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.

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Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases

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.

https://www.pnas.org/content/pnas/108/12/5003.full.pdf

Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma

Gold nanoparticles have been used in biomedical applications since their first colloidal syntheses more than three centuries ago. However, over the past two decades, their beautiful colors and unique electronic properties have also attracted tremendous attention due to their historical applications in art and ancient medicine and current applications in enhanced optoelectronics and photovoltaics. In spite of their modest alchemical beginnings, gold nanoparticles exhibit physical properties that are truly different from both small molecules and bulk materials, as well as from other nanoscale particles. Their unique combination of properties is just beginning to be fully realized in range of medical diagnostic and therapeutic applications. This critical review will provide insights into the design, synthesis, functionalization, and applications of these artificial molecules in biomedicine and discuss their tailored interactions with biological systems to achieve improved patient health. Further, we provide a survey of the rapidly expanding body of literature on this topic and argue that gold nanotechnology-enabled biomedicine is not simply an act of ‘gilding the (nanomedicinal) lily’, but that a new ‘Golden Age’ of biomedical nanotechnology is truly upon us. Moving forward, the most challenging nanoscience ahead of us will be to find new chemical and physical methods of functionalizing gold nanoparticles with compounds that can promote efficient binding, clearance, and biocompatibility and to assess their safety to other biological systems and their long-term term effects on human health and reproduction (472 references).

The golden age: gold nanoparticles for biomedicine

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.

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Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation.

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

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Cell-free nucleic acids as biomarkers in cancer patients

C95 We evaluated and validated a novel platform that uses the branched-chain DNA amplification system for analysis of gene expression panels in clinical samples. We hypothesized that the clinical utility of an assay depends on its accuracy and reproducibility. The largest source for the development of effective diagnostic and biomarkers consists of formalin-fixed and paraffin embedded (FFPE) tissues. However, until recently, measurement of RNA in FFPE tissues has been unreliable. In contrast to other methods, the bDNA assay does not require RNA isolation from tissue homogenate, avoids measurement errors caused by enzymatic preamplification and has a simple workflow. To assess the reproducibility and sensitivity of the bDNA assay, samples from 10 prostate cancer xenografts and 4 FFPE tissue blocks were assayed by the bDNA assay and qPCR as a reference method. The validity and gene-specific sensitivity of the assay were further determined by analyzed a set prostate cancer specific genes in archival tissue blocks. To calculate the error of the bDNA assay, mixed effects statistical models were used to partition the overall variance into components attributable to xenograft, sample and assay. For FFPE tissues, the coefficient of reliability for 5 genes was significantly higher for the bDNA assay (93.0 - 100%) than for qPCR (82.4% - 95%0). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, and were similar to those from qPCRFROZEN and qPCRFFPE. The sensitivity of the bDNA assay in tissue homogenates was approximate 10-fold higher than in an equivalent amount of purified RNA. In macrodissected tissues from 9 - 13 year old blocks with poor RNA quality, the bDNA assay correctly identified the overexpression of known cancer genes. In conclusion, the bDNA assay is considerably more reliable and sensitive than qPCR and appears to be well suited for clinical analysis of FFPE tissues with diagnostic gene expression biomarker panels.

Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues: reproducible measurement of gene expression panels related to therapeutic responsiveness in clinical samples

The primary objective of this study is to show the activation and analyze the regulation of the MEK- S6 kinase pathway in high-grade ovarian cancer Phospho-ERK (pERK), a direct substrate of MEK and 2 phosphorylation sites on the ribosomal protein, S6, Ser235/236, and Ser240/244, which are both targeted by the MEK and PI3-kinase/AKT pathways, were analyzed in 13 cell lines, 28 primary cancers and 8 cases of cancer cells from ascites In primary cancers, ERK and S6 phosphorylation was measured by immunohistochemistry (IHC) pERK, pS6, pAKT, and p4EBP1 were also measured by Western blotting (WB) The regulation of S6 phosphorylation by the MEK and PI3-kinase pathways was determined in ovarian cancer cell lines We observed frequent pERK expression in primary ovarian cancers (100% by WB, 75% by IHC) but not in ovarian cancer cells from ascites (25% of cases by WB) The activation of the AKT pathway, measured by pAKT expression occurred in 7 cases of primary ovarian cancer by WB, but in none of the ascites samples In ovarian cancer cell lines, the MEK pathway had a greater effect on S6 phosphorylation in cells without hyperactive AKT signaling Our data suggest that MEK is a potential drug target in high-grade ovarian cancer, however, cancer cells with hyperactive AKT and cancer cells in ascites may be less responsive to MEK inhibition The phosphorylation of S6 as a specific biomarker for either MEK or PI3-kinase pathway activation should be used with caution

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April Allen

Papers

Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues: reproducible measurement of gene expression panels related to therapeutic responsiveness in clinical samples

Activation of the MEK-S6 pathway in high-grade ovarian cancers.