Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells’ exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells’ exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.     

Tubular cell-derived exosomal miR-150-5p contributes to renal fibrosis following unilateral ischemia-reperfusion injury by activating fibroblast in vitro and in vivo

Unilateral ischemia reperfusion injury (UIRI) with longer ischemia time is associated with an increased risk of acute renal injury and chronic kidney disease. Exosomes can transport lipid, protein, mRNA, and miRNA to corresponding target cells and mediate intercellular information exchange. In this study, we aimed to investigate whether exosome-derived miRNA mediates epithelial-mesenchymal cell communication relevant to renal fibrosis after UIRI. The secretion of exosomes increased remarkably in the kidney after UIRI and in rat renal tubular epithelium cells (NRK-52E) after hypoxia treatment. The inhibition of exosome secretion by Rab27a knockout or GW4869 treatment ameliorates renal fibrosis following UIRI in vivo. Purified exosomes from NRK-52E cells after hypoxia treatment could activate rat kidney fibroblasts (NRK-49F). The inhibition of exosome secretion in hypoxic NRK-52E cells through Rab27a knockdown or GW4869 treatment abolished NRK-49F cell activation. Interestingly, exosomal miRNA array analysis revealed that miR-150-5p expression was increased after hypoxia compared with the control group. The inhibition of exosomal miR-150-5p abolished the ability of hypoxic NRK-52E cells to promote NRK-49F cell activation in vitro, injections of miR-150-5p enriched exosomes from hypoxic NRK-52E cells aggravated renal fibrosis following UIRI, and renal fibrosis after UIRI was alleviated by miR-150-5p-deficient exosome in vivo. Furthermore, tubular cell-derived exosomal miR-150-5p could negatively regulate the expression of suppressor of cytokine signaling 1 to activate fibroblast. Thus, our results suggest that the blockade of exosomal miR-150-5p mediated tubular epithelial cell-fibroblast communication may provide a novel therapeutic target to prevents UIRI progression to renal fibrosis.     

Myotube-derived exosomal miRNAs downregulate Sirtuin1 in myoblasts during muscle cell differentiation

It has recently been established that exosomes can mediate intercellular cross-talk under normal and pathological conditions through the transfer of specific miRNAs. As muscle cells secrete exosomes, we addressed the question of whether skeletal muscle (SkM) exosomes contained specific miRNAs, and whether they could act as “endocrine signals” during myogenesis. We compared the miRNA repertoires found in exosomes released from C2C12 myoblasts and myotubes and found that 171 and 182 miRNAs were exported into exosomes from myoblasts and myotubes, respectively. Interestingly, some miRNAs were expressed at higher levels in exosomes than in their donor cells and vice versa, indicating a selectivity in the incorporation of miRNAs into exosomes. Moreover miRNAs from C2C12 exosomes were regulated during myogenesis. The predicted target genes of regulated exosomal miRNAs are mainly involved in the control of important signaling pathways for muscle cell differentiation (e.g., Wnt signaling pathway). We demonstrated that exosomes from myotubes can transfer small RNAs (C. elegans miRNAs and siRNA) into myoblasts. Moreover, we present evidence that exosome miRNAs secreted by myotubes are functionally able to silence Sirt1 in myoblasts. As Sirt1 regulates muscle gene expression and differentiation, our results show that myotube–exosome miRNAs could contribute to the commitment of myoblasts in the process of differentiation. Until now, myokines in muscle cell secretome provided a conceptual basis for communication between muscles. Here, we show that miRNA exosomal transfer would be a powerful means by which gene expression is orchestrated to regulate SkM metabolic homeostasis.     

