Rapid Detection of Exosomal MicroRNAs Using Virus‐Mimicking Fusogenic Vesicles

Exosomal microRNAs (miRNAs) are important biomarkers for clinical diagnosis and disease treatment monitoring. However, most approaches for exosomal miRNA detection are time‐consuming, laborious, and expensive. Herein, we report a virus‐mimicking fusogenic vesicle (Vir‐FV) that enables rapid, efficient, and high‐throughput detection of exosomal miRNAs within 2 h. Fusogenic proteins on Vir‐FVs can specifically target the sialic‐acid‐containing receptors on exosomes, inducing efficient fusion of Vir‐FVs and exosomes. Upon vesicle content mixing, the molecular beacons encapsulated in Vir‐FVs specifically hybridize with the target miRNAs in the exosomes, generating fluorescence. Combined with flow cytometry, the Vir‐FVs can not only detect exosomal miRNAs but also distinguish tumor exosomes from normal exosomes by sensing the tumor‐related miRNAs, paving the way towards the rapid and efficient detection of exosomal miRNAs for diagnosis and prognosis prediction of diseases.     

Plasma Exosomes of Patients with Breast and Ovarian Tumors Contain an Inactive 20S Proteasome

Exosomes are directly involved in governing of physiological and pathological conditions of an organism through the transfer of information from producing to receiving cells. It can be assumed that exosomes are one of the key players of tumor dissemination since they are very stable and small enough to penetrate from various tissues into biological fluids and then back, thus interacting with tissue target cells. We evaluated the enzymatic activity and the level of 20S proteasome in tissue and exosomes of healthy females (n = 39) and patients with ovarian (n = 50) and breast (n = 108) tumors to reveal the critical role of exosomal cargo in the mediation of different types of metastases. Exosomes from plasma and ascites were isolated and characterized in according to International Society for Extracellular Vesicles guidelines. The level of 20S proteasome in tissue and exosomes was determined using Western blot analysis. Chymotrypsin- and caspase-like (ChTL and CL, respectively) peptidase activities of the proteasomes were determined using fluorogenic Suc-LLVY-AMC and Cbz-LLG-AMC substrates, respectively. We observed increased levels of 20S proteasome in ovarian cancer tissue and luminal B subtype breast cancer tissue as well as in plasma exosomes from cancer patients. Moreover, the level of the 20S proteasome in plasma exosomes and ascites exosomes in patients with ovarian tumors is comparable and higher in ovarian cancer patients with low volume ascites than in patients with moderate and high-volume ascites. We also found increased ChTL and CL activities in breast cancer and ovarian cancer tissues, as well as in peritoneal metastases in ovarian cancer, while proteasomal activity in exosomes from plasma of healthy females and all patients, as well as from ascites of ovarian tumor patients were lower than detection limit of assay. Thus, regardless of the type of tumor metastasis (lymphogenous or peritoneal), the exosomes of cancer patients were characterized by an increased level of 20S proteasome, which do not exhibit enzymatic activity.     

Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.     

Toehold‐initiated Rolling Circle Amplification for Visualizing Individual MicroRNAs In Situ in Single Cells

The ability to quantitate and visualize microRNAs (miRNAs) in situ in single cells would greatly facilitate the elucidation of miRNA‐mediated regulatory circuits and their disease associations. A toehold‐initiated strand‐displacement process was used to initiate rolling circle amplification of specific miRNAs, an approach that achieves both stringent recognition and in situ amplification of the target miRNA. This assay, termed toehold‐initiated rolling circle amplification (TIRCA), can be utilized to identify miRNAs at physiological temperature with high specificity and to visualize individual miRNAs in situ in single cells within 3 h. TIRCA is a competitive candidate technique for in situ miRNA imaging and may help us to understand the role of miRNAs in cellular processes and human diseases in more detail.     

Sequence-based analysis of 5′UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5′UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5′UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p’s binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5′UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5′UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.     

