In the last 10 years the field of molecular diagnostics has witnessed an explosion of interest in the use of nanomaterials in assays for gases, metal ions, and DNA and protein markers for many diseases. Intense research has been fueled by the need for practical, robust, and highly sensitive and selective detection agents that can address the deficiencies of conventional technologies. Chemists are playing an important role in designing and fabricating new materials for application in diagnostic assays. In certain cases assays based upon nanomaterials have offered significant advantages over conventional diagnostic systems with regard to assay sensitivity, selectivity, and practicality. Some of these new methods have recently been reviewed elsewhere with a focus on the materials themselves or as subclassifications in more generalized overviews of biological applications of nanomaterials.1-7 We intend to review some of the major advances and milestones in the field of detection systems based upon nanomaterials and their roles in biodiagnostic screening for nucleic acids, * To whom correspondence should be addressed. Phone: 847-4913907. Fax: 847-467-5123. E-mail: chadnano@northwestern.edu. Nathaniel L. Rosi earned his B.A. degree at Grinnell College (1999) and his Ph.D. degree from the University of Michigan (2003), where he studied the design, synthesis, and gas storage applications of metal−organic frameworks under the guidance of Professor Omar M. Yaghi. In 2003 he began postdoctoral studies as a member of Professor Mirkin’s group at Northwestern University. His current research focuses on the rational assembly of DNA-modified nanostructures into larger-scale materials.

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Nanostructures in Biodiagnostics

MicroRNAs (miRNAs) are small noncoding RNAs that typically inhibit the translation and stability of messenger RNAs (mRNAs), controlling genes involved in cellular processes such as inflammation, cell-cycle regulation, stress response, differentiation, apoptosis, and migration. Thus, miRNAs have been implicated in the regulation of virtually all signaling circuits within a cell, and their dysregulation has been shown to play an essential role in the development and progression of cancer. Here, after a brief description of miRNA genomics, biogenesis, and function, we discuss the effects of miRNA dysregulation in the cellular pathways that lead to the progressive conversion of normal cells into cancer cells and the potential to develop new molecular miRNA-targeted therapies.

MicroRNAs in cancer.

Dendrimers in biomedical applications--reflections on the field.

Circulating nucleic acids have been shown to have potential as non-invasive diagnostic markers in cancer. We therefore investigated whether microRNAs also have diagnostic utility by comparing levels of tumour-associated MIRN155 (miR-155), MIRN210 (miR-210) and MIRN21 (miR-21) in serum from diffuse large B-cell lymphoma (DLBCL) patients (n = 60) with healthy controls (n = 43). Levels were higher in patient than control sera (P = 0.009, 0.02 and 0.04 respectively). Moreover, high MIRN21 expression was associated with relapse-free survival (P = 0.05). This is the first description of circulating microRNAs and suggests that microRNAs have potential as non-invasive diagnostic markers for DLBCL and possibly other cancers.

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Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma.

Early studies have shown how aberrantly expressed microRNAs are a hallmark of several diseases like cancer. MicroRNA expression profiling was shown to be associated with tumour development, progression and response to therapy, suggesting their possible use as diagnostic, prognostic and predictive biomarkers. Moreover, based on the increasing number of studies demonstrating that microRNAs can function as potential oncogenes or oncosuppressor genes, with the goal to improve disease response and increase cure rates, miRNA-based anticancer therapies have recently been exploited, either alone or in combination with current targeted therapies. The advantage of using microRNA approaches is based on its ability to concurrently target multiple effectors of pathways involved in cell differentiation, proliferation and survival. Here, we review our current knowledge about the involvement of microRNAs in cancer, and their potential as diagnostic, prognostic and therapeutic tools.

