GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer

Colorectal cancer (CRC) is the second leading cause of cancer‐related deaths worldwide. However, a biomarker for a sensitive and simple diagnostic test and highly effective target therapy of CRC is still clinically unavailable. This study is to investigate the evidence and significance of plasma GPC1 positive exosomes as a biomarker of CRC. Results showed that GPC1⁺ exosomes were successfully isolated from tissues and plasma. The percentage of GPC1⁺ exosomes and the GPC1 protein expression in exosomes from tumour tissues and plasma of CRC patients before surgical treatment was significantly elevated compared to that in the peritumoural tissues and the plasma of healthy controls. miR‐96‐5p and miR‐149 expression in tumour tissues and plasma of CRC patients as well as in the GPC1⁺ exosomes from CRC patients were significantly decreased compared to that in the peritumoural tissues and the plasma of healthy controls. Two months after surgical treatment, levels of all tested markers significantly normalized. Overexpression of miR‐96‐5p and miR‐149 significantly decreased GPC1 expression in HT‐29 and HCT‐116 cells, xenograft tumours, plasma in mice bearing HT‐29 and HCT‐116 tumours, and the secretion of GPC1⁺ exosomes from the HT‐29 and HCT‐116 cells and xenograft tumours. Overexpression of miR‐96‐5p and miR‐149 significantly decreased cell viability and increased cell apoptosis in HT‐29 and HCT‐116 cells, and inhibited the growth of xenograft HT‐29 and HCT‐116 tumours. In conclusion, the increased plasma GPC1⁺ exosomes and reduced plasma miR‐96‐5p and miR‐149 expression are specific markers for the diagnosis of CRC and targets for the therapy of CRC.     

Quantifying compartment‐associated variations of protein abundance in proteomics data

Quantitative mass spectrometry enables to monitor the abundance of thousands of proteins across biological conditions. Currently, most data analysis approaches rely on the assumption that the majority of the observed proteins remain unchanged across compared samples. Thus, gross morphological differences between cell states, deriving from, e.g., differences in size or number of organelles, are often not taken into account. Here, we analyzed multiple published datasets and frequently observed that proteins associated with a particular cellular compartment collectively increase or decrease in their abundance between conditions tested. We show that such effects, arising from underlying morphological differences, can skew the outcome of differential expression analysis. We propose a method to detect and normalize morphological effects underlying proteomics data. We demonstrate the applicability of our method to different datasets and biological questions including the analysis of sub‐cellular proteomes in the context of Caenorhabditis elegans aging. Our method provides a complementary perspective to classical differential expression analysis and enables to uncouple overall abundance changes from stoichiometric variations within defined group of proteins.     

Chiroptical spectroscopy and metabolomics for blood‐based sensing of pancreatic cancer

To enable the early diagnosis of pancreatic cancer, the search for and definition of reliable biomarkers remain a subject of great interest, with the specificity and sensitivity of the currently used biomarkers being below the required values. We tested a novel diagnostic approach for pancreatic cancer based on the specific molecular signature of blood plasma components. To acquire more detailed structural information, structure‐sensitive chiroptical methods (electronic circular dichroism and Raman optical activity) were supplemented by conventional Raman and infrared spectroscopies. The obtained spectra were subsequently processed by linear discriminant analysis yielding high values of specificity and sensitivity. In addition, to monitor not only large biomolecules as potential biomarkers but also those of low molecular weight, we conducted an analysis of blood plasma samples by using metabolomics. The achieved results suggest a panel of promising biomarkers for a reliable detection of pancreatic cancer.     

外泌体及其在肿瘤诊疗中的意义

外泌体(exosome)是由多种活细胞分泌的囊泡小体,其中含有蛋白质和RNA等多种组分。这种机体内普遍存在的纳米级被膜结构能够参与细胞间的物质交换和信息交流,在多种生理和病理过程中发挥重要作用。外泌体在外周血、尿液、唾液、腹水、羊水等体液中具有很高的丰度,而不同组织来源的外泌体在组成和功能方面存在差异,同时这种差异受到细胞外基质和微环境的动态调控。肿瘤来源或肿瘤相关的外泌体是调控肿瘤发生发展的重要机制,对肿瘤外泌体的分析和检测可以辅助肿瘤的早期诊断、疗效评价和预后分析。此外,外泌体及其修饰加工产物还可以作为基因或药物的有效载体,用于肿瘤治疗。关于外泌体的研究是肿瘤学的一个新兴领域,在转化医学的研究模式下,将极大推动肿瘤学研究进展,为肿瘤临床诊断和治疗带来新的契机。     

Cellular context in epigenetics: quantitative multicolor imaging and automated per-cell analysis of miRNAs and their putative targets

This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis.     

