increased with bacterial infection time, while expression of TNF-α increased, and that of IL-10 decreased, significantly. Moreover, artificial infection studies demonstrated that injection of serum exosomes isolated from infected fish increased expression of TNF-α, IL-6, and IL-8, which is consistent with the increase in proinflammatory cytokines induced by V. harveyi infection. To further investigate the mechanisms by which exosomes increase proinflammatory cytokine production, we performed miRNA expression profiling and found that 26 differentially expressed miRNAs were associated with bacterial infection and immune responses; of these, miR-133-3p was considerably more abundant in serum exosomes from infected fish. Bioinformatics analysis suggested that miR-133-3p inhibits NF-κB signaling pathways by targeting PP2A and affecting cytokine release. We also found that miR-133-3p increased expression of TNF-α, IL-6, and IL-8 in fish blood and kidney, whereas an miR-133-3p inhibitor showed the opposite results. Thus, the data suggest that serum exosomes participate in innate immunity in teleost fish by promoting inflammatory responses to bacterial infection. Exosome-mediated transfer of miR-133-3p increases expression of proinflammatory cytokines in C. semilaevis, resulting in excessive inflammatory responses during V. harveyi infection. These data may lead to development of methods and strategies that control skin ulceration in Chinese tongue sole.

[Proteomics of serum exosomes in children in the acute stage of Kawasaki disease: a prospective study]. / æ€¥æ€§æœŸå·å´Žç— æ‚£å„¿è¡€æ¸ å¤–æ³Œä½“è›‹ç™½è´¨ç»„å­¦çš„å‰çž»æ€§ç ”ç©¶.

OBJECTIVES: To study the biological processes and functions of serum exosomes in children in the acute stage of Kawasaki disease (KD), so as to provide new biomarkers for the early diagnosis of KD. METHODS: In this prospective study, 13 children with KD who were treated in Children's Hospital of Soochow University from June 2019 to August 2020 were enrolled as the KD group, and 13 children who were hospitalized due to bacterial infection during the same period were enrolled as the control group. Whole blood was collected on the next morning after admission, serum samples were obtained by centrifugation, and exosomes were extracted through ultracentrifugation. Serum exosomes were analyzed by label-free quantitative proteomics, and differentially expressed proteins (DEPs) were screened out for functional enrichment analysis. A protein-protein interaction (PPI) network was plotted, and unique proteins were validated by targeted proteomics. RESULTS: A total of 131 DEPs were screened out for the two groups, among which 27 proteins were detected in both groups. There were 48 unique DEPs in the KD group, among which 23 were upregulated and 25 were downregulated, and these proteins acted on "complement and coagulation cascades" and "the MAPK signaling pathway". Validation by targeted proteomics showed that FGG, SERPING1, C1R, C1QA, IGHG4, and C1QC proteins were quantifiable in the KD group. A total of 29 proteins were only expressed in the control group, among which 12 were upregulated and 17 were downregulated. Four proteins were quantifiable based on targeted proteomics, i.e., VWF, ECM1, F13A1, and TTR. A PPI network was plotted for each group. In the KD group, FGG and C1QC had close interaction with other proteins, while in the control group, VWF had close interaction with other proteins. CONCLUSIONS: The serum exosomes FGG and C1QC in children in the acute stage of KD are expected to become the biomarkers for the early diagnosis of KD. For children with unexplained fever, detection of FGG, C1QC1, and VWF may help with etiological screening.

Serum-derived miR-574-5p-containing exosomes contribute to liver fibrosis by activating hepatic stellate cells.

AIM: To investigate the association of serum exosomes miR-574-5p with liver fibrosis, and explore the effect and mechanism of serum exosomes on HSC activation. MATERIALS AND METHODS: Using serum samples collected from healthy adults and patients with liver cirrhosis, we extracted human serum exosomes via ultra-high-speed centrifugation, and co-cultured them with hepatic stellate cells (HSCs) line LX2. LX-2-mediated intake of human serum exosomes was examined by confocal microscopy. To induce liver fibrosis, we administered 20% CCl4 to mice intraperitoneally and adopted an exoEasy MIDI kit to extract serum exosomes.Liver fibrosis-related molecules were determined via qRT-PCR, Western blot, Masson staining, and Immunohistochemical staining. RESULTS: Significantly high miR-574-5p levels were expressed in serum exosomes and were positively correlated with the expression of miR-574-5p, collagen deposition, and α-SMA expression in liver tissues of mice during liver fibrosis. Compared to healthy subjects, serum exosomes from cirrhosis patients were associated with higher expression of miR-574-5p. MiR-574-5p mimic promoted the expression of α-SMA and COL1A1 mRNA and protein in LX-2, whereas miR-574-5p inhibitor exerted no effect. CONCLUSION: This article demonstrates that miR-574-5p expression in serum exosomes is positively correlated with collagen deposition and HSC activation in liver tissues during liver fibrosis.Serum exosomes potentially activate HSC through the transfer of miR-574-5p to HSC during liver fibrosis.