https://www.embopress.org/doi/pdf/10.1002/emmm.201100209

MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review

Recent studies indicate that microRNAs (miRNAs) are mechanistically involved in the development of various human malignancies, suggesting that they represent a promising new class of cancer biomarkers. However, previously reported methods for measuring miRNA expression consume large amounts of tissue, prohibiting high-throughput miRNA profiling from typically small clinical samples such as excision or core needle biopsies of breast or prostate cancer. Here we describe a novel combination of linear amplification and labeling of miRNA for highly sensitive expression microarray profiling requiring only picogram quantities of purified microRNA. Comparison of microarray and qRT-PCR measured miRNA levels from two different prostate cancer cell lines showed concordance between the two platforms (Pearson correlation R2 = 0.81); and extension of the amplification, labeling and microarray platform was successfully demonstrated using clinical core and excision biopsy samples from breast and prostate cancer patients. Unsupervised clustering analysis of the prostate biopsy microarrays separated advanced and metastatic prostate cancers from pooled normal prostatic samples and from a non-malignant precursor lesion. Unsupervised clustering of the breast cancer microarrays significantly distinguished ErbB2-positive/ER-negative, ErbB2-positive/ER-positive, and ErbB2-negative/ER-positive breast cancer phenotypes (Fisher exact test, p = 0.03); as well, supervised analysis of these microarray profiles identified distinct miRNA subsets distinguishing ErbB2-positive from ErbB2-negative and ER-positive from ER-negative breast cancers, independent of other clinically important parameters (patient age; tumor size, node status and proliferation index). In sum, these findings demonstrate that optimized high-throughput microRNA expression profiling offers novel biomarker identification from typically small clinical samples such as breast and prostate cancer biopsies.

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Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies

We have shown that smoking impacts bronchial airway gene expression and that heterogeneity in this response associates with smoking-related disease risk. In this study, we sought to determine whether microRNAs (miRNAs) play a role in regulating the airway gene expression response to smoking. We examined whole-genome miRNA and mRNA expression in bronchial airway epithelium from current and never smokers (n = 20) and found 28 miRNAs to be differentially expressed (P < 0.05) with the majority being down-regulated in smokers. We further identified a number of mRNAs whose expression level is highly inversely correlated with miRNA expression in vivo. Many of these mRNAs contain potential binding sites for the differentially expressed miRNAs in their 3'-untranslated region (UTR) and are themselves affected by smoking. We found that either increasing or decreasing the levels of mir-218 (a miRNA that is strongly affected by smoking) in both primary bronchial epithelial cells and H1299 cells was sufficient to cause a corresponding decrease or increase in the expression of predicted mir-218 mRNA targets, respectively. Further, mir-218 expression is reduced in primary bronchial epithelium exposed to cigarette smoke condensate (CSC), and alteration of mir-218 levels in these cells diminishes the induction of the predicted mir-218 target MAFG in response to CSC. These data indicate that mir-218 levels modulate the airway epithelial gene expression response to cigarette smoke and support a role for miRNAs in regulating host response to environmental toxins.

https://www.pnas.org/content/pnas/106/7/2319.full.pdf

MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium.

Effective diagnosis and surveillance of complex multi-factorial disorders such as cancer can be improved by screening of easily accessible biomarkers. Highly stable cell free Circulating Nucleic Acids (CNA) present as both RNA and DNA species have been discovered in the blood and plasma of humans. Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators. Towards this goal microRNAs (miRNAs) representing a class of naturally occurring small non-coding RNAs of 19–25 nt in length have emerged as an important set of markers that can associate their specific expression profiles with cancer development. In this study we investigate some of the pre-analytic considerations for isolating plasma fractions for the study of miRNA biomarkers. We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins. In order to assess the relative proportions of this cell-derived class, we have fractionated whole blood into plasma and its ensuing sub-fractions. Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class. A map of differentially expressed profiles is presented and the intrinsic variability of circulating miRNA species investigated in subsets of healthy males and females.

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Impact of cellular miRNAs on circulating miRNA biomarker signatures.

Dendritic Nucleic Acid Probes for DNA Biosensors

Inflammatory Bowel Disease – comprised of Crohn's Disease and Ulcerative Colitis (UC) - is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis.

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Robert Getts

Papers

Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies

MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium.

Impact of cellular miRNAs on circulating miRNA biomarker signatures.

Dendritic Nucleic Acid Probes for DNA Biosensors

Genome-wide maps of circulating miRNA biomarkers for ulcerative colitis.