Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency.

BACKGROUND: Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. METHODS: The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. RESULTS: An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. CONCLUSION: This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.

Capillary electrophoresis-laser-induced fluorescence (CE-LIF)-based immunoassay for quantifying antibodies against cyclic citrullinated peptides.

We developed an assay for diagnosing rheumatoid arthritis (RA) by capillary electrophoresis with the laser-induced fluorescence detection (CE-LIF) of antibodies against cyclic citrullinated peptides (CCPs) using fluorescein isothiocyanate-labeled F-CCP11A and F-CCP. The CCPs were incubated overnight with a purified antibody against CCP and then injected into the CE-LIF system to estimate complex concentration. The immunocomplexes and free peptides were separated within 5 min; the LOD and LOQ values of the immunoassay are 0.1 µg mL-1 and 0.3 µg mL-1, respectively. The calibration curves for the immunocomplex had a coefficient of determination of 0.98. The reproducibility of the CE-LIF based immunoassay when using F-CCP11A and F-CCP was less than 5% and the accuracy ranged between 89 and 103%. The applicability of the assay was evaluated using serum samples from RA patients and healthy control subjects. We found that the sensitivity and specificity for RA diagnosis were 100% and 83.3%, respectively, for F-CCP11A and 100% and 90%, respectively, for F-CCP. These results demonstrate that the CE-LIF method can be useful for quantitative immunoassay development.

MIR-27a regulates the TGF-ß signaling pathway by targeting SMAD2 and SMAD4 in lung cancer.

The transforming growth factor-ß (TGF-ß) signaling pathway is associated with carcinogenesis and various biological processes. SMAD2 and SMAD4, which are putative tumor suppressors, have an important role in TGF-ß signaling. The aberrant expression of these genes is implicated in some cancers. However, the mechanisms of SMAD2 and SMAD4 dysregulation are poorly understood. In this study, we observed that miR-27a was upregulated in lung cancer cell lines and patients. In addition, SMAD2 and SMAD4 genes were identified as targets of miR-27a by several target prediction databases and experimental validation. Functional studies revealed that miR-27a overexpression decreased SMAD2 and SMAD4 mRNA and protein levels. Furthermore, miR-27a contributed to cell proliferation and invasion by inhibiting TGF-ß-induced cell cycle arrest. These results suggest that miR-27a may function as an oncogene by regulating SMAD2 and SMAD4 in lung cancer. Thus, miR-27a may be a potential target for cancer therapy.

An improvement of miRNA extraction efficiency in human plasma.

MicroRNAs (miRNAs) are short noncoding RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play important roles in the pathogenesis of diseases. Numerous studies are aimed at discovering biomarkers of diseases or determining miRNA functions by monitoring circulating miRNAs in various biological sources such as plasma and urine. However, the analysis of miRNA in such fluids presents problems related to accuracy and reproducibility because of their low levels in biological fluids. Therefore, better extraction kits and more sensitive detection systems have been developed for improved and reproducible analysis of circulating miRNAs. However, new extraction methods are also needed to improve the yield of miRNAs for their reliable analysis from biological fluids. The combination of yeast transfer RNA (tRNA) and glycogen as carrier molecules and incubation durations were optimized to maximize extraction efficiency. The extraction recovery using a combination of yeast tRNA and glycogen was approximately threefold more than that by using glycogen or yeast tRNA alone. In addition, reproducible and accurate analysis of miRNAs can be carried out after extraction using a combination of yeast tRNA and glycogen without an impact on plasma components. Graphical abstract Steps of miRNA extraction in plasma.

Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay.

An assay for protein kinase C delta (PKCδ) activity based on the quantification of a synthetic substrate using capillary electrophoresis with laser-induced fluorescence detection was developed. The peptides labeled with fluorescein isothiocyanate F-ERK (where ERK is extracellular signal-regulated kinase) and the phosphorylated form, P-F-ERK, were utilized for the method development and validation. The migration time of F-ERK and P-F-ERK were 6.3 ± 0.1 and 8.7 ± 0.2 min, respectively. LOD and LOQ values of F-ERK were 2 and 6 ng/mL and those of P-F-ERK were 4 and 12 ng/mL. The correlation coefficients obtained from two standard curves were approximately 0.99. The reproducibility and accuracy of the method for F-ERK ranged 1.5-4.7 and 86-109%, respectively, and those for P-F-ERK were 1.6-6.1 and 93-109%, respectively. The activity of PKCδ was studied in vitro using the human gastric cancer cell line MKN-1. The use of PKCδ inhibitor candidates, including GÓ§6983, bisindolylmaleimide II, staurosporine, and rottlerin in the assay resulted in IC50 values of 50 nM, 15 nM, 795 nM, and 4 µM, respectively. Comparison of our assay with a commercial PKC kit revealed that our assay is more adaptable to differing enzyme isoforms. This method has potential for high throughput screening for kinase inhibitors as part of a drug discovery program.