PIWI-interacting RNAs are aberrantly expressed and may serve as novel biomarkers for diagnosis of lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is the main subtype of primary lung cancer and is a leading cause of cancer-related death worldwide. PIWI-interacting RNAs (piRNAs) are a type of small non-coding RNAs that may play crucial roles in cancer progression and serve as biomarkers for tumor detection. This study aimed to explore the expression profiles and diagnostic values of piRNAs in LUAD. METHODS: Small RNA sequencing was performed to investigate tissue piRNA profiles of LUAD. The expression of selected upregulated piRNAs were detected in tissues and serum exosome samples by quantitative real-time polymerase chain reaction (qRT-PCR). Serum exosomes were identified by transmission electron microscope, nanoparticle tracking analysis, and western blot analysis. Receiver operating characteristic (ROC) curve was adopted to quantify the diagnostic potentials of piRNAs in LUAD. Finally, a piRNA panel was developed by multivariate logistic regression model. RESULTS: We identified that 76 piRNAs were overexpressed and 9 piRNAs were underexpressed in LUAD tissues compared with adjacent non-tumor tissues. Among the top 10 overexpressed piRNAs, 4 piRNAs (piR-hsa-26925, piR-hsa-5444, piR-hsa-30636, and piR-hsa-8757) were verified by qRT-PCR to be significantly upregulated in LUAD tissues. Moreover, piR-hsa-26925 and piR-hsa-5444 had a significantly higher level in serum exosome samples of LUAD patients than those of healthy controls. We finally established a 2-piRNA panel composed of piR-hsa-26925 and piR-hsa-5444, which showed higher diagnostic performance for LUAD with an AUC of 0.833. CONCLUSIONS: Our finding revealed the abnormally expressed piRNAs in LUAD, and serum exosomal piR-hsa-26925 and piR-hsa-5444 could serve as potential biomarkers for LUAD diagnosis.

Serum exosomal lncRNA XIST is a potential non-invasive biomarker to diagnose recurrence of triple-negative breast cancer.

Exosomal lncRNAs secreted by cancer cells can serve as potential biomarkers in the diagnosis and prognosis of various tumours. Here, we are committed to explore the diagnostic and prognostic value of serum exosomal XIST secreted by tumour cells to predict recurrence in patients with triple-negative breast cancer (TNBC). Significant increments in XIST and exo-XIST from tumour tissues and blood serum were found in reoccurring TNBC patients by comparison with non-recurrences. Levels of serum exo-XIST were only significantly increased in TNBC recurrence and no association with other clinicopathological parameters. Additionally, serum exo-XIST levels could be served as an assessment of change in the load of triple-negative breast cancer. Expressions of exo-XIST were markedly decreased after resection of the primary breast tumours and obviously elevated at the time of recurrence. Finally, an obvious association was identified between serum exo-XIST levels and a poorer overall survival (OS) in TNBC patients. Levels of serum exo-XIST may serve as a diagnostic and prognostic biomarker to predict the recurrent TNBC-loading status.

Ischaemic preconditioning-induced serum exosomes protect against myocardial ischaemia/reperfusion injury in rats by activating the PI3K/AKT signalling pathway.

Ischaemia/reperfusion (I/R) injury can lead to severe arrhythmia and aggravate myocardial damage. Exosomes are small-membrane vesicles that play a protective role in myocardial I/R injury. This study aimed to explore the protective effects of ischaemic preconditioning (IPC)-induced serum exosomes (IPC-Exo) on myocardial I/R injury in rats and its underlying mechanism. Serum exosomes were extracted from IPC rats and quantified using a bicinchoninic acid assay kit. IPC-Exo (50 µg) was injected into the infarcted myocardium immediately after ligation. Rats were randomly divided into Sham, I/R, IPC-Exo + I/R, I/R + LY294002, and I/R + IPC-Exo + LY294002 groups. Haemodynamic parameters were measured by physiological recording. Transthoracic echocardiography was used to detect cardiac function. The serum levels of creatine kinase isomer-MB, lactate dehydrogenase, aspartate transaminase, tumour necrosis factor-alpha, interleukin (IL)-1ß, and IL-10 were detected by enzyme-linked immunosorbent assay. Triphenyl tetrazolium chloride staining was used to measure the myocardial infarct size. Apoptosis in myocardial tissues was detected by TUNEL staining. Western blotting was used to detect the levels of PI3K/AKT and apoptosis-related proteins. Our results showed that treatment with IPC-Exo ameliorated cardiac function and reduced inflammatory factor production, cardiomyocyte apoptosis, and myocardial infarct size. Moreover, IPC-Exo treatment promoted the protein expression of Bcl-2, p-PI3K, and p-AKT but inhibited that of caspase-3 and Bax. However, treatment with LY294002 significantly reversed that IPC-Exo-induced increase in p-PI3K and p-AKT levels, improvement of haemodynamics, and decrease of inflammatory factor production and apoptosis in the I/R + IPC-Exo group. Taken together, our results suggest that IPC-Exo may alleviate I/R injury via activating the PI3K/AKT signalling pathway.

