Differentially Expressed microRNAs in MIA PaCa-2 and PANC-1 Pancreas Ductal Adenocarcinoma Cell Lines are Involved in Cancer Stem Cell Regulation.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, and thus better understanding of its molecular pathology is crucial for us to devise more effective treatment of this deadly disease. As cancer cell line remains a convenient starting point for discovery and proof-of-concept studies, here we report the miRNA expression characteristics of two cell lines, MIA PaCa-2 and PANC-1, and discovered three miRNAs (miR-7-5p, let-7d, and miR-135b-5p) that are involved in cancer stem cells (CSCs) suppression. After transfection of each miRNA's mimic into PANC-1 cells which exhibits higher stemness feature than MIA-PaCa-2 cells, partial reduction of CSC surface markers and inhibition of tumor sphere formation were observed. These results enlighten us to consider miRNAs as potential therapeutic agents for pancreatic cancer patients via specific and effective inhibition of CSCs.

Genome-wide study of salivary microRNAs as potential noninvasive biomarkers for detection of nasopharyngeal carcinoma.

BACKGROUND: Recent studies reported that blood-based microRNAs (miRNAs) could detect cancers and predict prognosis have opened a new field of utilizing circulating miRNAs as cancer biomarkers. In this pilot study, we conducted for the first time, to our knowledge, the evaluation of the applicability of salivary miRNAs as novel biomarkers for nasopharyngeal carcinoma (NPC) detection. METHODS: Microarray miRNA expression profiling was performed on saliva samples from 22 newly diagnosed NPC patients and 25 healthy controls, and 12 significantly down-regulated miRNAs were selected for quantitative real-time-PCR (qRT-PCR) validation and further analysis. Their target genes enriched by gene ontology and pathway analysis were used to construct regulatory and interaction networks. The receiver operating characteristic analyses (ROC) and logistic regression were calculated to assess discriminatory accuracy. RESULTS: Twelve dysregulated miRNAs screened by microarray that showed the same expression patterns with qRT-PCR analysis. Through bioinformatics analysis, the most prominent hub gene probably regulated by the 12 down-regulated miRNAs is found to be TP53. The ROC including the 12 miRNAs separated NPC patients from healthy controls with very high accuracy (areas under the receiver operating characteristic curve [AUC] = 0.999, sensitivity = 100.00%, specificity = 96.00%). Furthermore, if only six significantly dysregulated miRNAs were selected for the ROC analysis, the accuracy is still impressive (AUC = 0.941, sensitivity = 95.45%, specificity = 80.00%). CONCLUSIONS: This study highlights the potential for salivary miRNAs as biomarkers for the detection of NPC. Meanwhile, differentially expressed miRNAs in saliva might play critical roles in NPC by regulating their target genes, which associated with some significant pathways, such as p53 signaling pathway.

Graphically encoded suspension array for multiplex immunoassay and quantification of autoimmune biomarkers in patient sera.

In precision medicine, clinical decisions and pharmaceutical evaluations tends to be made upon parallel analysis of multiple protein biomarkers. Currently, the growing needs of high-throughput multiplex immunoassay is partially satisfied by spectrally encoded bead flow suspension arrays and other platforms, yet there is still room for progress in terms of encoding capacity, decoding accuracy, ease-of-manufacture/operation, and cost-effectiveness, for which graphical suspension arrays could make substantial contributions. Here we described a suspension array system made up of graphically encoded silica particles, an automated microplate imager and an in-house data processing program. The micro-fabricated, highly uniform planar particles provide a code space of 128-plex with further extendibility. The derived multiplex immunoassay reaches sub-picogram per milliliter sensitivity level (lowest LoD = 80 fg/ml) with wide dynamic range, as well as high precision and accuracy. The potential of clinical diagnostics was demonstrated by parallel measurement of three serum biomarkers for type 1 diabetes patients. Importantly, use of standard microplates as assay vessel extends its power to high-throughput applications, such as disease screening or drug discovery.

Digitally encoded silica microparticles for multiplexed nucleic acid detection.

