Wei Liao

Electrochemical Sensor for Multiplex Biomarkers Detection

Purpose: Multiplexing assay of biomarkers at the point-of-care is an elusive goal for molecular diagnostics. Experimental Design: Here, we report an electrochemical (EC) sensor for oral cancer detection based on the simultaneous detection of two salivary biomarkers: interleukin (IL)-8 mRNA and IL-8 protein. Results: Under the multiplexing mode, the limit of detection of salivary IL-8 mRNA reaches to 3.9 fM and 7.4 pg/mL for IL-8 protein in saliva. Multiplex assay of these 2 biomarkers directly from 28 cancer and 28 matched control saliva samples shows significant difference between the two groups. From the receiver operating characteristic analysis, the EC sensor yields around 90% sensitivity and specificity for both IL-8 mRNA and IL-8 protein, which are very close to the data measured by traditional assays (ELISA and PCR) with the same group of saliva. Combined IL-8 mRNA and protein show better AUC compared with single biomarker. Conclusions: We show, for the first time, concurrently multiplexing detection of salivary mRNA and protein biomarkers using point-of-care EC sensor.

Bio/Abiotic Interface Constructed from Nanoscale DNA Dendrimer and Conducting Polymer for Ultrasensitive Biomolecular Diagnosis

For sensors detecting immobilized biomarkers, the interface between the surface and the fluid medium plays an important role in determining the levels of signal and noise in the electrochemical detection process. When protein is directly immobilized on the metal electrode, denaturation of the protein by surface-protein interaction results in low activity and low signal level. A conducting polymer-based interface can prevent the protein conformation change and alleviate this problem. A DNA dendrimer is introduced into the interfacial film on the sensor surface to further improve the sensor performance. DNA dendrimer is a nanoscale dendrite constructed of short DNA sequences, which can be easily incorporated into the abiotic conducting polymer matrix and is biocompatible with most biological species. In this work, DNA dendrimer and polypyrrole (DDPpy) form the bio/abiotic interface on electrochemical sensors. Detection of two salivary protein markers (IL-8 and IL-1beta) and one mRNA salivary marker (IL-8) is used to demonstrate the efficiency of the DDPpy sensor. A limit of detection (LOD) of protein of 100-200 fg mL(-1) is achieved, which is three orders of magnitude better than that without the DNA dendrimer interface. An LOD of 10 aM is established for IL-8 mRNA. The typical sample volume used in the detection is 4 microL, thus the LOD reaches only 25 target molecules (40 yoctomole).

Electrochemical detection of low-copy number salivary RNA based on specific signal amplification with a hairpin probe

We developed a technique for electrochemical detection of salivary mRNA employing a hairpin probe (HP). Steric hindrance (SH) suppresses unspecific signal and generates a signal-on amplification process for target detection. The stem-loop configuration brings the reporter end of the probe into close proximity with the surface and makes it unavailable for binding with the mediator. Target binding opens the hairpin structure of the probe, and the mediator can then bind to the accessible reporter. Horseradish peroxidase is utilized to generate electrochemical signal. This signal-on process is characterized by a low basal signal, a strong positive readout and a large dynamic range. The SH is controlled via hairpin design and electrical field. By applying electric field control to HPs, the limit of detection of RNA is about 0.4 fM, which is 10 000-fold more sensitive than conventional linear probes. Endogenous Interleukin-8 mRNA is detected with the HP, and good correlation with the quantitative PCR technique is obtained. The resultant process allows a simple setup and by reducing the number of steps it is suited for the point-of-care detection of specific nucleic acid sequences from complex body fluids such as saliva.

Exon 11 Skipping of E-Cadherin RNA Downregulates Its Expression in Head and Neck Cancer Cells

E-cadherin is an important tumor suppressor gene whose expression is lost when cells acquire a metastatic phenotype. We analyzed the role of E-cadherin missplicing as a mechanism of its downregulation by analyzing a misspliced E-cadherin transcript that lacks exon 11 of this gene. This results in a frameshift and a premature termination codon that targets this transcript for degradation. Tumor tissues, including breast (20%, n = 9), prostate (30%, n = 9) and head and neck (75%, n = 8) cancer, express the exon 11-skipped transcripts (vs. nonmalignant controls) and its levels inversely correlate with E-cadherin expression. This is a novel mechanism of E-cadherin downregulation by missplicing in tumor cells, which is observed in highly prevalent human tumors. In the head and neck cancer model, nontumorigenic keratinocytes express exon 11–skipped splice product two- to sixfold lower than the head and neck tumor cell lines. Mechanistic studies reveal that SFRS2 (SC35), a splicing factor, as one of the regulators that increases missplicing and downregulates E-cadherin expression. Furthermore, this splicing factor was found to be overexpressed in 5 of 7 head and neck cell lines and primary head and neck tumors. Also, methylation of E-cadherin gene acts as a regulator of this aberrant splicing process. In 2 head and neck cell lines, wild-type transcript expression increased 16- to 25-folds, whereas the percentage of exon 11-skipped transcripts in both the cell lines decreased five- to 30-folds when cells were treated with a hypomethylating agent, azacytidine. Our findings reveal that promoter methylation and an upregulated splicing factor (SFRS2) are involved in the E-cadherin missplicing in tumors. Mol Cancer Ther; 10(9); 1751–9. ©2011 AACR.