Exploration of small RNA biomarkers for testicular injury in the serum exosomes of rats.

Testicular injury is often observed in drug development. Serum hormones are usually used as noninvasive biomarkers for testicular injury; however, their sensitivities are low. Therefore, it is difficult to monitor testicular injury in drug development. In recent years, molecules in body fluid exosomes have attracted attention as biomarkers for diseases. In this study, small RNAs in serum exosomes were analyzed to identify noninvasive biomarkers of testicular injury in rats, which are often used in preclinical drug development. The rat models of testicular injury were prepared by a single oral administration of 2000 mg/kg ethylene glycol monomethyl ether, in which spermatocyte degeneration and Sertoli cell vacuolation were observed, or 400 mg/kg carbendazim, in which Sertoli cell vacuolation and seminiferous tubule dilation were observed. Serum exosomal small RNA-seq analysis of these models was performed. The analysis identified 3 small RNAs that fluctuated in common between the models, and miR-423-5p and miR-128-3p were selected as candidate markers. For evaluating these candidate markers in other testicular injury models, the models were prepared by a single oral administration of 60 mg/kg 1,3-dinitrobenzene or 500 mg/kg nitrofurazone, and spermatocyte degeneration and Sertoli cell vacuolation were observed. In qPCR analysis, these exosomal miRNAs were upregulated in all models except for the 1,3-dinitrobenzene model, in which severe hemolysis was observed. By contrast, these miRNAs in whole serum extracts did not significantly change in any of the models. In conclusion, we identified miR-423-5p and miR-128-3p in serum exosomes as noninvasive biomarkers for testicular injury in rats.

Serum exosomal microRNA-34a as a potential biomarker in epithelial ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is a leading cause of cancer-related death in women, and thus an accurate diagnosis of the predisposition and its early detection is necessary. The aims of this study were to determine whether serum exosomal microRNA-34a (miR-34a) in ovarian cancer could be used as a potential biomarker. METHODS: Exosomes from OC patients' serum were collected, and exosomal miRNAs were extracted. The relative expression of miR-34a was calculated from 58 OC samples by quantitative real-time polymerase chain reaction. RESULTS: Serum exosomal miR-34a levels were significantly increased in early-stage OC patients compared with advanced-stage patients. Its levels were significantly lower in patients with lymph node metastasis than in those with no lymph node metastasis. Furthermore, its levels in the recurrence group were significantly lower than those in the recurrence-free group. CONCLUSIONS: Serum exosomal miR-34a could be a potential biomarker for improving the diagnostic efficiency of OC.

Downregulation of serum exosomal miR-320d predicts poor prognosis in hepatocellular carcinoma.

BACKGROUND: MicroRNAs (miRNAs) is a class of functional regulator of tumorigenesis of human cancer including hepatocellular carcinoma (HCC). However, the potential clinical significance of serum exosomal miR-320d in HCC has not been elucidated. METHODS: Real-time reverse transcription PCR was used to detect the expression pattern of serum exosomal miR-320d in patients with HCC, and the correlation between the deregulation of serum exosomal miR-320d and the clinical outcome of HCC was explored. The biological function of exosomal miR-320d in HCC was also investigated. RESULTS: Our results showed that the expression levels of exosomal miR-320d were remarkably reduced in the serum samples of HCC patients and the culture medium of HCC cell lines compared with their respective controls. Serum exosomal miR-320d could differentiate the HCC patients from healthy controls with high accuracy. In addition, its level was remarkably increased in the HCC patients who had received surgical treatment. Moreover, reduced serum exosomal miR-320d was associated with advanced tumor stage, positive lymph node metastasis, and poorly differentiated tumors. HCC patients with lower serum exosomal miR-320d had shorter overall and disease-free survival. Low serum exosomal miR-320d was identified to be an independent unfavorable prognostic factor for HCC. Finally, overexpression of miR-320d inhibited the proliferation and invasion of HCC cells, and BMI1 was demonstrated to be a direct target of miR-320d. CONCLUSION: Taken together, serum exosomal miR-320d could be a potential non-invasive biomarker for the diagnosis and prognosis of HCC.

