Exosomal Small RNA Research Articles

Exosomal miR-7-25207 Increases Subgroup J Avian Leukosis Virus Titers by Targeting the Akt-CyclinQ1 and PRC1-YAF2 Dual Pathways.

Subgroup J avian leukosis virus (ALV-J) is a major pathogen in poultry, causing substantial economic losses to the poultry industry worldwide. Exosomal small RNAs derived from virus-infected cells or biological fluids can serve as viral transmission vectors. However, the role and mechanism of exosomal miRNA in ALV-J infection are unclear. In this study, we demonstrated that exosomal microRNA-7-25207 (miR-7-25207) could increase the titers of ALV-J. Exosomes isolated from ALV-J-infected DF-1 cells (Exo-ALV-J) contained partial viral proteins from ALV-J and could transmit the infection to uninfected DF-1 cells, leading to productive infection. Additionally, the RNA expression profile of exosomes was altered following ALV-J infection. miRNA analysis revealed that the expression of exosomal miR-7-25207 increased. Overexpression of miR-7-25207 significantly increased the titers of ALV-J in transfected cells. Furthermore, miR-7-25207 directly suppressed the expression of Akt and PRC1. Akt, in turn, directly inhibited CyclinQ1 expression, while PRC1 directly interfered with YAF2 expression. In conclusion, ALV-J infection activates the expression of miR-7-25207, which is subsequently delivered via exosomes to uninfected cells, increasing ALV-J titers by targeting Akt-CyclinQ1 and PRC1-YAF2 dual pathways. These findings suggest that exosomal miR-7-25207 may serve as a potential biomarker for clinical parameters in ALV-J infection.

Exosomal Small RNA Sequencing Profiles in Plasma from Subjects with Latent Mycobacterium tuberculosis Infection.

Despite huge efforts, tuberculosis (TB) is still a major public health threat worldwide, with approximately 23% of the human population harboring a latent TB infection (LTBI). LTBI can reactivate and progress to active and transmissible TB disease, contributing to its spread within the population. The challenges in diagnosing and treating LTBI patients have been major factors contributing to this phenomenon. Exosomes offer a novel avenue for investigating the process of TB infection. In this study, we conducted small RNA sequencing to investigate the small RNA profiles of plasma exosomes derived from individuals with LTBI and healthy controls. Our findings revealed distinct miRNA profiles in the exosomes between the two groups. We identified 12 differentially expressed miRNAs through this analysis, which were further validated via qRT-PCR using the same exosomes. Notably, six miRNAs (hsa-miR-7850-5p, hsa-miR-1306-5p, hsa-miR-363-5p, hsa-miR-374a-5p, hsa-miR-4654, has-miR-6529-5p, and hsa-miR-140-5p) exhibited specifically elevated expression in individuals with LTBI. Gene ontology and KEGG pathway analyses revealed that the targets of these miRNAs were enriched in functions associated with ferroptosis and fatty acid metabolism, underscoring the critical role of these miRNAs in regulating the intracellular survival of Mycobacterium tuberculosis (Mtb). Furthermore, our results indicated that the overexpression of miR-7850-5p downregulated the expression of the SLC11A1 protein in both Mtb-infected and Mtb-uninfected THP1 cells. Additionally, we observed that miR-7850-5p promoted the intracellular survival of Mtb by suppressing the expression of the SLC11A1 protein. Overall, our findings provide valuable insights into the role of miRNAs and repetitive region-derived small RNAs in exosomes during the infectious process of Mtb and contribute to the identification of potential molecular targets for the detection and diagnosis of latent tuberculosis.

Inhibition of miR-199b-5p reduces pathological alterations in osteoarthritis by potentially targeting Fzd6 and Gcnt2

Osteoarthritis (OA) is a degenerative disease with a high prevalence in the elderly population, but our understanding of its mechanisms remains incomplete. Analysis of serum exosomal small RNA sequencing data from clinical patients and gene expression data from OA patient serum and cartilage obtained from the GEO database revealed a common dysregulated miRNA, miR-199b-5p. In vitro cell experiments demonstrated that miR-199b-5p inhibits chondrocyte vitality and promotes extracellular matrix degradation. Conversely, inhibition of miR-199b-5p under inflammatory conditions exhibited protective effects against damage. Local viral injection of miR-199b-5p into mice induced a decrease in pain threshold and OA-like changes. In an OA model, inhibition of miR-199b-5p alleviated the pathological progression of OA. Furthermore, bioinformatics analysis and experimental validation identified Gcnt2 and Fzd6 as potential target genes of MiR-199b-5p. Thus, these results indicated that MiR-199b-5p/Gcnt2 and Fzd6 axis might be a novel therapeutic target for the treatment of OA.