Exosomal miR-1260b derived from non-small cell lung cancer promotes tumor metastasis through the inhibition of HIPK2

Tumor-derived exosomes (TEXs) contain enriched miRNAs, and exosomal miRNAs can affect tumor growth, including cell proliferation, metastasis, and drug resistance through cell-to-cell communication. We investigated the role of exosomal miR-1260b derived from non-small cell lung cancer (NSCLC) in tumor progression. Exosomal miR-1260b induced angiogenesis by targeting homeodomain-interacting protein kinase-2 (HIPK2) in human umbilical vein endothelial cells (HUVECs). Furthermore, exosomal miR-1260b or suppression of HIPK2 led to enhanced cellular mobility and cisplatin resistance in NSCLC cells. In patients with NSCLC, the level of HIPK2 was significantly lower in tumor tissues than in normal lung tissues, while that of miR-1260b was higher in tumor tissues. HIPK2 and miR-1260b expression showed an inverse correlation, and this correlation was strong in distant metastasis. Finally, the expression level of exosomal miR-1260b in plasma was higher in patients with NSCLC than in healthy individuals, and higher levels of exosomal miR-1260b were associated with high-grade disease, metastasis, and poor survival. In conclusion, exosomal miR-1260b can promote angiogenesis in HUVECs and metastasis of NSCLC by regulating HIPK2 and may serve as a prognostic marker for lung cancers.Subject terms: Non-small-cell lung cancer, Metastasis, miRNAs     

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression

Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease, characterized by increased ovarian androgen biosynthesis, chronic anovulation and polycystic ovaries. The objective of this study was to identify the altered miRNA expression profiles in follicular fluid derived exosomes isolated from PCOS patients and to investigate the molecular functions of exosomal miR-424-5p. Herein, small RNA sequencing showed that twenty-five miRNAs were differentially expressed between control and PCOS group. The alterations in the miRNA profile were related to the endocrine resistance, cell growth and proliferation, cellular senescence and insulin signaling pathway. Among these differentially expressed miRNAs, we found that the expression of miR-424-5p was significantly decreased in PCOS exosomes and primary granulosa cells (GCs). Exosome-enriched miR-424-5p significantly promoted GCs senescence and suppressed cell proliferation. Similar to the results obtained in the cells transfected with miR-424-5p mimic, miR-424-5p mimic significantly decreased cell proliferation ability and induced senescence, but treatment with miR-424-5p inhibitor got the opposite results. In addition, cell division cycle associated 4 (CDCA4) gene displayed an inverse expression pattern to those of miR-424-5p, was identified as the direct target of miR-424-5p. Overexpression of CDCA4 reversed the effects of exosomal miR-424-5p on GCs via activation of Rb/E2F1 signaling pathway. These results demonstrate that exosomal miR-424-5p inhibits GCs proliferation and induces cellular senescence in PCOS through blocking CDCA4-mediated Rb/E2F1 signaling. Our findings provide new information on abnormal follicular development in PCOS.     

Blockage of transferred exosome-shuttled miR-494 inhibits melanoma growth and metastasis

There is emerging evidence of bioactive material transport by exosomes in melanoma. However, the functions of exosome content underlying such cancer progression remain largely unknown. We aimed at determining whether exosome secretion contributes to cellular microRNA-494 (miR-494) loss and investigated the roles of miR-494 in melanoma progression. The exosomes from blood serum and cell culture conditioned media were separated by ultracentrifugation. A short hairpin RNA was used to silence rab27a for inhibiting exosome release. To address the functional role of exosomal miR-494, we assessed cell proliferation, migration, invasion capabilities, and cell apoptosis. Finally, subcutaneous xenograft and lung-metastasis models were constructed to determine the effect of exosomal miR-494 in vivo. Based on long noncoding RNA microarray analysis of melanocyte and melanoma-derived exosomes from the Gene Expression Omnibus database, we discovered that miR-494 was enriched in melanoma-derived exosomes. And miR-494 was increased in exosomes secreted from melanoma patients’ serum and A375 cells. Rab27a depletion reduced exosome secretion and rescued the abundance of cellular miR-494. Functional studies revealed that knockdown of rab27a and subsequent accumulation of miR-494 significantly suppressed the malignant phenotypes of melanoma cells via inducing cell apoptosis. Nude mice experiments confirmed that tumor growth and metastasis were suppressed by increasing miR-494 accumulation after rab27a depletion. In conclusion, blocking transferred exosome-shuttled miR-494 is a potential therapeutic option for melanoma.     