Potential application of miRNAs as diagnostic and therapeutic tools in chronic pancreatitis

Chronic pancreatitis (CP) is a progressive inflammatory disease typified by end‐stage fibrosis. This disease can also increase the risk of pancreatic cancer. The associated diagnosis, pain and other complications further add to the burden of disease management. In recent years, significant progress has been achieved in identifying miRNAs and their physiological functions, including mRNA repression and protein expression control. Given the extensive effort made on miRNA research, a close correlation has been discovered between certain types of miRNAs and disease progression, particularly for tissue fibrosis. Designing miRNA‐related tools for disease diagnosis and therapeutic treatments presents a novel and potential research frontier. In the current review, we discuss various miRNAs closely interacting with CP, as well as the possible development of targeted miRNA therapies in managing this disease.     

Clinical utility of plasma miR‐371a‐3p in germ cell tumors

Germ cell tumours predominantly of the testis ((T)GCTs) are remarkably chemotherapy sensitive. However, a small proportion of patients fail to be cured with cisplatin‐based combination chemotherapy. miR‐371a‐3p is a new liquid biopsy biomarker for (T)GCTs. The aim of this study was to evaluate clinical utility of plasma miR‐371a‐3p level in patients starting systemic chemotherapy. Patients were included before the first cycle (N = 180) and second cycle (N = 101) of systemic first line chemotherapy, treated between July 2010 and May 2017. Plasma miR‐371a‐3p levels were measured with the ampTSmiR test and compared to disease characteristics and outcome. Pretreatment plasma miR‐371a‐3p levels were increased in 51.7% of cases and associated with number of metastatic sites, presence of lung, retroperitoneal, and mediastinal lymph node metastases, S – stage, IGCCCG risk group, and response to therapy. Patients with a negative pretreatment plasma level had better progression‐free survival (PFS) and overall survival (OS) compared to patients being positive for miR‐371a‐3p (hazard ratio [HR] = 0.26, 95% confidence interval [CI] 0.09‐0.71, P = 0.02 for PFS and HR = 0.21, 95% CI 0.07‐0.67, P = 0.03 for OS, respectively). Patients negative for miR‐371a‐3p in both samples had a superior PFS (HR = 0.10, 95% CI 0.01‐21.49, P = 0.02) and OS (HR = 0.08, 95% CI 0.01‐27.81, P = 0.008) compared to patients with miR‐371a‐3p positive in both samples (multivariate analyses were non‐significant). In total 68% of the patients were S0. This study demonstrates clinical value of plasma miR‐371a‐3p level in chemotherapy naïve (T)GCT patients starting first line of chemotherapy to predict prognosis.     

Identification and Analysis of Powdery Mildew‐Responsive miRNAs in Wheat

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most consistently damaging diseases of common wheat worldwide and greatly affects crop productivity. Recently, several plant microRNAs (miRNAs) have been reported as gene expression regulators related to various adverse environments. However, up to now, less is known on the roles of miRNAs in powdery mildew infection response of wheat. In this study, miRNA expression patterns were investigated for identifying Bgt‐responsive miRNAs in wheat leaves using a plant miRNA microarray platform. A total of 79 miRNAs from 24 families were detected in wheat leaves. Among those, seven miRNAs were further validated to be involved in wheat powdery mildew response and two of them have never been reported. In addition, their target expression profiles showed a negative correlation with that of the seven miRNAs in mock‐ and Bgt‐infected samples furtherly proved, which in turn as the robust evidence, that those seven powdery mildew‐responsive miRNAs are highly reliable. These findings could extend the current view about miRNAs as ubiquitous regulators under stress conditions.     

Expression of VEGFA‐regulating miRNAs and mortality in wet AMD

MicroRNAs (miRNAs) regulate gene expression; many of them act in the retinal pigment epithelium (RPE), and RPE degeneration is known to be a critical factor in age‐related macular degeneration (AMD). Repeated injections with anti‐VEGFA (vascular endothelial growth factor A) are the only effective therapy in wet AMD. We investigated the correlation between the expression of 18 miRNAs involved in the regulation of the VEGFA gene in serum of 76 wet AMD patients and 70 controls. Efficacy of anti‐VEGFA treatment was evaluated by counting the number of injections delivered up to 12 years. In addition, we compared the relative numbers of deaths in patient with AMD and control groups. We observed a decreased expression of miR‐34‐5p, miR‐126‐3p, miR‐145‐5p and miR‐205‐5p in wet AMD patients as compared with controls. These miRNAs are involved in the regulation of angiogenesis, cytoprotection and protein clearance. No miRNA was significantly correlated with the treatment outcome. Wet AMD patients had greater mortality than controls, and their survival was inversely associated with the number of anti‐VEGFA injections per year. No association was observed between miRNA expression and mortality. Our study emphasizes the need to clarify the role of miRNA regulation in AMD pathogenesis.     