Inhibition of miR-199b-5p reduces pathological alterations in osteoarthritis by potentially targeting Fzd6 and Gcnt2.

Osteoarthritis (OA) is a degenerative disease with a high prevalence in the elderly population, but our understanding of its mechanisms remains incomplete. Analysis of serum exosomal small RNA sequencing data from clinical patients and gene expression data from OA patient serum and cartilage obtained from the GEO database revealed a common dysregulated miRNA, miR-199b-5p. In vitro cell experiments demonstrated that miR-199b-5p inhibits chondrocyte vitality and promotes extracellular matrix degradation. Conversely, inhibition of miR-199b-5p under inflammatory conditions exhibited protective effects against damage. Local viral injection of miR-199b-5p into mice induced a decrease in pain threshold and OA-like changes. In an OA model, inhibition of miR-199b-5p alleviated the pathological progression of OA. Furthermore, bioinformatics analysis and experimental validation identified Gcnt2 and Fzd6 as potential target genes of MiR-199b-5p. Thus, these results indicated that MiR-199b-5p/Gcnt2 and Fzd6 axis might be a novel therapeutic target for the treatment of OA.

Circulating exosomal microRNAs as biomarkers of lupus nephritis.

Disruption in the delicate symphony of genes, microRNA (miRNA), or protein expression can result in the dysregulation of the immune system, leading to the devastating consequences such as lupus nephritis (LN). The capacity of exosomes to transport miRNAs between cells and modify the phenotype of recipient cells implies their involvement in persistent kidney inflammation. This study unveils identifying two previously undiscovered exosomal miRNAs in the serum of LN patients, offering potential solutions to the current challenges in LN diagnosis and management. Initially, we used a reagent-based kit to isolate serum exosomes from patients with Systemic lupus erythematosus (SLE) and used Trizol method for total RNA extraction. Subsequently, we employed small RNA sequencing to screen for differential expression profiles of exosomal small RNAs. The RT-qPCR method was used to individually validate samples in both the screening and validation cohorts, enabling the identification of candidate small RNAs; specific to LN. We assessed the diagnostic potency using receiver operating characteristic (ROC) curve, and explored the biological roles of miRNAs using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Compared to SLE patients without LN, SLE patients accompanied by LN exhibited significantly spiked levels of exosomal hsa-miR-4796-5p and hsa-miR-7974. The duo of miRNAs, hsa-miR-4796-5p and hsa-miR-7974, exhibited promising potential as biomarkers for diagnosing LN, with an AUC exceeding 0.8. Correlation analysis revealed a strong positive association between these miRNAs and proteinuria, as well as the SLE Disease Activity Index (SLEDAI) score. Moreover, the levels of two miRNAs in LN patients were significantly elevated in comparison to other autoimmune nephritis conditions, such as immunoglobulin A nephropathy (IgAN) and diabetic nephropathy (DN). Furthermore, the bioinformatics analysis indicated that this miRNAs duo can play a pivotal role in the regulation of immune processes by modulating signal pathways, such as the mTOR and PI3K-Akt signaling pathway. This study provides a new ground that serum exosomal miRNAs can effectively identify and predict LN in SLE patients.

Exosomal miR-181d-5p Derived from Rapamycin-Conditioned MDSC Alleviated Allograft Rejection by Targeting KLF6.

Immune rejection and side effects of long-term administration of immunosuppressants are the two major obstacles to allograft acceptance and tolerance. The immunosuppressive extracellular vesicles (EVs)-based approach has been proven to be effective in treating autoimmune/inflammatory disorders. Herein, the anti-rejection advantage of exosomes (Rapa-Exo) from rapamycin-conditioned myeloid-derived suppressor cells (MDSCs) over exosomes (Exo-Nor) from the untreated MDSCs is shown. The exosomal small RNA sequencing and loss-of-function assays reveal that the anti-rejection effect of Rapa-Exo functionally relies on miR-181d-5p. Through target prediction and double-luciferase reporter assay, Kruppel-like factor (KLF) 6 is identified as a direct target of miR-181d-5p. Finally, KLF6 knockdown markedly resolves inflammation and prolongs the survival of corneal allografts. Taken together, these findings support that Rapa-Exo executes an anti-rejection effect, highlighting the immunosuppressive EVs-based treatment as a promising approach in organ transplantation.

