Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery

Lv, Lin‐Li; Cao, Yuhan; Liú, Dan; Xu, Min; Liu, Hong; Tang, Ri‐Ning; Ma, Kun-Ling; Liu, Bi‐Cheng

doi:10.7150/ijbs.6100

Cited by 177 publications

(141 citation statements)

References 32 publications

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“…Exosomes are small vesicles of about 40-100 nm that are secreted from cells into extracellular fluid, and are found in different bodily fluids, such blood, blood derivatives, urine, and amniotic fluid. Exosomes are produced by all cell types and have a molecular phenotype, which largely reflects that of a parent cell (43,44) The contents of exosomes reflect the origin and the physiological status of the source cells, and thus exosomal RNAs, such as DENND1A.V2, can serve as biomarkers for various diseases. These data further confirm our findings in that DENND1A.V2 mRNA is increased in PCOS, and provide the basis for a potential noninvasive diagnostic for PCOS.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

McAllister

Modi

Miller³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

191

157

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Polycystic ovary syndrome (PCOS), characterized by increased ovarian androgen biosynthesis, anovulation, and infertility, affects 5-7% of reproductive-age women. Genome-wide association studies identified PCOS candidate loci that were replicated in subsequent reports, including DENND1A, which encodes a protein associated with clathrin-coated pits where cell-surface receptors reside. However, these studies provided no information about functional roles for DENND1A in the pathogenesis of PCOS. DENND1A protein was located in the cytoplasm as well as nuclei of theca cells, suggesting a possible role in gene regulation. DENND1A immunostaining was more intense in the theca of PCOS ovaries. Using theca cells isolated and propagated from normal cycling and PCOS women, we found that DENND1A variant 2 (DENND1A.V2) protein and mRNA levels are increased in PCOS theca cells. Exosomal DENND1A.V2 RNA was significantly elevated in urine from PCOS women compared with normal cycling women. Forced overexpression of DENND1A.V2 in normal theca cells resulted in a PCOS phenotype of augmented CYP17A1 and CYP11A1 gene transcription, mRNA abundance, and androgen biosynthesis. Knock-down of DENND1A.V2 in PCOS theca cells reduced androgen biosynthesis and CYP17A1 and CYP11A1 gene transcription. An IgG specific to DENND1A.V2 also reduced androgen biosynthesis and CYP17 and CYP11A1 mRNA when added to the medium of cultured PCOS theca cells. We conclude that the PCOS candidate gene, DENND1A, plays a key role in the hyperandrogenemia associated with PCOS. These observations have both diagnostic and therapeutic implications for this common disorder. P olycystic ovary syndrome (PCOS) is one of the most common endocrinopathies affecting 5-7% of reproductive age women world-wide (1). PCOS is associated with hyperandrogenemia/ hyperandrogenism, anovulation, infertility, and a characteristic ovarian morphology consisting of multiple small subcortical follicular "cysts" embedded in bilaterally enlarged ovaries (2-5). The presence of elevated circulating concentrations of testosterone results primarily from increased production of androgens by the ovaries, and is a classic endocrine phenotype of women with PCOS. Although there has been debate about the diagnostic criteria for PCOS, hyperandrogenemia/hyperandrogenism and anovulation, not explained by other causes, is a hallmark of the disorder, and is included as a key element in all "consensus" diagnosis schemes (6-10).There is consensus that the ovarian theca cells are the primary source of excess androgen biosynthesis in women with PCOS (11-13). Studies on freshly isolated thecal tissue or cultures of theca cells derived from normal and PCOS women have demonstrated that PCOS theca secretes greater amounts of androgen than theca tissue or cells from regularly ovulating women (12,(14)(15)(16)(17)(18)(19). Increased androgen biosynthesis in PCOS theca cells results from increased expression of the key enzymes involved in androgen biosynthesis, steroid-17-α-hydroxylase/17,20 lyase (encoded by the CYP17...