A robust high throughput suspension array was proposed to analyze biomolecules. To exploit its capacity for nucleic acid detection, we designed a multiplex nested asymmetric PCR (MNAS PCR) that can produce single stranded DNA (ssDNA) efficiently without complicated optimization. Multiplexed HPV genotyping was demonstrated with high selectivity, high sensitivity and rapid hybridization within 20 minutes.

Rapid and multiplex microRNA detection on graphically encoded silica suspension array.

MicroRNA (miRNA), an 18-24-nucleotide noncoding RNA molecule, has become an ideal class of biomarker candidates for clinical diagnosis of cancers. By now, a number of detection methods for miRNAs have been developed on planar arrays and suspension arrays. In this work, we describe a hybridization-triggered fluorescence strategy for label-free and multiplex miRNA detection on graphically encoded silica suspension array. The total RNA is directly applied for analysis with an 8-mer Universal Tag which can be selectively captured by the capture probe via base-stacking effects. Benefiting from base-stacking effects, this novel method exhibits superb discrimination ability toward the 5' and 3' end single-nucleotide alteration. Mature miRNAs can be distinguished from their corresponding pre-miRNAs easily. Moreover, the estimated detection limit of 5 amol is comparable to some of the most sensitive methods. All these mentioned characteristics offer exciting possibilities for discovery and clinical applications.

Label-free microRNA profiling not biased by 3' end 2'-O-methylation.

Accurate quantification of miRNA expression level is essential to the study of its biology, and many cutting-edge technologies have been developed to accommodate this need. Yet most of them were designed primarily for the "regular" RNAs such as animal miRNAs and may overlook the fact that plant miRNAs and many other small noncoding RNAs are 2'-O-methylated at the 3' end nucleotide. According to our experimental data and previous reports, this structural variation is detrimental to the effectiveness of the commonly used enzymatic labeling methods, leading to strongly biased results (~24-fold difference). Herein, we demonstrate that our Stacking-Hybridized Universal Tag (SHUT) microarray assay is well suited for unbiased profiling of both normal and methylated small RNA species. The detected signals of small RNAs with 2'-hydroxyl and 2'-O-methyl 3' ends are highly consistent (no significant difference at α = 0.01 level). For specificity, the presented method edges over others by its unique ability to discriminate single-base difference at or near the 5' end. Notably, as compared to many delicate techniques, this enzyme-free and label-free approach requires much less reagent and manipulation, benefiting the SHUT-based applications with more efficient workflow and highly reproducible results.

Label-free high-throughput microRNA expression profiling from total RNA.

MicroRNAs (miRNAs) are key biological regulators and promising disease markers whose detection technologies hold great potentials in advancing fundamental research and medical diagnostics. Currently, miRNAs in biological samples have to be labeled before being applied to most high-throughput assays. Although effective, these labeling-based approaches are usually labor-intensive, time-consuming and liable to bias. Besides, the cross-hybridization of co-existing miRNA precursors (pre-miRNAs) is not adequately addressed in most assays that use total RNA as input. Here, we present a hybridization-triggered fluorescence strategy for label-free, microarray-based high-throughput miRNA expression profiling. The total RNA is directly applied to the microarray with a short fluorophore-linked oligonucleotide Universal Tag which can be selectively captured by the target-bound probes via base-stacking effects. This Stacking-Hybridized Universal Tag (SHUT) assay has been successfully used to analyze as little as 100 ng total RNA from human tissues, and found to be highly specific to homogenous miRNAs. Superb discrimination toward single-base mismatch at the 5' or 3' end has been demonstrated. Importantly, the pre-miRNAs generated negligible signals, validating the direct use of total RNA.

A reverse transcription-free real-time PCR assay for rapid miRNAs quantification based on effects of base stacking.

A rapid and reverse transcription-free real-time PCR microRNA assay was developed based on effects of base stacking. This microRNA assay has been shown to be highly specific to homogenous miRNAs, and the procedure can be completed within 30 min starting from total RNA.