Abstract P3154: Characterized Expression Of MicroRNAs In Plasma Exosome In Patients With Coronary Chronic Total Occlusion

Backgrounds: Even with similar coronary artery occlusion, chronic total occlusion (CTO) has different clinical features from acute myocardial infarction (AMI). MicroRNAs (miRNA; miR) have been regarded as biomarker to identify heterogenous patients, as well as new therapeutic targets in cardiovascular disease. We aimed to identify exosomal miRNA important for CTO. Methods: Patients with percutaneous coronary intervention (PCI) of CTO (n=30) and AMI (n=30) were performed miRNA expression profiling via exosomal small RNA sequencing in the plasma for these groups. Receiver operating characteristic (ROC) analysis, gene ontology, and functional pathway were analyzed in the miRNA exhibiting >4 absolute fold changes with FDR < 0.05. Results: We detected 10 miRNAs -including miR-21-5p, miR-206, miR-215-5p, and miR-1-3p upregulated in CTO and 3 miRNAs -including miR-9-5p, miR-3529-3p, and miR-127-3p downregulated in CTO as compared to AMI (FDR q < 0.05). Gene enrichment and pathway analysis showed that the target genes of the 10 upregulated miRNAs were involved in regulation of osteoblast proliferation and interleukin-4 and interleukin-13 signaling. In contrast, the target genes of the 3 down-regulated miRNAs were enriched in heart valve development and blood vessel development. We selected 7 miRNAs -including miR-21-5p, miR-206, miR-215-5p, miR-1-3p, miR-9-5p, miR-3529-3p, and miR-127-3p which were related to major pathway term in gene ontology and validated by quantitative real-time polymerase chain reaction (qRT-PCR). In qRT-PCR analysis, the expression levels of miR-9-5p and miR-127-3p of plasma exosome were both lower in CTO than those of AMI, similar to the results of exosomal small RNA sequencing. The ROC curve were analyzed in the 2 miRNAs and for the integrative miRNA expression score showed high accuracy in distinguishing between the CTO and AMI (AUC = 0.948) with 79% specificity and 97% sensitivity. Conclusions: We identified significantly different miRNA expression patterns between the CTO and AMI. The levels of these miRNAs in plasma exosomes may be promising markers that distinguish CTO from AMI.

Abstract P3168: Identification Of Exosome MicroRNA In Association With Severe Calcification In Coronary Chronic Total Occlusion

Backgrounds: Coronary artery calcification (CAC) is prevalent in patients with coronary chronic total occlusion (CTO). We investigated the association between plasma miRNAs and coronary artery calcification in patients with CTO. Methods: Blood samples were collected from 18 patients who underwent percutaneous coronary intervention (PCI) of CTO. Exosomal small RNA sequencing was performed from plasma to quantify miRNA expression. Calcium volume was quantified in computed tomography (CT) in CTO. Gene ontology and functional pathway were analyzed in the miRNAs that significantly correlates with calcium volume. Results: Calcium volume was positively correlated with miR-1-3p and miR-708-3p (p <0.05), and was negatively correlated with 4 miRNAs -including let-7d-5p (p <0.05). Gene enrichment and pathway analysis showed that the target genes of the 2 positively correlated miRNAs were involved in regulation of osteoblast differentiation and osteopontin signaling pathway. In contrast, the target genes of the 4 negatively correlated miRNAs were enriched in acetylcholine-mediated vasodilation involved in regulation of systemic arterial blood pressure, blood vessel development and response to sterol. Multiple linear regression showed that miR-1-3p and let-7d-5p could be key predictors of the calcium volume of CTO (adjusted R 2 = 0.798 and p <0.001). Conclusions: We identified miR-1-3p and let-7d-5p could be related with CAC in patients with CTO.

Pancreatic stellate cell-derived exosomal tRF-19-PNR8YPJZ promotes proliferation and mobility of pancreatic cancer through AXIN2.

The pancreatic stellate cells (PSCs) play an important role in the development of pancreatic cancer (PC) through mechanisms that remain unclear. Exosomes secreted from PSCs act as mediators for communication in PC. This study aimed to explore the role of PSC-derived exosomal small RNAs derived from tRNAs (tDRs) in PC cells. Exosomes from PSCs were extracted and used to detect their effects on PC cell proliferation, migration and invasion. Exosomal tDRs profiling was performed to identify PSC-derived exosomal tDRs. ISH and qRT-PCR were used to examine the tRF-19-PNR8YPJZ levels and clinical value in clinical samples. The biological function of exosomal tRF-19-PNR8YPJZ was determined using the CCK-8, clone formation, wound healing and transwell assays, subcutaneous tumour formation and lung metastatic models. The relationship between the selected exosomal tRF-19-PNR8YPJZ and AXIN2 was determined by RNA sequencing, luciferase reporter assay. PSC-derived exosomes promoted the proliferation, migration, and invasion of PC cells. Novel and abundant tDRs are found to be differentially expressed in PANC-1 cells after treatment with PSC-derived exosomes, such as tRF-19-PNR8YPJZ. PC tissue samples showed markedly higher levels of tRF-19-PNR8YPJZ than normal controls. Patients with PC exhibiting high tRF-19-PNR8YPJZ expression had a highly lymph node invasion, metastasis, perineural invasion, advanced clinical stage and poor overall survival. Exosomal tRF-19-PNR8YPJZ from PSCs targeted AXIN2 in PC cells and decreased its expression, thus activating the Wnt pathway and promoting proliferation and metastasis. Exosomal tRF-19-PNR8YPJZ from PSCs promoted proliferation and metastasis in PC cells via AXIN2.

Abstract 867: Identifying plasma exosomal small non-coding RNAs as potential biomarkers for lung cancer early detection

Abstract Introduction: Lung cancer is the second most common cancer and the leading cause of cancer death in the US and worldwide. The National Lung Screening Trial (NLST) through thoracic low-dose computed tomography (LDCT) scans has shown that screening decreases mortality by 20%, but nearly 40% of patients in the NLST had a positive screen at one point, with 96.4% of these being false positive. It is urgent to develop new non-invasive methods with high efficacy, such as blood molecular biomarkers as companion diagnostic tools to complement LDCT screening. Recent studies have shown that small non-coding RNAs (ncRNAs) play important roles in cancer development, and exosomes are actively secreted extracellular vesicles that contain proteins, lipids, and nucleic acids, including miRNAs and other classes of small ncRNAs. Our study aimed to screen and identify noval plasma exosomal small ncRNAs for lung cancer early detection. Methods: A supersensitive human plasma exosome isolation and purification as well as exosome small RNA sequencing were conducted on 233 human subjects including 117 lung cancer early-stage patients and 116 non-cancer subjects (54 benign and 62 normal samples). Sequencing reads were first processed by trimming off the adapters and low-quality bases using cutadapt. Following trimming, the sequences were aligned to the human genome (Genome Reference Consortium GRCh38) and mapped to different small ncRNA databases using STAR. Read counts for each RNA category (miRNA, piRNA, snRNA, snoRNA, tRNA, tsRNA) were calculated from the mapping results. Differential gene expression analysis was performed by the DESeq2 bioconductor package. The genes with Benjamini and Hochberg q-value < 0.05 were called differentially expressed. Based on the differential gene lists, diagnostic models were built by 10-fold cross validated Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression. Results: A total of 5470 small ncRNAs were initially identified, including 1094 miRNA, 2172 piRNA, 1185 snRNA, 376 snoRNA, 582 tRNA, 61 tsRNA. Using DESeq2 with an FDR q-value < 0.05 with a cut off of 1.5-fold change, we found 508 small ncRNAs significantly differentially expressed between cancer and non-cancer groups, consisting of 231 small ncRNAs up-regulated and 277 down-regulated in tumor. By diagnostic modeling, a set of 16 small ncRNAs were identified as promising biomarkers, which could differentiate patients with cancer from non-cancer with an area under the receiver operating (ROC) curve (AUC) of 96.71%. Using the same sixteen-smallRNA signature to distinguish cancer from benign, the AUC achieved 97.23%; while the same panel could distinguish cancer from normal with an AUC of 96.26%. Conclusion: This preliminary study demonstrated the potential diagnostic value of the exosomal small ncRNA biomarkers in lung cancer early detection. The results will be evaluated by more cases in the future study. Citation Format: Yuanyuan Fu, Masaki Nasu, Mayumi Jijiwa, Yu Chen, Zitong Gao, Isam Ibrahim, Youping Deng. Identifying plasma exosomal small non-coding RNAs as potential biomarkers for lung cancer early detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 867.

Abstract 3333: Exosomal non-coding RNAs as a biomarker to improve early ovarian cancer detection

Abstract Purpose: There is no consensus on screening for ovarian cancer in high-risk women because no effective biomarker or screening method exists, especially for early-stage cancer patients. The available methods, such as monitoring of CA125 levels, transvaginal ultrasound, and combination of pelvic examination are associated with high false-positive results, leading to additional unnecessary imaging tests, emotional burdens, and risk of invasive surgery for women without cancer. An effective, reliable, and non-invasive method for early detection of ovarian cancer is thus critically needed. In this research, weThus, we aim to identify a distinctive signature of exosomal small RNAs to serve as a novel biomarker for detecting early-stage ovarian cancer in high-risk population. Methods: Women of all ages and ethnical backgrounds were included in this study. Blood samples were collected in the clinic from women with germline BRCA mutations. A total of 30 patients with ovarian cancer before surgery, and 30 age-matched (mean age = 55) healthy women as controls were used. Among the 30 thirty cases, 11 patients had early (stage I) ovarian cancer. Identification of differential exosome small non-coding RNA copy numbers in plasma samples from BRCA1 mutation carriers with and without ovarian cancer was performed by next-generation RNA-Sseq. We randomly split 60 samples into two groups: training set (20 cancer, 20 normal) and , testing set (10 cancer, 10 normal). We apply 10-fold cross-validation to determine hyperparameter of five machine learning models and use the ensemble learning method to evaluate the predictive value of the small RNAs in the exosome by integrating the five machine learning models. Results: The ensemble learning model achieved the testing accuracy of 85% with the sensitivity of 70% and the specificity of 100% by using small RNA and CA125 abundance level as input features. While uUsing CA125 alone, the testing accuracy equals to 90% with the sensitivity of 80% and the specificity of 100%. For the 11 eleven early-stage patient samples, our ensemble learning model made 2 false negative predictions with a total accuracy of 95.1% (sensitivity=81.8%, specificity=100%) while using CA125 alone had five 5 false negative predictions with a total accuracy of 87.8% (sensitivity=54.5%, specificity=100%). All 30 control samples had been correctly classified. Conclusions: We found that ensemble learning model using combining both exosome small RNAs and CA125 will not improve the prediction accuracy for late-stage cancer patients compared to CA125 alone but will significantly improve the prediction accuracy for early-stage patients. Our research findings may indicate a two-pronged strategy in assessing ovarian cancer risks using existing CA125 test followed by small RNA features to detect early-stage ovarian cancer. We are now collecting a larger number of early-stage cancer patient samples for validation. Citation Format: Yuliang Cao, Cun Han, Jianting Sheng, Samuel Mok, Stephen T.C. Wong. Exosomal non-coding RNAs as a biomarker to improve early ovarian cancer detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3333.

Analysis on Exosomal Small RNA Derived from Periodontal Pathogen-infected Ca9-22 Oral Cancer Cells

Analysis on Exosomal Small RNA Derived from Periodontal Pathogen-infected Ca9-22 Oral Cancer Cells

Comparative Profiling of Circulating Exosomal Small RNAs Derived From Peruvian Patients With Tuberculosis and Pulmonary Adenocarcinoma.