RNAPro•SAL: a device for rapid and standardized collection of saliva RNA and proteins.

The stabilization and processing of salivary transcriptome and proteome biomarkers is a critical challenge due to the ubiquitous nature of nucleases and proteases as well as the inherent instability of these biomarkers. Furthermore, extension of salivary transcriptome and proteome analysis to point-of-care and remote sites requires the availability of self-administered ambient temperature collection and storage tools. To address these challenges, a self-contained whole saliva collection and extraction system, RNAPro•SAL, has been developed that provides rapid ambient temperature collection along with concurrent processing and stabilization of extracellular RNA (exRNA) and proteins. The system was compared to the University of California, Los Angeles (UCLA) standard clinical collection process (standard operating procedure, SOP). Both systems measured total RNA and protein, and exRNA IL-8, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin and ribosomal protein S9 (RPS9) by qPCR. Proteome analysis was measured by EIA analysis of interleukin-8 (IL-8), and β-actin, as well as total protein. Over 97% of viable cells were removed by both methods. The system compared favorably to the labor-intensive clinical SOP, which requires low-temperature collection and isolation, yielding samples with similar protein and exRNA recovery and stability.

Identification of Histone Epigenetic Modifications with Chromatin Immunoprecipitation PCR Array in Chronic Lymphocytic Leukemia

Histone epigenetic modifications are one of the frequently observed epigenetic alterations in leukemic cells. To efficiently identify histone acetylation status of a number of gene promoters in chronic lymphocytic leukemia (CLL) specimens, we performed a combination of chromatin immunoprecipitation (ChIP) and PCR array. ChIP by an antipan acetylated H4 histone antibody was followed by a PCR amplification of gene promoters (n=84) to determine their histone acetylation status. A subset of genes with differential histone acetylation status was identified in CLL specimens, and their RNA expression level was correlated with real time quantitative RT-PCR analysis (qRT-PCR). Globally CLL specimens have more hypo-acetylated or epigenetically silenced gene promoters as compared to normal peripheral-blood mononuclear cells. Promoters of E-cadherin, Fos, CDKN2B, c-Jun, BAX genes were found to be histone hypo-acetylated while the promoters of cyclin D1, CDK4, Myc, and TGF beta were hyper-acetylated. The histone acetylation status correlated well with qRT-PCR analysis of a number of CLL specimens. Based on the ChIP-PCR array epigenetically silenced genes and transcriptionaly active genes can be identified. Histone deacetylase inhibitors (HDACi) induce apoptosis in CLL specimens and to determine changes in gene transcription induced by HDACi the ChIP-PCR array was performed with HDACi treatment. Interestingly the HDACi exposure resulted in histone hyper-acetylation of a subset of genes with increased expression of CDKN2B, c-Jun and BAX RNA while promoters of pro-growth and survival genes such as BCL-2, NFKB1, beta-catenin and CDK4 underwent promoter histone hypo-acetylation and downregulation of their RNA expression. The ChIP-PCR array assay can be utilized to detect histone modifications in the genome of leukemic cells and reflects the transcriptional status of the genes.

Discovery and Validation of Salivary Extracellular RNA Biomarkers for Noninvasive Detection of Gastric Cancer

BACKGROUND Biomarkers are needed for noninvasive early detection of gastric cancer (GC). We investigated salivary extracellular RNA (exRNA) biomarkers as potential clinical evaluation tools for GC. METHODS Unstimulated whole saliva samples were prospectively collected from 294 individuals (163 GC and 131 non-GC patients) who underwent endoscopic evaluation at the Samsung Medical Center in Korea. Salivary transcriptomes of 63 GC and 31 non-GC patients were profiled, and mRNA biomarker candidates were verified with reverse transcription quantitative real-time PCR (RT-qPCR). In parallel, microRNA (miRNA) biomarkers were profiled and verified with saliva samples from 10 GC and 10 non-GC patients. Candidate biomarkers were validated with RT-qPCR in an independent cohort of 100/100 saliva samples from GC and non-GC patients. Validated individual markers were configured into a best performance panel. RESULTS We identified 30 mRNA and 15 miRNA candidates whose expression pattern associated with the presence of GC. Among them, 12 mRNA and 6 miRNA candidates were verified with the discovery cohort by RT-qPCR and further validated with the independent cohort (n = 200). The configured biomarker panel consisted of 3 mRNAs (SPINK7, PPL, and SEMA4B) and 2 miRNAs (MIR140-5p and MIR301a), which were all significantly down-regulated in the GC group, and yielded an area under the ROC curve (AUC) of 0.81 (95% CI, 0.72-0.89). When combined with demographic factors, the AUC of the biomarker panel reached 0.87 (95% CI, 0.80-0.93). CONCLUSIONS We have discovered and validated a panel of salivary exRNA biomarkers with credible clinical performance for the detection of GC. Our study demonstrates the potential utility of salivary exRNA biomarkers in screening and risk assessment for GC.

Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells.