Circulating microRNAs as stable blood-based markers for cancer detection
29 Jul 2008-Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences)-Vol. 105, Iss: 30, pp 10513-10518
TL;DR: It is shown here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity and established the measurement of tumor-derived mi RNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
Abstract: Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small (≈22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
Citations
More filters
TL;DR: It is demonstrated that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses, and can serve as potential biomarkers for the detection of various cancers and other diseases.
Abstract: Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.
4,184 citations
TL;DR: Identification of extracellular Ago2–miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation, and reveals two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma mi RNAs.
Abstract: MicroRNAs (miRNAs) circulate in the bloodstream in a highly stable, extracellular form and are being developed as blood-based biomarkers for cancer and other diseases. However, the mechanism underlying their remarkable stability in the RNase-rich environment of blood is not well understood. The current model in the literature posits that circulating miRNAs are protected by encapsulation in membrane-bound vesicles such as exosomes, but this has not been systematically studied. We used differential centrifugation and size-exclusion chromatography as orthogonal approaches to characterize circulating miRNA complexes in human plasma and serum. We found, surprisingly, that the majority of circulating miRNAs cofractionated with protein complexes rather than with vesicles. miRNAs were also sensitive to protease treatment of plasma, indicating that protein complexes protect circulating miRNAs from plasma RNases. Further characterization revealed that Argonaute2 (Ago2), the key effector protein of miRNA-mediated silencing, was present in human plasma and eluted with plasma miRNAs in size-exclusion chromatography. Furthermore, immunoprecipitation of Ago2 from plasma readily recovered non–vesicle-associated plasma miRNAs. The majority of miRNAs studied copurified with the Ago2 ribonucleoprotein complex, but a minority of specific miRNAs associated predominantly with vesicles. Our results reveal two populations of circulating miRNAs and suggest that circulating Ago2 complexes are a mechanism responsible for the stability of plasma miRNAs. Our study has important implications for the development of biomarker approaches based on capture and analysis of circulating miRNAs. In addition, identification of extracellular Ago2–miRNA complexes in plasma raises the possibility that cells release a functional miRNA-induced silencing complex into the circulation.
2,900 citations
Cites background or methods or result from "Circulating microRNAs as stable blo..."
...By using quantitative RT-PCR (qRT-PCR), we assayed the vesicle pellets and the vesicle-poor supernatants for three well characterized circulating miRNAs: miR-16, miR-92a, and let-7a (1, 8, 24)....
[...]
...This, along with the finding that miRNAs are remarkably stable in plasma despite high circulating RNase activity (1), suggests that miRNAs may be developed into a powerful new class of blood-based biomarkers....
[...]
...We determined miRNA copies by absolute quantification and normalized between samples using synthetic Caenorhabditis elegans miRNA oligonucleotides that were spiked into each sample after RNase inactivation (1, 25)....
[...]
...We reliably detected 128 and 136 miRNAs in unfractionated plasma and serum, respectively (Tables S1 and S2), which was comparable to our profiling analysis in a previous study (1)....
[...]
TL;DR: An update on canonical and non-canonical miRNA biogenesis pathways and various mechanisms underlying miRNA-mediated gene regulations and the current knowledge of the dynamics of miRNA action and of the secretion, transfer, and uptake of extracellular miRNAs is provided.
Abstract: MicroRNAs (miRNAs) are a class of non-coding RNAs that play important roles in regulating gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3' untranslated region (3' UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interaction of miRNAs with other regions, including the 5' UTR, coding sequence, and gene promoters, have also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription. The interaction of miRNAs with their target genes is dynamic and dependent on many factors, such as subcellular location of miRNAs, the abundancy of miRNAs and target mRNAs, and the affinity of miRNA-mRNA interactions. miRNAs can be secreted into extracellular fluids and transported to target cells via vesicles, such as exosomes, or by binding to proteins, including Argonautes. Extracellular miRNAs function as chemical messengers to mediate cell-cell communication. In this review, we provide an update on canonical and non-canonical miRNA biogenesis pathways and various mechanisms underlying miRNA-mediated gene regulations. We also summarize the current knowledge of the dynamics of miRNA action and of the secretion, transfer, and uptake of extracellular miRNAs.
2,538 citations
Cites background from "Circulating microRNAs as stable blo..."
...Contrary to cellular RNA species, extracellular miRNAs are highly stable, resisting degradation at room temperature for up to 4 days and in deleterious conditions such as boiling, multiple freeze-thaw cycles, and high or low pH (117, 124)....
[...]
TL;DR: Findings are discussed with a specific focus on the clinical utility of cell-free nucleic acids as blood biomarkers for cancer screening, prognosis and monitoring of the efficacy of anticancer therapies.
Abstract: 1described the presence of cell-free nucleic acid (cfNA) in human blood for the first time. This attracted little attention in the scientific community and it was not until 1994 that the importance of cfNA was recognized as a result of the detection of mutated RAS gene fragments in the blood of cancer patients 2,3 (TIMELINE). In 1996, microsatellite alterations on cell-free DNA (cfDNA) were shown in cancer patients 4
2,427 citations
TL;DR: Evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities is presented, indicating that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNA.