Tuberculosis (TB) is one of the most fatal infectious diseases, caused by the aerobic bacteria Mycobacterium tuberculosis. It is estimated that one-third of the world’s population is infected with the latent (LTB) version of this disease, with only 5-10% of infected individuals developing its active (ATB) form. Pulmonary adenocarcinoma (PA) is the most common and diverse form of primary lung carcinoma. The simultaneous or sequential occurrence of TB and lung cancer in patients has been widely reported and is known to be an issue for diagnosis and surgical treatment. Raising evidence shows that patients cured of TB represent a group at risk for developing PA. In this work, using sRNA-sequencing, we evaluated the expression patterns of circulating small RNAs available in exosomes extracted from blood samples of Peruvian patients affected by latent tuberculosis, active tuberculosis, or pulmonary adenocarcinoma. Differential expression analysis revealed a set of 24 microRNAs perturbed in these diseases, revealing potential biomarker candidates for the Peruvian population. Most of these miRNAs are normally expressed in healthy lung tissue and are potential regulators of different shared and unique KEGG pathways related to cancers, infectious diseases, and immunology.

Digital-resolution and highly sensitive detection of multiple exosomal small RNAs by DNA toehold probe-based photonic resonator absorption microscopy.

Small noncoding RNAs (snRNA) have been emerging as promising diagnostic biomarkers for detecting early stage cancer. Currently existing methods for snRNA detection, including northern blot, reverse transcription-polymerase chain reaction, microarrays and RNA-Seq, are limited to time-consuming, low sensitivity, expensive instrumentation or complex analysis of data. Herein, we present a rapid quantitative analysis of multiple liver cancer-associated exosomal snRNA by a nucleic acid toehold probe-based photonic resonator absorption microscopy (PRAM) assay, with digital resolution and high sensitivity. The assay relies on the use of three toehold probe-encoded gold nanoparticles (AuNPs) and addressable photonic crystal (PC) sensing chips. The presence of target snRNA will initiate toehold-mediated strand displacement reactions that trigger the capture of gold particles onto the PC surface, which is subsequently imaged by PRAM for digital counting of detected snRNA molecules. We achieved highly sensitive and selective detection of three snRNA targets in buffer with a 30min assay protocol, with detection limits of 4.56fM, 4.68fM and 0.69 pM. Having confirmed our assay's performance for detection of snRNA targets spiked into exosomal RNA extracts, we demonstrated its capability for quantitative detection of the same targets from patient blood plasma samples. The approach offers a rapid, simple workflow that operates at room temperature with a single step without enzymatic amplification, while the detection instrument can be implemented as a low-cost portable system for point of care environments.

Exosomal miR-181a-2-3p derived from citreoviridin-treated hepatocytes activates hepatic stellate cells trough inducing mitochondrial calcium overload

Increasing evidences indicate the vital role of exosomes-mediated intercellular communication in the pathogenesis of liver fibrosis. However, the underlying mechanisms are still not clearly defined. In this study, we found that citreoviridin (CIT), a mycotoxin and ectopic ATP synthase (e-ATPS) inhibitor, induced liver fibrosis in mice. The exosomes derived from CIT-treated L-02 hepatocytes activated hepatic stellate cells (HSC) LX-2. With exosomal small RNA sequencing, we found 156 differentially expressed miRNAs in the exosomes from CIT-treated L-02 cells, and the predicted target genes of exosomal miRNAs were enriched in calcium signaling pathway. The exosomes from CIT-treated L-02 cells induced mitochondrial calcium accumulation in LX-2 cells. And pharmacological inhibition of mitochondrial calcium uptake relieved exosomes-activated fibrogenic response in LX-2 cells. The miR-181a-2-3p that was predicted to target-regulate mitochondrial calcium uptake 1 (MICU1) was significantly increased in the exosomes from CIT-treated L-02 cells. Exosomes-induced reduction of MICU1, mitochondrial calcium overload and activation of LX-2 cells were reversed by AntagomiR-181a-2-3p. In this study, we pointed out that exosomal miR-181a-2-3p from CIT-treated hepatocytes induced mitochondrial calcium accumulation and activated HSC subsequently through inhibiting the expression of MICU1, shedding new light on the mechanism underlying liver fibrosis and CIT hepatotoxicity.

MicroRNAs in Serum Exosomes as Circulating Biomarkers for Postmenopausal Osteoporosis.