Evaluation of inhibition of miRNA expression induced by anti-miRNA oligonucleotides.

MicroRNAs (miRNAs) are short RNA molecules that control the expression of mRNAs associated with various biological processes. Therefore, deregulated miRNAs play an important role in the pathogenesis of diseases. Numerous studies aimed at developing novel miRNA-based drugs or determining miRNA functions have been conducted by inhibiting miRNAs using anti-miRNA oligonucleotides (AMOs), which inhibit the function by hybridizing with miRNA. To increase the binding affinity and specificity to target miRNA, AMOs with various chemical modifications have been developed. Evaluating the potency of these various types of AMOs is an essential step in their development. In this study, we developed a capillary electrophoresis with laser-induced fluorescence (CE-LIF) method to evaluate the potency of AMOs by measuring changes in miRNA levels with fluorescence-labeled ssDNA probes using AMO-miR-23a, which inhibits miR-23a related to lung cancer. In order to eliminate interference by excess AMOs during hybridization of the ssDNA probe with the miR-23a, the concentration of the ssDNA probe was optimized. This newly developed method was used to compare the potency of two different modified AMOs. The data were supported by the results of a luciferase assay. This study demonstrated that CE-LIF analysis could be used to accurately evaluate AMO potency in biological samples.

Highly sensitive immunoassay for the diagnosis of acute myocardial infarction using silica spheres encapsulating a quantum dot layer.

Commercial ELISA kits for substance P (SubP), which are helpful for the clinical diagnosis of acute myocardial infarction, are limited in efficacy because of low sensitivity. A highly sensitive immunoassay was developed using silica spheres encapsulating a quantum dot-layer (SQS) and labeling antibodies, on a Parylene A-modified plate. The high sensitivity was possible by taking advantage of the enhanced photoluminescence of the SQS and dense immobilization of SubP on a Parylene A-modified plate. Glutaraldehyde was used for cross-linking of SQS to the anti-SubP antibody and SubP to the Parylene A coating. The SQS-linked immunosorbent assay (SQSLISA) was optimized and validated. The dynamic range for the assay was 1-10000 pg/mL with a linear correlation factor of 0.9992 when the competitive SQSLISA was employed. The intra- and interday accuracies were 93-100% and 87-122%, respectively. The reproducibility was lower than 11%. The developed method was applied to clinical samples collected from healthy controls (n = 30) and acute myocardial infarction (n = 16) and it displayed a high correlation with the commercial ELISA kit, with a limit of detection that was 30-fold lower. Clinical sample analysis confirmed that SubP is a promising diagnostic marker for acute myocardial infarction. The SQSLISA is expected to be a practical and useful assay tool.

Quantitative analysis of a ubiquitin-dependent substrate using capillary electrophoresis with dual laser-induced fluorescence.

Protein degradation by the ubiquitin-proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS-dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)-fusion of the ubiquitin substrate ribophorin 1 (GFP-RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP-RPN1 and RFP in cell lysates were performed in an untreated capillary (75 µm × 50 cm) and 100 mM Tris-CHES buffer (pH 9.0) containing 10 mM SDS. GFP-RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP-RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP-RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS-dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.

Potential role of Hsp25 in calcium-modulated cardiomyocytes.

Cardiac muscle contraction is initiated by the entry of Ca²âº ions from the extracellular spaces and the Ca²âº stores in the sarcoplasmic reticulum into the myoplasm. Calcium signaling is the most important factor in cardiac cell homeostasis. In this study, we investigated the effect of caffeine, an inducer of intracellular Ca²âº accumulation, on HL-1 cardiomyocytes by using a proteomic approach. Following the separation of the cell lysates and visualization of the protein spots using two-dimensional gel electrophoresis and silver staining, respectively, we identified 24 differentially expressed protein spots in the caffeine-treated group as compared with the controls by using MALDI-TOF/TOF MS. Of these 24 spots, 8 proteins were up-regulated and 16 proteins were down-regulated. These differentially expressed proteins are predominantly involved in cellular metabolism, cellular organization, and ion/protein transport. Furthermore, we found that Hsp25, one of the differentially expressed proteins, is modified by caffeine treatment. Depletion of Hsp25 transcripts by siRNA increased caffeine-mediated signaling, including ERK activation, and decreased the Ca²âº transient peak and expression of calsequestrin 2 in HL-1 cardiomyocytes. These results suggest that proteins having various functions are involved in the regulation of Ca²âº homeostasis, and that Hsp25 plays an important role in regulating cardiac function during caffeine response.