Abstract: Circulating microRNAs (miRNA) are relatively stable in plasma and are a new class of disease biomarkers. Here we present evidence that high-density lipoprotein (HDL) transports endogenous miRNAs and delivers them to recipient cells with functional targeting capabilities. Cellular export of miRNAs to HDL was demonstrated to be regulated by neutral sphingomyelinase. Reconstituted HDL injected into mice retrieved distinct miRNA profiles from normal and atherogenic models. HDL delivery of both exogenous and endogenous miRNAs resulted in the direct targeting of messenger RNA reporters. Furthermore, HDL-mediated delivery of miRNAs to recipient cells was demonstrated to be dependent on scavenger receptor class B type I. The human HDL-miRNA profile of normal subjects is significantly different from that of familial hypercholesterolemia subjects. Notably, HDL-miRNA from atherosclerotic subjects induced differential gene expression, with significant loss of conserved mRNA targets in cultured hepatocytes. Collectively, these observations indicate that HDL participates in a mechanism of intercellular communication involving the transport and delivery of miRNAs.
2,410 citations
References
More filters
TL;DR: It is shown that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location, and it is proposed that this RNA is called “exosomal shuttle RNA” (esRNA).
Abstract: Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).
10,484 citations
"Circulating microRNAs as stable blo..." refers background in this paper
...Exosomes are 50- to 90-nm (24), membrane-bound particles that have been reported to be abundant in plasma (25) and that have recently been shown (in cell culture studies) to contain miRNAs (26)....
[...]
TL;DR: A new, bead-based flow cytometric miRNA expression profiling method is used to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers, and finds the miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours.
Abstract: Recent work has revealed the existence of a class of small non-coding RNA species, known as microRNAs (miRNAs), which have critical functions across various biological processes. Here we use a new, bead-based flow cytometric miRNA expression profiling method to present a systematic expression analysis of 217 mammalian miRNAs from 334 samples, including multiple human cancers. The miRNA profiles are surprisingly informative, reflecting the developmental lineage and differentiation state of the tumours. We observe a general downregulation of miRNAs in tumours compared with normal tissues. Furthermore, we were able to successfully classify poorly differentiated tumours using miRNA expression profiles, whereas messenger RNA profiles were highly inaccurate when applied to the same samples. These findings highlight the potential of miRNA profiling in cancer diagnosis.
9,470 citations
"Circulating microRNAs as stable blo..." refers background in this paper
...We established a list of likely blood-based miRNA biomarker candidates for prostate cancer by (i) compiling a list of miRNAs expressed in human prostate cancer specimens based on published miRNA expression profiling data (7, 18) and (ii) filtering out miRNAs detected in healthy donor-derived plasma in our miRNA cloning experiment (Fig....
[...]
...We hypothesized that miRNAs could be an ideal class of blood-based biomarkers for cancer detection because: (i) miRNA expression is frequently dysregulated in cancer (5, 6), (ii) expression patterns of miRNAs in human cancer appear to be tissue-specific (7), and (iii) miRNAs have unusually high stability in formalin-fixed tissues (8–10)....
[...]
...(7), who profiled a diverse range of human cancer types....
[...]
Journal Article•
TL;DR: The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery as discussed by the authors.
Abstract: MicroRNA (miRNA) alterations are involved in the initiation and progression of human cancer. The causes of the widespread differential expression of miRNA genes in malignant compared with normal cells can be explained by the location of these genes in cancer-associated genomic regions, by epigenetic mechanisms and by alterations in the miRNA processing machinery. MiRNA-expression profiling of human tumours has identified signatures associated with diagnosis, staging, progression, prognosis and response to treatment. In addition, profiling has been exploited to identify miRNA genes that might represent downstream targets of activated oncogenic pathways, or that target protein- coding genes involved in cancer.
6,306 citations
Journal Article•
TL;DR: I MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators as discussed by the authors, and have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer.
Abstract: I MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators. They regulate diverse biological processes, and bioinformatic data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumour suppressors and oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer.
6,064 citations
TL;DR: A novel microRNA quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis, which enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases.
Abstract: A novel microRNA (miRNA) quantification method has been developed using stem–loop RT followed by TaqMan PCR analysis. Stem–loop RT primers are better than conventional ones in terms of RT efficiency and specificity. TaqMan miRNA assays are specific for mature miRNAs and discriminate among related miRNAs that differ by as little as one nucleotide. Furthermore, they are not affected by genomic DNA contamination. Precise quantification is achieved routinely with as little as 25 pg of total RNA for most miRNAs. In fact, the high sensitivity, specificity and precision of this method allows for direct analysis of a single cell without nucleic acid purification. Like standard TaqMan gene expression assays, TaqMan miRNA assays exhibit a dynamic range of seven orders of magnitude. Quantification of five miRNAs in seven mouse tissues showed variation from less than 10 to more than 30 000 copies per cell. This method enables fast, accurate and sensitive miRNA expression profiling and can identify and monitor potential biomarkers specific to tissues or diseases. Stem–loop RT–PCR can be used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs). Furthermore, the concept of stem–loop RT primer design could be applied in small RNA cloning and multiplex assays for better specificity and efficiency.
4,599 citations
"Circulating microRNAs as stable blo..." refers methods in this paper
...To quantitate specific miRNAs, we used TaqMan quantitative RT-PCR (qRT-PCR) assays (12) to measure three miRNAs (miR15b, miR-16, and miR-24) in plasma from three healthy individuals....
[...]