Postmenopausal osteoporosis (PMOP) is the most common skeletal disease in postmenopausal women and has become a global public health issue. Emerging evidence demonstrated the important relationship between microRNAs and PMOP. However, miRNAs have not yet been reported in PMOP. Hence, the present study aimed to investigate the differences in miRNA expression profiles in PMOP with fragility fractures to identify the key circulating miRNAs in serum exosomes and to validate these molecules as potential biomarkers. Postmenopausal women with osteoporotic fracture and normal bone mass were enrolled. Serum exosomes were isolated by traditional differential ultracentrifugation from participants. Isolated exosomes were identified by electron microscopy, western blotting and nanoparticle-tracking analysis and then examined for exosomal small RNA sequencing. The expression of miRNAs was compared by sRNA deep sequencing and bioinformatics analysis. Three miRNAs (mir-324-3p, mir-766-3p and mir-1247-5p) were found to be associated with BMD of L1-L4, FN (femur neck) and TH (total hip), while mir-330-5p and mir-3124-5p were associated with BMD of FN and TH. Furthermore, mir-330-5p was found to promote the ALP activity of hBMSCs, while mir-3124-5p showed the opposite result. The results showed that serum exosomal miRNAs were differentially expressed in postmenopausal osteoporosis patients with fragility fractures. Our study provides the first evidence that exosomal miRNA profiling revealed aberrant circulating miRNA in postmenopausal osteoporosis. Mir-324-3p, mir-766-3p, mir-1247-5p, mir-330-5p and mir-3124-5p, which were associated with bone mineral density (BMD), may serve as candidate diagnostic biomarkers as well as potentially contribute to pathophysiology of PMOP.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles inhibit shoulder stiffness via let-7a/Tgfbr1 axis

Shoulder stiffness (SS) is a common shoulder disease characterized by increasing pain and limited range of motion. SS is considered to be an inflammatory and fibrotic disorder pathologically. However, there is no consensus on the most effective conservative treatment for fibrosis. Given that human Bone Marrow Mesenchymal Stem Cell-derived extracellular vesicles (BMSC-EVs) displayed promising therapeutic effects for various tissues, we investigated the therapeutic effect of BMSC-EVs on fibrosis in a mice immobilization model and two cell models. By conducting a series of experiments, we found that BMSC-EVs can significantly inhibit the fibrogenic process both in vitro and in vivo. In detail, BMSC-EVs suppressed the aberrant proliferation, high collagen production capacity, and activation of fibrotic pathways in TGF-β-stimulated fibroblasts in vitro. Besides, in vivo, BMSC-EVs reduced cell infiltration, reduced fibrotic tissue in the shoulder capsule, and improved shoulder mobility. In addition, via exosomal small RNA sequencing and qPCR analysis, let-7a-5p was verified to be the highest expressed miRNA with predicted antifibrotic capability in BMSC-EVs. The antifibrotic capacity of BMSC-EVs was significantly impaired after the knockdown of let-7a-5p. Moreover, we discovered that the mRNA of TGFBR1 (the membrane receptor of transforming growth factor β) was the target of let-7a-5p. Together, these findings elucidated the antifibrotic role of BMSC-EVs in shoulder capsular fibrosis. This study clarifies a new approach using stem cell-derived EVs therapy as an alternative to cell therapy, which may clinically benefit patients with SS in the future.

A signature of saliva-derived exosomal small RNAs as predicting biomarker for esophageal carcinoma: a multicenter prospective study

BackgroundThe tRNA-derived small RNAs (tsRNAs) are produced in a nuclease-dependent manner in responses to variety of stresses that are common in cancers. We focus on a cancer-enriched tsRNA signature to develop a salivary exosome-based non-invasive biomarker for human esophageal squamous cell carcinoma (ESCC).MethodsCancer-enriched small RNAs were identified by RNA sequencing of salivary exosomes obtained from ESCC patients (n = 3) and healthy controls (n = 3) in a pilot study and further validated in discovery cohort (n = 66). A multicenter prospective observational study was conducted in two ESCC high-incidence regions (n = 320 and 200, respectively) using the newly developed biomarker signature.ResultsThe tsRNA (tRNA-GlyGCC-5) and a previously undocumented small RNA were specifically enriched in salivary exosomes of ESCC patients, ESCC tissues and ESCC cells. The bi-signature composed of these small RNAs was able to discriminate ESCC patients from the controls with high sensitivity (90.50%) and specificity (94.20%). Based on the bi-signature Risk Score for Prognosis (RSP), patients with high-RSP have both shorter overall survival (OS) (HR 4.95, 95%CI 2.90–8.46) and progression-free survival (PFS) (HR 3.69, 95%CI 2.24–6.10) than those with low-RSP. In addition, adjuvant therapy improved OS (HR 0.47, 95%CI 0.29–0.77) and PFS (HR 0.36, 95%CI 0.21–0.62) only for patients with high but not low RSP. These findings are consistent in both training and validation cohort.ConclusionsThe tsRNA-based signature not only has the potential for diagnosis and prognosis but also may serve as a pre-operative biomarker to select patients who would benefit from adjuvant therapy.Trial registrationA prospective study of diagnosis biomarkers of esophageal squamous cell carcinoma, ChiCTR2000031507. Registered 3 April 2016 - Retrospectively registered.