Growing trend of CE at the omics level: the frontier of systems biology--an update.

Omics is the study of proteins, peptides, genes, and metabolites in living organisms. Systems biology aims to understand the system through the study of the relationship between elements such as genes and proteins in biological system. Recently, systems biology emerged as the result of the advanced development of high-throughput analysis technologies such as DNA sequencers, DNA arrays, and mass spectrometry for omics studies. Among a number of analytical tools and technologies, CE and CE coupled to MS are promising and relatively rapidly developing tools with the potential to provide qualitative and quantitative analyses of biological molecules. With an emphasis on CE for systems biology, this review summarizes the method developments and applications of CE for the genomic, transcriptomic, proteomic, and metabolomic studies focusing on the drug discovery and disease diagnosis and therapies since 2009.

Endothelin-1- and isoproterenol-induced differential protein expression and signaling pathway in HL-1 cardiomyocytes.

It is well known that the two chemical compounds endothelin-1 (ET-1) and isoproterenol (ISO) can individually induce cardiac hypertrophy through G protein-coupled receptors in cardiomyocytes. However, the cardiac hypertrophy signaling pathway activated by ET-1 and ISO is not well defined. Therefore, we investigated the protein expression profile and signaling transduction in HL-l cardiomyocyte cells treated with ET-1 and ISO. Following separation of the cell lysates by using 2-DE and silver staining, we identified 16 protein spots that were differentially expressed as compared to the controls. Of these 16 spots, three changed only after treatment with ET-1, whereas four changed only after treatment with ISO, suggesting that these two stimuli could induce different signaling pathways. In order to reveal the differences between ET-1- and ISO-induced signaling, we studied the different events that occur at each step of the signaling pathways, when selected biocomponents were blocked by inhibitors. Our results indicated that ET-1 and ISO used different pathways for phosphorylation of glycogen synthase kinase-3ß (GSK3ß). ET-1 mainly used the mitogen-activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways to activate GSK3ß, whereas under ISO stimulation, only the phosphatidylinositol-3-kinase/AKT pathway was required to trigger the GSK3ß pathway. Furthermore, the strength of the GSK3ß signal in ISO-induced cardiac hypertrophy was stronger than that in ET-1-induced cardiac hypertrophy. We found that these two agonists brought about different changes in the protein expression of HL-1 cardiomyocytes through distinct signaling pathways even though the destination of the two signaling pathways was the same.

Capillary electrophoretic mobility shift assay for binding of DNA with NFAT3, a transcription factor from H9c2 cardiac myoblast cells.

Nuclear factor of activated T-cells (NFAT) is a transcription factor involved in the development of cardiac and skeletal muscle and the nervous system. NFAT is activated by calcium signal pathway and translocated into the nucleus. The quantification of binding between NFAT and NFAT-specific DNA gives important information about cardiac hypertrophy. We investigated the binding of NFAT3 in nuclear extracts from H9c2 cells to its specific DNA by capillary electrophoretic mobility shift assay. The binding reaction time required for stable formation of the DNA-NFAT3 complex was 3 h and the separation of the complex and free DNA was achieved within 10 min by CE. The formation of NFAT3-DNA complex was confirmed by the competitive reaction. Comparison of the ratios of complex/free DNA peak area for 1 µM endothelin-1 (ET-1)-treated cells and control cells showed the NFAT3 translocation into the nucleus promoted by ET-1. The binding constant between NFAT3 and DNA was estimated to be 7.7×10(9) M(-1) at 4°C.

Growing trend of CE at the omics level: the frontier of systems biology.

In a novel attempt to comprehend the complexity of life, systems biology has recently emerged as a state-of-the-art approach for biological research in contrast to the reductionist approaches that have been used in molecular cell biology since the 1950s. Because a massive amount of information is required in many systems biology studies of life processes, we have increasingly come to depend on techniques that provide high-throughput omics data. CE and CE coupled to MS have served as powerful analytical tools for providing qualitative and quantitative omics data. Recent systems biology studies have focused strongly on the diagnosis and treatment of diseases. The increasing number of clinical research papers on drug discovery and disease therapies reflects this growing interest among scientists. Since such clinical research reflects one of the ultimate purposes of bioscience, these trends will be sustained for a long time. Thus, this review mainly focuses on the application of CE and CE-MS in diagnosis as well as on the latest CE methods developed. Furthermore, we outline the new challenges that arose in 2008 and later in elucidating the system-level functions of the bioconstituents of living organisms.