Serum exosomal miR-210 as a potential biomarker for clear cell renal cell carcinoma

Exosomal microRNAs (miRNAs) are suggested to reflect molecular changes occurring in their cells of origin and are potential indicators in the early detection of cancers. This study aimed to determine whether certain exosomal miRNAs from tumor tissue can be used as noninvasive biomarkers for clear cell renal cell carcinoma (ccRCC). Based on ccRCC miRNA expression profiles and the literature, we selected six miRNAs (miR-210, miR-224, miR-452, miR-155, miR-21, and miR-34a) and analyzed their expression in tissues, sera, and serum exosomes through quantitative real-time polymerase chain reaction in hypoxia-induced (with CoCl₂) renal cell lines. miR-210, miR-224, miR-452, miR-155, and miR-21 were upregulated in tumor tissues compared with normal tissues. Serum miR-210 and miR-155 levels were higher in patients with ccRCC than in healthy controls (HCs). Furthermore, only exosomal miR-210 was significantly upregulated in patients with ccRCC than in HCs. Moreover, receiver operating characteristic (ROC) curve analysis revealed an area under the ROC curve of 0.8779 (95% confidence interval, 0.7987-0.9571) and a sensitivity and specificity of 82.5% and 80.0%, respectively. Moreover, exosomal miR-210 was upregulated at an advanced stage, and Fuhrman grade and metastasis decreased significantly one month after surgery. Acute hypoxia exposure activates miR-210 and release of exosomes with upregulated miR-210 in both normal and tumor RCC cell lines and interferes with vacuole membrane protein 1 mRNA expression, especially in the metastatic ccRCC cell line. In conclusion, Serum exosomal miR-210 originating from tumor tissue has potential as a novel noninvasive biomarker for the detection and prognosis of ccRCC.     

肝细胞癌患者血清外泌体microRNA表达鉴定

目的:探讨肝癌细胞外泌体中差异表达的microRNAs(miRNAs)在肝细胞癌(HCC)诊断中的应用价值。方法:通过高通量测序筛选肝癌细胞外泌体中差异表达的miRNAs。实时定量PCR验证差异表达分子;检测差异表达的miRNAs在健康人(Health)、慢性乙型肝炎患者(CHB)、肝硬化患者(LC)及乙型肝炎病毒阳性的肝细胞癌患者(HCC)血清外泌体中的表达。结果:高通量测序筛选到肝癌细胞外泌体中差异表达的miRNA共88种,其中58种表达上调,30种表达下调。选择其中8种差异表达的miRNAs进行q RT-PCR验证,结果显示,此8种miRNAs在细胞上清外泌体、细胞内、癌与癌旁组织中的表达趋势与测序结果一致。miR-221-3p和miR-224-5p在HCC组外泌体中的表达水平显著高于Health组、CHB组和LC组(P0.01),miR-124-3p和let-7a-5p在HCC组外泌体中的表达水平显著低于其他各组(P0.05)。四个组中,miR-21-5p、miR-191-5p、miR-34a-5p和miR-122-5p的表达水平不存在显著性差异(P0.05)。结论:血清外泌体中的miR-221-3p、miR-224-5p、miR-124-3p和let-7a-5p可能成为肝细胞癌的候选标志物。     

microRNA-16-5p-containing exosomes derived from bone marrow-derived mesenchymal stem cells inhibit proliferation,migration, and invasion,while promoting apoptosis of colorectal cancer cells by downregulating ITGA2

Colorectal cancer (CRC) is a form of cancer developing from either the colon or rectum. Nowadays, research supports the functionality of exosome expressing microRNAs (miRNAs) as potential biomarker for various cancers including CRC. This study was performed with the intent of investigating the roles of both bone marrow-derived mesenchymal stem cells (BMSCs) and exosomal miR-16-5p in CRC by regulating integrin α2 (ITGA2). A microarray-based analysis was conducted to screen the CRC-associated differentially expressed genes (DEGs) as well as potential regulatory miRNAs. Next, the role of miR-16-5p in terms of its progression in association with CRC was determined. Subsequently, CRC cells were exposed to exosomes secreted by BMSCs transfected with miR-16-5p, isolated and cocultured with CRC cells in an attempt to identify the role of exosomes. Effects of BMSCs-derived exosomes overexpressing miR-16-5p on biological functions of CRC cells and tumorigenicity were all subsequently detected. Effects of miR-16-5p treated with CRC cells in regard to CRC in vivo were also measured. ITGA2 was overexpressed, while miR-16-5p was poorly expressed in CRC cells and miR-16-5p targeted ITGA2. The in vitro experiments revealed that the BMSCs-derived exosomes overexpressing miR-16-5p inhibited proliferation, migration, and invasion, while simultaneously stimulating the apoptosis of the CRC cells via downregulation of ITGA2. Furthermore, the results of in vivo experiments confirmed that the BMSCs-derived exosomes overexpressing miR-16-5p repressed the tumor growth of CRC. Collectively, BMSCs-derived exosomes overexpressing miR-16-5p restricted the progression of CRC by downregulating ITGA2.     