A Next-Generation Sequencing of Plasma Exosome-Derived microRNAs and Target Gene Analysis with a Microarray Database of Thermally Injured Skins: Identification of Blood-to-Tissue Interactions at Early Burn Stage.

BackgroundPlasma exosome-derived microRNA (miRNA) profiles following thermal injury and their relationship with gene expression derangements in burned skin remain unexplored. This study focused on the identification of key miRNA-mRNA axes in potential blood-to-tissue interactions at early burn stage.MethodsPlasma exosomes were obtained from 6 severe burn patients 4–7 days post injury and 6 healthy volunteers. Next-generation sequencing (NGS) of exosomal small RNAs presented the differentially expressed miRNAs (DEMs). Target genes of the DEMs were predicted in the mirDIP database. Dataset GSE8056 was enrolled to acquire differentially expressed genes (DEGs) in burned skin compared to normal skin. Overlap between the DEGs and target genes of the DEMs were focus genes. The protein–protein interaction (PPI) network and enrichment analyses of the focus genes demonstrated hub genes and suggested underlying mechanisms and pathways. The hub genes and upstream DEMs were selected to construct key miRNA-mRNA axes.ResultsThe NGS of plasma exosome-derived small RNAs identified 85 DEMs (14 downregulated miRNAs and 71 upregulated miRNAs) with 12,901 predicted target genes. Dataset GSE8056 exhibited 1861 DEGs in partial-thickness burned skins 4–7 days postburn. The overlap between DEGs and target genes of DEMs displayed 1058 focus genes. The top 9 hub genes (CDK1, CCNB1, CCNA2, BUB1B, PLK1, KIF11, AURKA, NUSAP1 and CDCA8) in the PPI network of the focus genes pointed to 16 upstream miRNAs in DEMs, including 4 downregulated miRNAs (hsa-miR-6848-3p, has-miR-4684-3p, has-miR-4786-5p and has-miR-365a-5p) and 12 upregulated miRNAs (hsa-miR-6751-3p, hsa-miR-718, hsa-miR-4754, hsa-miR-6754-3p, hsa-miR-4739, hsa-miR-6739-5p, hsa-miR-6884-3p, hsa-miR-1224-3p, hsa-miR-6878-3p, hsa-miR-6795-3p, hsa-miR-550a-3p, and hsa-miR-550b-3p). A key miRNA-mRNA network in potential blood-to-tissue interactions at early burn stage was therefore constructed.ConclusionAn NGS and bioinformatic analysis in the study identified key miRNA-mRNA axes in potential blood-to-tissue interactions at early burn stage, suggesting plasma exosome-derived miRNAs may impact on the alteration patterns of gene expressions in a burn wound.

Systematic Profiling of Exosomal Small RNA Epigenetic Modifications by High-Performance Liquid Chromatography-Mass Spectrometry.

Exosomes are nanosized extracellular vesicles that have a critical role in intercellular communication and tumor microenvironment regulation. Extensive research has shown that exosomal small RNAs contribute to metastasis in multiple tumor types and that abnormal epigenetic modifications in nucleic acids also have an association with diverse diseases. However, the content of modified nucleosides on exosomal small RNAs has not been quantitatively reported. Because of the trace amounts of exosomes and matrix complexity, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) as a powerful tool for label-free sensitive and simultaneous determinations of six important modified nucleosides on small RNAs inside exosomes. This system performed well using only approximately 107-108 particles of exosomes to obtain modified nucleoside levels between 0.001 and 0.03, and the most striking result was that the content of m6A in exosomal small RNAs was continuously higher than that in the cells being analyzed. We hope that this conclusion helps establish a greater degree of deciphering accuracy on exosomes, which has considerable application potential in the diagnosis and prognosis of diseases.