Sensitivity enhancement of CE and CE-MS for the analysis of peptides by a dynamic pH junction.

An analytical method of CE-MS and CE with an online preconcentration technique induced by a dynamic pH junction, addition of organic solvent and large volume injection was developed for sensitive determination of peptides in biological samples. Leucine enkephalin, methionine enkephalin, dynorphin A, ß-endorphin and angiotensin II were used as model peptides. The optimal online preconcentration conditions were obtained at a sample matrix consisting of 100 mM borate buffer (pH 10.0) with 50% v/v acetonitrile and a BGE containing 1 M formic acid at pH 2.0, along with a 25-cm injection length. Under the optimized conditions, a 4.0×10(3)-1.1×10(4)-fold increase in peak intensity was achieved without degrading the peak shape. This online preconcentration method was applied to analyze the intracellular angiotensin II within the peptides extracted from HL1 cells and approximately increase of 1×10(4)-fold sensitivity was achieved compared to normal condition. Thus, the developed method could be applied to the analysis of various peptides for peptidomics study in biological samples.

MiR-195 and miR-497 suppress tumorigenesis in lung cancer by inhibiting SMURF2-induced TGF-ß receptor I ubiquitination.

SMURF2 is a member of the HECT family of E3 ubiquitin ligases that have important roles as a negative regulator of transforming growth factor-ß (TGF-ß) signaling through ubiquitin-mediated degradation of TGF-ß receptor I. However, the regulatory mechanism of SMURF2 is largely unknown. In this study, we identified that micro(mi)R-195 and miR-497 putatively target SMURF2 using several target prediction databases. Both miR-195 and miR-497 bind to the 3'-UTR of the SMURF2 mRNA and inhibit SMURF2 expression. Furthermore, miR-195 and miR-497 regulate SMURF2-dependent TßRI ubiquitination and cause the activation of the TGF-ß signaling pathway in lung cancer cells. Upregulation of miR-195 and miR-497 significantly reduced cell viability and colony formation through the activation of TGF-ß signaling. Interestingly, miR-195 and miR-497 also reduced the invasion ability of lung cancer cells when cells were treated with TGF-ß1. Subsequent in vivo studies in xenograft nude mice model revealed that miR-195 and miR-497 repress tumor growth. These findings demonstrate that miR-195 and miR-497 act as a tumor suppressor by suppressing ubiquitination-mediated degradation of TGF-ß receptors through SMURF2, and suggest that miR-195 and miR-497 are potential therapeutic targets for lung cancer.

Kinetic analysis of the MAPK and PI3K/Akt signaling pathways.

Computational modeling of signal transduction is currently attracting much attention as it can promote the understanding of complex signal transduction mechanisms. Although several mathematical models have been used to examine signaling pathways, little attention has been given to crosstalk mechanisms. In this study, an attempt was made to develop a computational model for the pathways involving growth-factor-mediated mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase/protein kinase B (PI3K/Akt). In addition, the dynamics of the protein activities were analyzed based on a set of kinetic data. The simulation approach integrates the information on several levels and predicts systems behavior. The in-silico analysis conducted revealed that the Raf and Akt pathways act independently.

Measurement of phosphorylated phospholamban levels in cardiomyocytes (HL-1) by immunoprecipitation.

Phospholamban (PLN) is a key regulatory protein involved in cardiac calcium signaling through the pumping of cytoplasmic Ca2+ into the sarcoplasmic reticulum (SR). Recent systems-level studies have focused on integrating quantitative data (e.g. protein expression levels) for a better understanding of cardiac systems biology. In this view, we developed a capillary electrophoresis (CE) based immunoprecipitation method for the measurement of phospho-PLN (ser 16) in cardiomyocytes (HL-1 cell line). Dose-dependent isoproterenol (Iso) treated cells were analyzed using CE, and the phospho-PLN levels were quantified using specific polyclonal antibodies. The CE method employed was accurate, quick and easier compare to other techniques and the results are useful for the subsequent computational systems biology research.

Validation of Neurotensin Receptor 1 as a Therapeutic Target for Gastric Cancer.