Exosomal microRNA-1246 from human umbilical cord mesenchymal stem cells potentiates myocardial angiogenesis in chronic heart failure

microRNAs (miRNAs) are of importance to chronic heart failure (CHF). However, the relevance of the exosomal miRNAs produced during CHF remains unknown. Our purpose here was to examine the relevance of exosomal microRNA-1246 (miR-1246) released from human umbilical cord mesenchymal stem cell (hucMSC) during CHF and the mechanism of action. Cardiac function, myocardial infarction area, apoptosis, and angiogenesis were all evaluated in a CHF rat model following treatment with hucMSC-derived exosomes (hucMSC-Exos). H9C2 and human umbilical vascular endothelial cells (HUVECs) were subjected to oxygen and glucose deprivation and exosome treatment to quantify the cell proliferation and apoptosis in H9C2 cells and the tube formation capacity of the HUVECs. A dual-luciferase activity reporter assay was conducted to validate the interaction between miR-1246 and serine protease 23 (PRSS23). HucMSCs treatment led to a reduction in H9C2 apoptosis and an increase in HUVEC angiogenesis, which were mitigated when hucMSCs were treated with a miR-1246 inhibitor. We also confirmed that PRSS23 is a putative target of miR-1246 and that miR-1246 attenuated hypoxia-induced myocardial tissue damage by targeting PRSS23 and inhibiting the activation of the Snail/alpha-smooth muscle actin signaling. Our findings suggest that exosomal miR-1246 from hucMSCs protects the heart from failure by targeting PRSS23.     

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.Subject terms: Ligand-gated ion channels, Metastasis, Melanoma, Ion channel signalling, miRNAs     

Cancer-associated fibroblast-derived exosomal microRNA-24-3p enhances colon cancer cell resistance to MTX by down-regulating CDX2/HEPH axis

MicroRNA-24-3p (miR-24-3p) has been implicated as a key promoter of chemotherapy resistance in numerous cancers. Meanwhile, cancer-associated fibroblasts (CAFs) can secret exosomes to transfer miRNAs, which mediate tumour development. However, little is known regarding the molecular mechanism of CAF-derived exosomal miR-24-3p in colon cancer (CC). Hence, this study intended to characterize the functional relevance of CAF-derived exosomal miR-24-3p in CC cell resistance to methotrexate (MTX). We identified differentially expressed HEPH, CDX2 and miR-24-3p in CC through bioinformatics analyses, and validated their expression in CC tissues and cells. The relationship among HEPH, CDX2 and miR-24-3p was verified using ChIP and dual-luciferase reporter gene assays. Exosomes were isolated from miR-24-3p inhibitor–treated CAFs (CAFs-exo/miR-24-3p inhibitor), which were used in combination with gain-of-function and loss-of-function experiments and MTX treatment. CCK-8, flow cytometry and colony formation assays were conducted to determine cell viability, apoptosis and colony formation, respectively. Based on the findings, CC tissues and cells presented with high expression of miR-24-3p and low expression of HEPH and CDX2. CDX2 was a target gene of miR-24-3p and could up-regulate HEPH. Under MTX treatment, overexpressed CDX2 or HEPH and down-regulated miR-24-3p reduced cell viability and colony formation and elevated cell apoptosis. Furthermore, miR-24-3p was transferred into CC cells via CAF-derived exosomes. CAF-derived exosomal miR-24-3p inhibitor diminished cell viability and colony formation and increased cell apoptosis in vitro and inhibited tumour growth in vivo under MTX treatment. Altogether, CAF-derived exosomal miR-24-3p accelerated resistance of CC cells to MTX by down-regulating CDX2/HEPH axis.     