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Section: Discussionmentioning

confidence: 99%

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

McAllister

Modi

Miller³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

191

157

View full text Add to dashboard Cite

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“…Intensification of miRNA expression signals has been reported in several recent studies. [73][74][75] However, the exosomal intensification effect on protein biomarkers was not as significant as its effect on miRNAs in this study. Further study is needed on the mechanism of miRNA and protein compartmentation into exosomes to explain the significant reduction of signal intensification of proteins compared to that of miRNAs.…”

Section: Discussionmentioning

confidence: 58%

Carcinogenic activity of PbS quantum dots screened using exosomal biomarkers secreted from HEK293 cells

Kim¹,

Kim²,

Lee³

et al. 2015

IJN

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Lead sulfide (PbS) quantum dots (QDs) have been applied in the biomedical area because they offer an excellent platform for theragnostic applications. In order to comprehensively evaluate the biocompatibility of PbS QDs in human cells, we analyzed the exosomes secreted from cells because exosomes are released during cellular stress to convey signals to other cells and serve as a reservoir of enriched biomarkers. PbS QDs were synthesized and coated with 3-mercaptopropionic acid (MPA) to allow the particles to disperse in water. Exosomes were isolated from HEK293 cells treated with PbS–MPA at concentrations of 0 µg/mL, 5 µg/mL, and 50 µg/mL, and the exosomal expression levels of miRNAs and proteins were analyzed. As a result, five miRNAs and two proteins were proposed as specific exosomal biomarkers for the exposure of HEK293 cells to PbS–MPA. Based on the pathway analysis, the molecular signature of the exosomes suggested that PbS–MPA QDs had carcinogenic activity. The comet assay and expression of molecular markers, such as p53, interleukin (IL)-8, and C-X-C motif chemokine 5, indicated that DNA damage occurred in HEK293 cells following PbS–MPA exposure, which supported the carcinogenic activity of the particles. In addition, there was obvious intensification of miRNA expression signals in the exosomes compared with that of the parent cells, which suggested that exosomal biomarkers could be detected more sensitively than those of whole cellular extracts.

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“…As exosomes exhibit cell surface markers and contain microRNA, they present potential targets to be used as biomarkers of cancer [10,11]. Promising results have been reported for ovarian cancer [12] and for non-small cell lung cancer [13,14].…”

Section: Introductionmentioning

confidence: 78%

Human urinary exosomes in bladder cancer patients: properties, concentrations and possible clinical application

Riches

Powis

Mullen

et al. 2016

Bladder

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OBJECTIVE:High grade bladder cancer is extremely aggressive. Early detection is thus an important challenge. Development of non-invasive diagnostic tools particularly using urine samples could be of importance in the diagnosis and surveillance of these patients. Exosomes are small vesicles present in the urine and have the potential to be used as biomarkers of cancer. Thus studies of the properties and concentrations of these particles in bladder cancer patients are of importance. MATERIALS AND METHODS:The concentration of exosomes present in urine was determined by nanoparticle tracking analysis using a Nanosight LM10 unit. Clinical urine samples were routinely collected and fixed using Preservcyt. The morphology of exosomes was studied in electron micrographs and characteristic exosome markers using Western blots. RESULTS:The exosome concentration of fixed samples stored at room temperature was constant for 48 hours and the same as fresh samples. Exosomes derived from patients presenting for a transurethral resection of their bladder tumor exhibited the exosome markers ALIX and TSG101 and also the classic cup shaped appearance in electron micrographs. The concentration of exosomes in patients presenting for transurethral resection of a bladder tumor was significantly greater than in patients presenting for check cystoscopy with no recurrence (median 77.2 × 10 8 per ml compared with 38.8 × 10 8 per ml, P < 0.001). A ROC analysis (area under the curve 77.4%) suggested that a suitable cut-off concentration of 85 × 10 8 per ml is associated with a sensitivity of 43% and specificity of 91% for diagnosing bladder cancer. CONCLUSIONS:Thus the concentration and properties of exosomes can be conveniently studied in fixed urine samples derived from bladder cancer patients. The characteristic properties of exosomes were preserved and increased numbers were found in patients presenting for transurethral resection of their tumor. With an appropriate cut-off value, urinary exosome concentrations may have utility in excluding a cancer recurrence when monitoring patients successfully treated for bladder cancer.

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Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery

Cited by 177 publications

References 32 publications

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

Carcinogenic activity of PbS quantum dots screened using exosomal biomarkers secreted from HEK293 cells

Human urinary exosomes in bladder cancer patients: properties, concentrations and possible clinical application

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