Differentially Expressed Extracellular Vesicle-Contained microRNAs before and after Transurethral Resection of Bladder Tumors

Bladder cancer (BC) is currently diagnosed and monitored by cystoscopy, a costly and invasive procedure. Potential biomarkers in urine, blood, and, more recently, extracellular vesicles (EVs), have been explored as non-invasive alternatives for diagnosis and surveillance of BC. EVs are nanovesicles secreted by most cell types containing diverse molecular cargo, including different types of small RNAs, such as microRNA (miRNA). In this study, we performed next-generation sequencing of EV-contained miRNA isolated from urine and serum of 41 patients with non-muscle invasive BC (27 stage Ta, 14 stage T1) and 15 non-cancer patients (NCP) with benign cystoscopy findings. MiRNA sequencing was also performed on serum supernatant samples for T1 patients. To identify potential BC-specific biomarkers, expression levels of miRNA in presurgery samples were compared to those at postsurgery check-ups, and to NCPs. Results showed that two miRNAs, urinary EV-contained miR-451a and miR-486-5p, were significantly upregulated in presurgery samples from T1 patients compared to postsurgery check-up samples. This was confirmed in a replica EV/RNA isolation and sequencing run of 10 T1 patients from the primary run; however, analyses revealed no differential expression of miRNAs in serum EVs, serum supernatant, or when comparing BC patients to NCPs. This is the first study to investigate EV-containing miRNA sequencing in pre- and postsurgery BC patient samples and our findings suggest that urinary EV-contained miR-451a and miR-486-5p may be potential biomarkers for recurrence-free survival of BC patients with stage T1 disease.     

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.     

microRNAs associated with the different human Argonaute proteins

MicroRNAs (miRNAs) are small noncoding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the 3′-untranslated region (3′-UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1–3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected.     

Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance

microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.     

MicroRNA Cargo of Extracellular Vesicles from Alcohol-exposed Monocytes Signals Naive Monocytes to Differentiate into M2 Macrophages

Membrane-coated extracellular vesicles (EVs) released by cells can serve as vehicles for delivery of biological materials and signals. Recently, we demonstrated that alcohol-treated hepatocytes cross-talk with immune cells via exosomes containing microRNA (miRNAs). Here, we hypothesized that alcohol-exposed monocytes can communicate with naive monocytes via EVs. We observed increased numbers of EVs, mostly exosomes, secreted by primary human monocytes and THP-1 monocytic cells in the presence of alcohol in a concentration- and time-dependent manner. EVs derived from alcohol-treated monocytes stimulated naive monocytes to polarize into M2 macrophages as indicated by increased surface expression of CD68 (macrophage marker), M2 markers (CD206 (mannose receptor) and CD163 (scavenger receptor)), secretion of IL-10, and TGFβ and increased phagocytic activity. miRNA profiling of the EVs derived from alcohol-treated THP-1 monocytes revealed high expression of the M2-polarizing miRNA, miR-27a. Treatment of naive monocytes with control EVs overexpressing miR-27a reproduced the effect of EVs from alcohol-treated monocytes on naive monocytes and induced M2 polarization, suggesting that the effect of alcohol EVs was mediated by miR-27a. We found that miR-27a modulated the process of phagocytosis by targeting CD206 expression on monocytes. Importantly, analysis of circulating EVs from plasma of alcoholic hepatitis patients revealed increased numbers of EVs that contained high levels of miR-27a as compared with healthy controls. Our results demonstrate the following: first, alcohol increases EV production in monocytes; second, alcohol-exposed monocytes communicate with naive monocytes via EVs; and third, miR-27a cargo in monocyte-derived EVs can program naive monocytes to polarize into M2 macrophages.     

Small RNA deep sequencing reveals a distinct miRNA signature released in exosomes from prion-infected neuronal cells

Prion diseases are transmissible neurodegenerative disorders affecting both humans and animals. The cellular prion protein, PrP^C, and the abnormal infectious form, PrP^Sc, are found associated with exosomes, which are small 50–130 nm vesicles released from cells. Exosomes also contain microRNAs (miRNAs), a class of non-coding RNA, and have been utilized to identify miRNA signatures for diagnosis of disease. While some miRNAs are deregulated in prion-infected brain tissue, the role of miRNA in circulating exosomes released during prion disease is unknown. Here, we investigated the miRNA profile in exosomes released from prion-infected neuronal cells. We performed the first small RNA deep sequencing study of exosomes and demonstrated that neuronal exosomes contain a diverse range of RNA species including retroviral RNA repeat regions, messenger RNA fragments, transfer RNA fragments, non-coding RNA, small nuclear RNA, small nucleolar RNA, small cytoplasmic RNA, silencing RNA as well as known and novel candidate miRNA. Significantly, we show that exosomes released by prion-infected neuronal cells have increased let-7b, let-7i, miR-128a, miR-21, miR-222, miR-29b, miR-342-3p and miR-424 levels with decreased miR-146 a levels compared to non-infected exosomes. Overall, these results demonstrate that circulating exosomes released during prion infection have a distinct miRNA signature that can be utilized for diagnosis and understanding pathogenic mechanisms in prion disease.     

Targeting miR-9 in Glioma Stem Cell-Derived Extracellular Vesicles: A Novel Diagnostic and Therapeutic Biomarker

BackgroundGlioblastoma (GBM) is a lethal brain tumor with no effective strategies in early diagnosis and treatment. This study was aimed to assess the miRNA expression profiles in EVs from CSF and tissue of glioblastoma patients to identify significantly upregulated miRNAs and investigate the underlying neoplastic mechanisms.MethodsEVs were measured by TEM and NTA assays. Differentially regulated miRNAs were measured using RNA sequencing in GBM CSF EVs and in GBM tissues compared with controls. RT-qPCR was employed to analyze miRNA and gene expression. Luciferase report assay was used to investigate gene target of miR-9. The proliferation ability was detected by EdU and CCK-8 experiment while cell migration was measured by transwell and wound healing assay.ResultsThe expression level of miR-9 was significantly higher in GBM CSF EVs and tissues than controls (p = 0.038). The area under curve for CSF EV miR-9 was 0.800 (95% CI: 0.583–1.000, p = 0.033). The expression of miR-9 was significantly higher in Glioma stem cells (GSCs) and GSC-derived EVs than in glioblastoma cells. GSC-derives EVs could promote GBM growth and migration Moreover, inhibition of miR-9 in GSCs showed the reverse anti-tumor effects through secreted EVs. MiR-9 could bind to the 3’UTR region of DACT3 and suppress its expression. The miR-9/DACT3 axis might attribute to GBM malignant phenotype.ConclusionMiR-9 in CSF EVs may act as a novel diagnostic biomarker for GBM and targeting miR-9 by GSC-derived EVs may be a specific and efficient strategy for GBM biotherapy.     

高通量miRNA活性谱检测发现BHK21细胞中miR-206高活性

用我们建立的活细胞miRNA活性谱检测技术测定BHK21、HEK293和Vero三种肾来源细胞系中58种miRNA活性谱,发现miR-206在BHK21细胞中具有特征性高活性.鉴于miR-206是典型的横纹肌特征性miRNA,进一步以小鼠成肌细胞C2Cl2及人胚肾细胞HEK293为对照,检测了BHK21细胞中miR-206的活性及表达水平.之后,用马血清诱导培养BHK21细胞,检测诱导前后BHK21细胞中骨骼肌肌球蛋白重链(Slow skeletal myosin heavy chain,MHC)表达情况,miR-206活性和表达水平以及受miR-206负调控的Connexin43(Cx43)的表达水平.结果发现,miR-206在BHK21细胞中活性和表达水平都明显高于C2C12细胞;马血清诱导后,BHK21细胞中MHC表达水平升高,miR-206活性和表达水平都升高,而Cx43表达水平下降.结果提示BHK21细胞具有成肌细胞特性.本研究首次发现BHK21细胞中miR-206的高活性,从miRNA角度证实了BHK21细胞来源于肾间质细胞,而不是肾实质细胞.研究结果还提示BHK21细胞有可能作为一种体外模型用于miR-206的功能研究.     

MicroRNAs are exported from malignant cells in customized particles

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.     

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。     

MicroRNAs associated with mitogen-activated protein kinase in human pancreatic cancer

Aberrant expression of microRNAs (miRNA) is associated with phenotypes of various cancers, including pancreatic cancer. However, the mechanism of the aberrant expression is largely unknown. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway plays a crucial role in gene expression related to the malignant phenotype of pancreatic cancer. Hence, we studied the role of MAPK in the aberrant expression of miRNAs in pancreatic cancer cells. The alterations in expression of 183 miRNAs induced by activation or inactivation of MAPK were assayed in cultured pancreatic cancer cells and HEK293 cells by means of the quantitative real-time PCR method. We found that four miRNAs, namely, miR-7-3, miR-34a, miR-181d, and miR-193b, were preferentially associated with MAPK activity. Among these miRNAs, miR-7-3 was upregulated by active MAPK, whereas the others were downregulated. Promoter assays indicated that the promoter activities of the host genes of miR-7-3 and miR-34a were both downregulated by alteration in MAPK activity. Exogenous overexpression of the MAPK-associated miRNAs had the effect of inhibition of the proliferation of cultured pancreatic cancer cells; miR-193b was found to exhibit the most remarkable inhibition. A search for target genes of miR-193b led to identification of CCND1, NT5E, PLAU, STARD7, STMN1, and YWHAZ as the targets. Translational suppression of these genes by miR-193b was confirmed by reporter assay. These results indicate that activation of MAPK may play a significant role in aberrant expression of miRNAs and their associated phenotypes in pancreatic cancer.     

Upregulation of miR-23a～27a～24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature in a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. Here, we show that the over expression of miR-23a∼27a∼24-2 cluster in HEK293T cells induces apoptosis by caspase-dependent as well as caspase-independent pathway as proved by the annexin assay, caspase activation, release of cytochrome-c and AIF (apoptosis inducing factor) from mitochondria. Furthermore, the over expressed cluster modulates the expression of a number of genes involved in apoptosis including FADD (Fas Associated protein with Death Domain). Bioinformatically, FADD is predicted to be the target of hsa-miR-27a and interestingly, FADD protein was found to be up regulated consistent with very less expression of hsa-miR-27a in HEK293T cells. This effect was direct, as hsa-miR-27a negatively regulated the expression of FADD 3′UTR based reporter construct. Moreover, we also showed that over expression of miR-23a∼27a∼24-2 sensitized HEK293T cells to TNF-α cytotoxicity. Taken together, our study demonstrates that enhanced TNF-α induced apoptosis in HEK293T cells by over expression of miR-23a∼27a∼24-2 cluster provides new insights in the development of novel therapeutics for cancer.