Exosomes Derived from Hypoxic Leukemia Cells Enhance Tube Formation in Endothelial Cells

Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.     

Exosomes from human umbilical cord mesenchymal stem cells inhibit ROS production and cell apoptosis in human articular chondrocytes via the miR-100-5p/NOX4 axis

Cyclic strain-induced chondrocyte damage is actively involved in the pathogenesis of osteoarthritis and arthritis. MicroRNAs (miRNAs) carried by exosomes have been implicated in various diseases. However, the role of miR-100-5p in cyclic strain-induced chondrocyte damage remains to be elucidated. miR-100-5p and NADPH oxidase 4 (NOX4) were silenced or overexpressed in human primary articular chondrocytes. PKH-67 Dye was used to trace exosome endocytosis. Reactive oxygen species (ROS) production was monitored using DCFH-DA. Cell apoptosis was measured using a flow cytometer. Quantitative RT-PCR and Western blots were used to evaluate gene expression. Cyclic strain promoted ROS production and apoptosis in primary articular chondrocytes in a time-dependent manner. HucMSCs-derived exosomal miR-100-5p inhibited cyclic strain-induced ROS production and apoptosis in primary articular chondrocytes. miR-100-5p directly targeted NOX4. Overexpressing NOX4 attenuated hucMSCs-derived exosomes-mediated protective effects in primary articular chondrocytes. Cyclic strain promotes ROS production and apoptosis in primary articular chondrocytes, which was abolished by hucMSCs-derived exosomal miR-100-5p through its target NOX4. The findings highlight the importance of miR-100-5p/NOX4 axis in primary articular chondrocytes injury and provide new insights into therapeutic strategies for articular chondrocytes injury and osteoarthritis.     

高通量测序技术分析急性髓系白血病血浆外泌体微RNA表达谱差异

急性髓系白血病（acute myeloid leukemia,AML）是一类造血干细胞的恶性克隆性疾病,目前的诊断方法不利于疾病的早期发现,且诊断结果重复性较差。已有大量研究显示,细胞外microRNA（miRNA或miR）富集在外泌体（exosome）中,且受其表面膜的保护而具有很好的稳定性,是理想的分子标志物。目前,多种实体肿瘤均已检测到肿瘤特异性外泌体miRNA(exosomal miRNA)。然而,在AML患者中未见此外泌体miRNA报道。本研究探讨急性髓系白血病血浆外泌体miRNA表达谱差异及新miRNA序列。采用solexa高通量测序技术对7例AML患者（AML组）及7例健康对照者（对照组）血浆外泌体miRNAs进行测序,利用Mireap预测软件进行新miRNAs分析,通过edger差异分析软件筛选组间差异miRNA,获得211个已知的差异表达miRNAs以及2个新miRNAs,选择4个差异表达的miRNAs：miR-155-5p、miR-335-5p、miR-451a及xxx-m0038 5p（新miRNA）,在两组（各23例）的血浆外泌体样本中,进行实时荧光定量PCR（qRT-PCR）验证,验证结果与测序结果一致。对差异表达的外泌体miRNA进行靶基因预测及其GO（Gene Ontology）和信号通路富集分析,发现靶基因聚集的生物学功能多数参与生物进展过程的调控。靶基因主要富集在FoxO、MAPK、Hippo信号通路以及HTLV-I感染等。结果显示,AML患者与健康对照者的血浆外泌体miRNA存在着差异性表达。差异性表达的miRNA特异性很高,对进一步阐明AML白血病发生与发展的分子机制、研发新的无创诊断方法、新的诊断标记物和有效治疗AML的方法具有十分重要和深远的意义。     

Cancer-associated fibroblasts-derived exosomes upregulate microRNA-135b-5p to promote colorectal cancer cell growth and angiogenesis by inhibiting thioredoxin-interacting protein