Knockout of beta‐2 microglobulin reduces stem cell‐induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR‐24/Bim pathway

Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M^‐UMSCs in which human leucocyte antigen (HLA) light chain β2‐microglobulin (B2M) was deleted. The therapeutic potential of B2M^‐UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M^‐UMSCs facilitated perfusion recovery and enhanced running capability, without inducing immune rejection. The beneficial effect was mediated by exosomes. Mechanistically, microRNA (miR) sequencing identified miR‐24 as a major component of the exosomes originating from B2M^‐UMSCs. We identified Bim as a potential target of miR‐24 through bioinformatics analysis, which was further confirmed by loss‐of‐function and gain‐of‐function approaches. Taken together, our data revealed that knockout of B2M is a convenient and efficient strategy to prevent UMSCs‐induced immune rejection, and it provides a universal clinical‐scale cell source for tissue repair and regeneration without the need for HLA matching in the future.     

Exosomal MiR‐500a‐3p promotes cisplatin resistance and stemness via negatively regulating FBXW7 in gastric cancer

Chemoresistance has been a major challenge in advanced gastric cancer (GC) therapy. Exosomal transfer of oncogenic miRNAs implicates important effects in mediating recipient cell chemoresistance by transmitting active molecules. In this study, we found that microRNA‐500a‐3p was highly expressed in cisplatin (DDP) resistant GC cells (MGC803/DDP and MKN45/DDP) and their secreted exosomes than that in the corresponding parental cells. MGC803/DDP‐derived exosomes enhance DDP resistance and stemness properties of MGC803 recipient cells via exosomal delivery of miR‐500a‐3p in vitro and in vivo through targeting FBXW7. However, reintroduction of FBXW7 in MGC803 cells reverses miR‐500a‐3p‐mediated DDP resistance as well as stemness properties. Furthermore, elevated miR‐500a‐3p in the plasma exosomes of GC patients is correlated with DDP resistance and thereby results in poor progression‐free prognosis. Our finding highlights the potential of exosomal miR‐500a‐3p as an potential modality for the prediction and treatment of GC with chemoresistance.     

Association of plasma exosomes with severity of organ failure and mortality in patients with sepsis

Current sepsis biomarkers may be helpful in determining organ failure and evaluating patient clinical course; however, direct molecular biomarkers to predict subsequent organ failure have not yet been discovered. Exosomes, a small population of extracellular vesicles, play an important role in the inflammatory response, coagulation process and cardiac dysfunction in sepsis. Nonetheless, the association of plasma exosome with severity and mortality of sepsis is not well known. Therefore, the overall levels of plasma exosome in sepsis patients were assessed and whether exosome levels were associated with organ failure and mortality was evaluated in the present study. Plasma level of exosomes was measured by ELISA. Among 220 patients with sepsis, 145 (66%) patients were diagnosed with septic shock. A trend of increased exosome levels in control, sepsis and septic shock groups was observed (204 µg/mL vs 525 µg/mL vs 802 µg/mL, P < 0.001). A positive linear relationship was observed between overall exosome levels and Sequential Organ Failure Assessment (SOFA) score in the study cohorts (r value = 0.47). When patients were divided into two groups according to best cut‐off level, a statistical difference in 28‐ and 90‐day mortality between patients with high and low plasma exosomes was observed. Elevated levels of plasma exosomes were associated with severity of organ failure and predictive of mortality in critically ill patients with sepsis.     

Serum miRNAs are potential biomarkers for the detection of disc degeneration,among which miR‐26a‐5p suppresses Smad1 to regulate disc homeostasis

Disc degeneration is a common clinical condition in which damaged discs cause chronic pain; however, a laboratory diagnosis method for its detection is not available. As circulating miRNAs have potential as biomarkers, their application in disc degeneration has not been explored. Here, we prepared serum miRNAs from a mouse disc degeneration model and performed miRNA‐Seq and quantitative PCR to characterize disc degeneration–associated miRNAs. We identified three miRNAs, including miR‐26a‐5p, miR‐122‐5p and miR‐215‐5p, undergoing perturbation during the pathogenesis of disc degeneration. Specifically, the levels of miR‐26a‐5p in the serum demonstrated steady increases in the model of disc degeneration, compared with those in the pre‐injury samples of younger age or compared with normal controls of the same age but without disc degeneration, whereas the miRNAs miR‐122‐5p and miR‐215‐5p exhibited lower expression in post‐injury samples than in their counterparts without the surgery. Moreover, we found that miR‐26a‐5p targets Smad1 expression, and Smad1 negatively regulates Vegfa expression in disc cells, and thus, miR‐26a‐5p promotes disc degeneration. In summary, we established a method that consistently profiles circulating miRNAs and identified multiple miRNAs as promising biomarkers for disc degeneration, among which miR‐26a‐5p enhances VEGF expression during disc degeneration through targeting Smad1 signalling.