MicroRNAs are short RNA molecules that regulate gene expression. They have been investigated as potential biomarkers because their expression levels are correlated with various diseases. However, the detection of microRNAs in the bloodstream remains difficult because current methods are not sufficiently selective or sensitive. Here, we show that a nanopore sensor based on the alpha-hemolysin protein selectively detected microRNAs at the single molecular level in plasma samples from lung cancer patients without the need for labelling or amplification. The sensor, which used a programmable oligonucleotide probe to generate a target-specific signature signal, was able to quantify sub-picomolar levels of cancer-associated microRNAs and to discriminate single nucleotide differences between microRNA family members. This approach could prove useful for quantitative microRNA detection, biomarker discovery, and the non-invasive early diagnosis of cancer.

Objective: Lung cancer is the leading cause of cancer-related deaths in both men and women. Since there is no validated population-based screening procedure, most patients with lung cancer are diagnosed at advanced stages with an overall five-year survival rate of only 15%. Therefore, diagnosis of lung cancer at an early stage is important for improving the outcome of patients. MicroRNAs (miRNAs), a family of 19- to 25-nucleotide and noncoding small RNAs that primarily function as gene regulators at post-transcriptional level, are frequently dysregulated in cancer. Recent studies demonstrate that the miRNAs in blood are present in notably stabile form and are readily detectible by various sensitive methods. This new finding has raised a concept that circulating miRNAs could be novel non-invasive biomarkers for cancer detection. The objective of this study is to investigate the potential of circulating microRNAs for lung cancer detection. Methods: First, we searched the miRNA microarray data of lung cancer from published literature, and selected 15 of miRNAs (miR-17, 21, 24, 106a, 125b, 128, 155, 182, 183, 197, 199b, 203, 205, 210 and 221) that were most frequently up-regulated in lung cancer tissues. Total RNA including miRNAs were isolated with mirVana PARIS kit (Ambion, TX), then polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. The levels of miRNAs were determined in 28 plasma samples from lung cancer patients and 16 of controls. Results: We found that the levels of miR-155, miR-182 and miR-197 in plasma of lung cancer patients were significantly elevated compared with controls. MiR-155 yielded an AUC (the areas under the ROC curve) of 0.8739 (95% CI:0.7489 to 0.9989, P<0.001), miR-182 yielded an AUC of 0.8426 (95% CI:0.7239 to 0.9614, P<0.001) and miR-197 yielded an AUC of 0.8037 (95% CI:0.6705 to 0.9369, P<0.001) in discriminating lung cancer from controls. Conclusions: MiR-155, miR-182 and miR-197 are significantly elevated in patient plasma with lung cancer and can be a potential non-invasive molecular biomarker for lung cancer screening and clinical follow-up. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-359.

Thioredoxin (Trx) is one of the two major thiol antioxidants playing essential roles in redox homeostasis and signaling. Despite the importance, there is a lack of method in monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1—a redox-sensitive red fluorescent protein. We utilized the resultant biosensor—TrxRFP1—to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

miRNAs are short noncoding RNA molecules that are important in regulating gene expression. Due to the correlation of their expression levels and various diseases, miRNAs are being investigated as potential biomarkers for molecular diagnostics. The fast-growing miRNA exploration demands rapid, accurate, low-cost miRNA detection technologies. This article will focus on two platforms of nanopore single-molecule approach that can quantitatively measure miRNA levels in samples from tissue and cancer patient plasma. Both nanopore methods are sensitive and specific, and do not need labeling, enzymatic reaction or amplification. In the next 5 years, the nanopore-based miRNA techniques will be improved and validated for noninvasive and early diagnosis of diseases.

In general, DNA methylation acts in concert with other epigenetic processes, including histone modifications, chromatin remodeling and microRNAs, to shape the overall chromatin structure of the nucleus and potentially modify its functional state. Aberrant DNA methylation events can occur in a number of human diseases but we are only just beginning to appreciate the scope and magnitude of this process in human health. As one example, in contrast to normal cells, the cancer methylome is characterized by reciprocal hypermethylation of specific regulatory regions of genes along with an overall decrease in the quantity of 5-methylcytosine throughout the remainder of the genome. Currently, near genome-wide technologies are available and have been utilized to examine the extent of DNA methylation in discovery-based studies involving several physiological and disease states. Although early in the process, DNA methylation is being explored as a biomarker to be used in clinical practice for early detection of disease, tumor classification and for predicting disease outcome or recurrence. This perspective focuses on the current and future states of the use of DNA methylation biomarkers in disease diagnosis, prognosis and classification, with a particular emphasis on cancer.