ObjectiveThe role of exosomes in human cancers has been identified, while the effect of cancer-associated fibroblasts (CAFs)-derived exosomes (CAF-exos) transmitting microRNAs (miRNAs) on colorectal cancer (CRC) remains largely unknown. We aim to explore the impact of CAF-derived exosomal miR-135b-5p on CRC progression by targeting thioredoxin-interacting protein (TXNIP).MethodsCRC tissues were collected to obtain CAF-exos, which were used to co-culture with LoVo and HT29 cells. The effect of miR-135b-5p and TXNIP on the in vivo growth, in vitro proliferation, apoptosis, migration, invasion and angiogenesis of CRC cells. miR-135b-5p and TXNIP expression in exosomes and CRC cells were detected and their targeting relationship was confirmed.ResultsMiR-135b-5p was upregulated whereas TXNIP was downregulated in CRC tissues and cells. The CAF-exos and CAF-exos upregulating miR-135b-5p promoted in vivo growth, in vitro proliferation, migration and invasion, and suppressed apoptosis of CRC cells, and also promoted the HUVEC angiogenesis. TXNIP was confirmed as a target of miR-135b-5p and overexpression of TXNIP could weaken the pro-CRC effect of exosomal miR-135b-5p,ConclusionCAF-exos upregulate miR-135b-5p to promote CRC cell growth and angiogenesis by inhibiting TXNIP.     

Deep Sequencing of RNA from Three Different Extracellular Vesicle (EV) Subtypes Released from the Human LIM1863 Colon Cancer Cell Line Uncovers Distinct Mirna-Enrichment Signatures

Secreted microRNAs (miRNAs) enclosed within extracellular vesicles (EVs) play a pivotal role in intercellular communication by regulating recipient cell gene expression and affecting target cell function. Here, we report the isolation of three distinct EV subtypes from the human colon carcinoma cell line LIM1863 – shed microvesicles (sMVs) and two exosome populations (immunoaffinity isolated A33-exosomes and EpCAM-exosomes). Deep sequencing of miRNA libraries prepared from parental LIM1863 cells/derived EV subtype RNA yielded 254 miRNA identifications, of which 63 are selectively enriched in the EVs - miR-19a/b-3p, miR-378a/c/d, and miR-577 and members of the let-7 and miR-8 families being the most prominent. Let-7a-3p*, let-7f-1-3p*, miR-451a, miR-574-5p*, miR-4454 and miR-7641 are common to all EV subtypes, and 6 miRNAs (miR-320a/b/c/d, miR-221-3p, and miR-200c-3p) discern LIM1863 exosomes from sMVs; miR-98-5p was selectively represented only in sMVs. Notably, A33-Exos contained the largest number (32) of exclusively-enriched miRNAs; 14 of these miRNAs have not been reported in the context of CRC tissue/biofluid analyses and warrant further examination as potential diagnostic markers of CRC. Surprisingly, miRNA passenger strands (star miRNAs) for miR-3613-3p*, -362-3p*, -625-3p*, -6842-3p* were the dominant strand in A33-Exos, the converse to that observed in parental cells. This finding suggests miRNA biogenesis may be interlinked with endosomal/exosomal processing.     

Circulating Exosomal miR-17-5p and miR-92a-3p Predict Pathologic Stage and Grade of Colorectal Cancer

Exosomes are extracellular membrane vesicles of 50- to 130-nm diameter secreted by most tumor cells. Exosomes can mediate the intercellular transfer of proteins and RNAs, including microRNAs (miRNAs), and promote both tumorigenesis and premetastatic niche formation. In this study, we performed exosomal RNA sequencing to identify candidate exosomal miRNAs that could be associated with colorectal cancer (CRC) and its distant metastasis. The expression profiles of exosomal miRNA, as secreted by isogenic human primary CRC cell line SW480 and highly metastatic cell line SW620, were analyzed and the potential targets related to tumorigenesis and metastatic progression were investigated. We found that 25 miRNAs had been up-regulated and 5 miRNAs had been down-regulated in exosomes purified from SW620 culture supernatant. Candidate miRNAs were further evaluated for CRC diagnosis using quantitative real-time polymerase chain reaction in CRC patients. Higher expression levels of circulating exosomal miR-17-5p and miR-92a-3p were significantly associated with pathologic stages and grades of the CRC patients. CONCLUSIONS: Circulating exosomal miR-17-5p and miR-92a-3p may provide a promising noninvasive prognostic biomarker for primary and metastatic CRC.