Gastric cancer is the fifth most common type of malignancy worldwide, and the survival rate of patients with advanced-stage gastric cancer is low, even after receiving chemotherapy. Here, we validated neurotensin receptor 1 (NTSR1) as a potential therapeutic target in gastric cancer. We compared NTSR1 expression levels in sixty different gastric cancer-tissue samples and cells, as well as in other cancer cells (lung, breast, pancreatic, and colon), by assessing NTSR1 expression via semi-quantitative real-time reverse transcription polymerase chain reaction, immunocytochemistry and western blot. Following neurotensin (NT) treatment, we analyzed the expression and activity of matrix metalloproteinase-9 (MMP-9) and further determined the effects on cell migration and invasion via wound-healing and transwell assays. Our results revealed that NTSR1 mRNA levels were higher in gastric cancer tissues than non-cancerous tissues. Both of NTSR1 mRNA levels and expression were higher in gastric cancer cell lines relative to levels observed in other cancer-cell lines. Moreover, NT treatment induced MMP-9 expression and activity in all cancer cell lines, which was significantly decreased following treatment with the NTSR1 antagonist SR48692 or small-interfering RNA targeting NTSR1. Furthermore, NT-mediated metastases was confirmed by observing epithelial-mesenchymal transition markers SNAIL and E-cadherin in gastric cancer cells. NT-mediated invasion and migration of gastric cancer cells were reduced by NTSR1 depletion through the Erk signaling. These findings strongly suggested that NTR1 constitutes a potential therapeutic target for the inhibition of gastric cancer invasion and metastasis.

Investigation of the estrogenic activities of pesticides from Pal-dang reservoir by in vitro assay.

Endocrine disruptors, when absorbed into the body, interfere with the normal function by mimicking or blocking the hormone system. To investigate compounds mimicking estrogen in the drinking water source of the residence of Seoul, the Pal-dang reservoir was monitored over a period of 5 years, between 2000 and 2004. Nine kinds of pesticide (carbaryl, DBCP, diazinon, fenitrothion, fenobucarb, flutolanil, iprobenphos, isoprothiolane and parathion) were found to exist in the river water sample. These compounds were detected at low concentrations in the water samples. The total concentration and those of each of these pesticides were below the permissible limits of the National Institute of Environmental Research (NIER), Korea. The estrogenic potencies of the nine pesticides were examined using an E-screen assay with MCF-7 BUS estrogen receptor (ER)-positive human breast cancer cells, with ER-negative MDA MB 231 cell lines also used to compare the results. From this, flutolanil and isoprothiolane were confirmed to have estrogenic activities as shown by the increasing MCF-7 BUS cell growth on their addition. In addition, the estrogen receptor alpha (ERalpha) protein, estrogen receptor-regulated progesterone receptor (PR) and pS2 mRNA levels on the addition of flutolanil and isoprothiolane were measured with MCF-7 BUS cells. It was observed that the levels of ERalpha protein decreased and those of the PR and pS2 genes increased on the addition of either flutolanil or isoprothiolane at concentrations of 10(-4) M, in the same manner as with the addition of 17beta-estradiol, which was used as the positive control. From these results, it was confirmed that flutolanil and isoprothiolane exhibit estrogenic activities, suggesting they might act through estrogen receptors.

Sample preparation for detection of low abundance proteins in human plasma using ultra-high performance liquid chromatography coupled with highly accurate mass spectrometry.

Proteomics is a valuable approach to discover biomarkers in human plasma for early diagnosis. However, detection of biomarkers in the plasma is still challenging because of its large protein content. In our study, we established albumin/IgG depletion methods for identification of low abundance proteins using two commercial kits with additional buffer conditions and various concentrations of cold acetone. Trypsin digestion, desalting, and data-dependent acquisition were also optimized. More than 80% depletion of albumin/IgG was achieved with two commercial kits and 98% depletion of albumin was obtained with 70% cold acetone. Recovery of four reference proteins, BNP (47-76), insulin, cytochrome c, and ubiquitin was obtained in all optimized methods. The best recovery of reference proteins was obtained using the ProteoExtract albumin/IgG removal kit with buffer A (61%-106%). After cold acetone precipitation, three reference proteins were recovered more than 48% except ubiquitin (12%). The number of identified proteins by Mascot was 28, 35, 17, and 34 for ProteoExtract, ProteoPrep, 70%, and 50% cold acetone, respectively. Furthermore, optimized methods detected MS/MS fragmentation patterns of elevated BNP in patient samples with cardiac disease. Our study provides the conditions for efficient biomarker discovery by minimal removing of high abundant proteins.