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

WNT1 Gene from WNT Signaling Pathway Is a Direct Target of miR-122 in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is an invasive form of hepatic cancer arising from the accumulation of multiple genetic alterations. In this study, the causal role of disturbed canonical Wnt/β-catenin pathway was approved, and some of HCC-driven important gene candidates were determined. MicroRNAs (miRNAs), small non-coding RNAs, are the key regulators of important cancer genes, and their participation in tumorigenesis has been shown. By reviewing literature, WNT1 gene with functional significance was selected to approve miRNAs as new subjects for targeted therapy.For proper and fast miRNA detection and also confirmation of the role of bioinformatics in obtaining practical data, we benefited from different bioinformatics tools such as TargetScan, miRanda, and DIANA. In order to use an HCC model, we used HepG2 cell line. Luciferase assay was applied to assess the ability of the selected miRNAs in targeting WNT1 3′-UTR. To overexpress the selected miRNA in HepG2 cell line, viral construct was prepared. Quantitative real-time PCR was performed to evaluate selected miRNA and target gene expression levels. miR-122 was selected according to data concerning various bioinformatics tools.miR-122 was downregulated and WNT1 gene expression was upregulated in HepG2 cell line. After viral construct transduction, miR-122 expression was elevated and WNT1 expression was notably declined. Finally, we introduced WNT1 gene as one of the important genes in HCC, and also, we showed that miR-122 can regulate WNT1 gene expression.Moreover, our study determines the potential of bioinformatics analyses in providing accurate and reliable data for miRNA: messenger RNA (mRNA) prediction.     

Capillary electrophoresis methods for microRNAs assays: A review

MicroRNAs (miRNAs) are short noncoding RNAs that conduct important roles in many cellular processes such as development, proliferation, differentiation, and apoptosis. In particular, circulating miRNAs have been proposed as biomarkers for cancer, diabetes, cardiovascular disease, and other illnesses. Therefore, determination of miRNA expression levels in various biofluids is important for the investigation of biological processes in health and disease and for discovering their potential as new biomarkers and drug targets. Capillary electrophoresis (CE) is emerging as a useful analytical tool for analyzing miRNA because of its simple sample preparation steps and efficient resolution of a diverse size range of compounds. In particular, CE with laser-induced fluorescence detection is a promising and relatively rapidly developing tool with the potential to provide high sensitivity and specificity in the analysis of miRNAs. This paper covers a short overview of the recent developments and applications of CE systems in miRNA studies in biological and biomedical areas.     

Trends in microRNA detection

MicroRNAs (miRNAs) are short, ~22 nucleotide length RNAs that perform gene regulation. Recently, miRNA has been shown to be linked with the onset of cancer and other diseases based on miRNA expression levels. It is important, therefore, to understand miRNA function as it pertains to disease onset; however, in order to fully understand miRNA’s role in a disease, it is necessary to detect the expression levels of these small molecules. The most widely used miRNA detection method is Northern blotting, which is considered as the standard of miRNA detection methods. This method, however, is time-consuming and has low sensitivity. This has led to an increase in the amount of detection methods available. These detection methods are either solid phase, occurring on a solid support, or solution phase, occurring in solution. While the solid-phase methods are adaptable to high-throughput screening and possess higher sensitivity than Northern blotting, they lack the ability for in vivo use and are often time-consuming. The solution-phase methods are advantageous in that they can be performed in vivo, are very sensitive, and are rapid; however, they cannot be applied in high-throughput settings. Although there are multiple detection methods available, including microarray technology, luminescence-based assays, electrochemical assays, etc., there is still much work to be done regarding miRNA detection. The current gaps of miRNA detection include the ability to perform multiplex, sensitive detection of miRNA with single-nucleotide specificity along with the standardization of these new methods. Current miRNA detection methods, gaps in these methods, miRNA therapeutic options, and the future outlook of miRNA detection are presented here.     

Macrophage-targeted nanomedicine for chronic diseases immunotherapy

Macrophage is the key innate immune effector in first-line defense against the pathogens, and can be polarized into different phenotypes to regulate a variety of immunological functions. However, the plasticity of macrophage is extraordinarily recruited, activated, and polarized under pathological conditions,playing paramount roles in occurrence, development, and prognosis of various chronic diseases, such as rheumatoid arthritis(RA), atherosclerosis(AS), and cancer. To this end, macrophage has ...