Serum Derived Exosomes From Pancreatic Cancer Patients Promoted Metastasis: An iTRAQ-Based Proteomic Analysis.

BACKGROUND: Pancreatic cancer (PC) is one of the most aggressive malignancies and has a poor prognosis despite being extensively researched. The role of serum-derived exosomes in tumorigenesis and the development of PC is still unclear. METHOD: The present study employed iTRAQ-based proteomic analysis to search for differences between the serum exosomes of PC patients and those from control patients. Then, bioinformatics methods were used to analyze the functions of the identified proteins, and the possible functions were verified through cell culture experiments. RESULTS: A total of 611 proteins were identified from exosomes, and 141 proteins were differentially expressed, with 91 up- and 50 down regulated proteins in PC cancer compared to healthy controls. Further analysis indicated that APOE serves as an important hub in the network. In addition, CRP, VWF, APOA2, NIN, and GSK3B potentially interact with many other proteins. We then tested the effect of patient serum-derived exosomes on pancreatic cancer cells and found that patient serum-derived exosomes, but not those from healthy controls, induced cell proliferation, migration, and EMT, supporting the role of exosomes in metastasis. CONCLUSION: Our data suggest that exosomes derived from PC patients may promote PC metastasis.

Metabolomics identifies serum and exosomes metabolite markers of pancreatic cancer.

INTRODUCTION: Pancreatic cancer (PC) is one of the most aggressive malignancies, and it's difficult to diagnosis PC at an early stage, which leads to the poor prognosis of PC. OBJECTIVES: To identifiy the possible prognosis or dignosis metabolite biomarkers in the serum exosome of PC patients. METHODS: We employed LC-DDA-MS based untargeted lipidomic analysis to search for potential candidate biomarkers in the serum exosome of PC patients. Then LC-MRM-MS based targeted lipid quantification was used to validate the trends of the candidate biomarkers in larger sample cohorts. RESULTS: About 270 lipids belonging to 20 lipid species were found significantly dysregulated between the serum exosome of PC patients and healthy controls. 61 of them were validated in larger samples size. We further analysis the correlation between these dysregulated lipids and other PC related factors, and results show that LysoPC 22:0, PC (P-14:0/22:2) and PE (16:0/18:1) are all associated with tumor stage, CA19-9, CA242 and tumor diameter. What's more, PE (16:0/18:1) is also found to be significantly correlated with the patient's overall survival. CONCLUSION: These data reveal dysregulated lipids in serum exosome of PC patients, which have potential to be biomarkers for diagnosis, or unveil pathological relationship between exosome and PC progress.

Serum Exosomes from Newborn Piglets Restrict Porcine Epidemic Diarrhea Virus Infection.

Exosomes are vehicles in the body fluid that participate in many biological processes, especially immune responses. In this study, we employed comparative proteome analysis to investigate the roles of serum exosomes during viral infection in neonates using porcine epidemic diarrhea virus (PEDV), a devastating enteric virus in newborn piglets, as a model virus. Serum exosomes were first isolated from newborn piglets infected with PEDV or mock-infected newborn piglets, followed by label-free LC-MS/MS-based comparative quantitative proteomic analysis. Among the 441 detected proteins, 10 complement proteins were found in the serum exosomes, and significantly decreased expression levels of the C3, C6, and CFB complements were measured in PEDV-infected serum exosomes compared to those in mock-infected serum exosomes. After confirmation by Western blot, we then investigated the function of these exosomes in PEDV infection and discovered that exosomes from mock-infected newborn piglets restricted PEDV infection. However, this inhibition disappeared after the exosomes were heat-inactivated, suggesting that complements are key antiviral molecules. Our findings improve the understanding of antiviral responses mediated by exosomes in neonatal piglets and facilitate the discovery of novel antiviral drugs.

Splicing variant of hepcidin mRNA.

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. In this study, we firstly revealed that a new alternative HAMP transcript was found in hepatoma-derived cell line HLF, which was identical to the wild-type preprohepcidin sequence except lacking of an internal 60 bases. In addition to HLF, most of hepatoma-derived cell lines have significant copy numbers of variant-type hepcidin mRNA by a copy-based-digital PCR. Furthermore, the copy number of hepcidin mRNA variant was significantly higher in serum exosomes of hepatocellular carcinoma patients. The quantification of exosomal hepcidin mRNA variant may serve as a potential new biomarker for HCC diagnosis.