TaqMan Low Density Array Analysis Research Articles

Regulation of serum microRNA expression by acupuncture in patients with diarrhea-predominant irritable bowel syndrome.

To screen for differentially expressed serum microRNAs (miRNAs) in patients with diarrhea-predominant irritable bowel syndrome (IBS-D) compared with healthy participants and explore the mechanism of acupuncture in the treatment of IBS-D based on miRNAs. IBS-D patients that met the Rome III diagnostic criteria and age- and sex-matched healthy participants were enrolled between April 2017 and December 2017. Serum miRNA levels were initially determined using a TaqMan low-density array (TLDA) in pooled samples. Markedly altered miRNAs in IBS-D patients were subsequently validated using quantitative real-time polymerase chain reaction (qRT-PCR) on individual samples. All IBS-D patients accepted the acupuncture therapy for 6 weeks. The disease severity was assessed using the IBS symptom severity scale (IBS-SSS) questionnaire before and after treatment. After acupuncture, the patients' serum was re-analyzed for altered expression of the miRNAs by qRT-PCR. TLDA and qRT-PCR analysis revealed six upregulated miRNAs (miR-1305, miR-575, miR-149-5p, miR-190a-5p, miR-135a-5p, and miR-148a-3p; P < 0.05) and two downregulated miRNAs (miR-194-5p, miR-127-5p; P < 0.05) in IBS-D patients compared with healthy controls. Post acupuncture treatment, total IBS-SSS scores, severity of abdominal pain, duration of abdominal pain, severity of abdominal distention, dissatisfaction with bowel habits and disruption in quality of life decreased significantly (P < 0.001). Furthermore, the upregulated miR-148a-3p levels in IBS-D patients also decreased significantly after acupuncture (P < 0.05). The over-expression or reduced expression of several miRNAs may contribute to IBS-D pathogenesis. Acupuncture might downregulate miR-148a-3p through multiple pathways to alleviate or relieve IBS-D symptoms. ChiCTR-IOR-17010860 (Chinese Clinical Trials Registry).

Circulating miR-145 as a marker of therapeutic response to anti-TNF therapy in patients with ankylosing spondylitis

Circulating miRNAs appear promising therapeutic and prognostic biomarkers. We aimed to investigate the predictive value of circulating miRNAs on the disease outcome following anti-TNF therapy in patients with ankylosing spondylitis (AS). Our study included 19 AS patients assessed at baseline (M0), after three (M3) and twelve months (M12) of therapy. Total RNA was isolated from plasma. A comprehensive analysis of 380 miRNAs using TaqMan Low Density Array (TLDA) was followed by a single assay validation of selected miRNAs. All AS patients had high baseline disease activity and an excellent response to anti-TNF therapy at M3 and M12. TLDA analysis revealed the dysregulation of 17 circulating miRNAs, including miR-145. Single assay validation confirmed that miR-145 is significantly downregulated at M3 compared to baseline. The decrease in the levels of miR-145 from M0 to M3 negatively correlated with the change in BASDAI from M0 to M3; and positively correlated with disease activity improvement from M3 to M12 as per BASDAI and ASDAS. The predictive value of the early change in miR-145 and levels of miR-145 at M3 were further validated by Receiver operating curves analysis. We show thatthe early change in circulating miR-145 may be a predictor for the future outcome ofAS patients treated with TNF inhibitors. Patients with a more significant decrease in miR-145 levels may show further significant improvement of disease activity after 12 months. Monitoring the expression of miR-145 in plasma in AS patients may, therefore, influence our therapeutic decision-making.

Plasma miR-193a-3p can be a potential biomarker for the diagnosis of diabetic nephropathy

Diabetic nephropathy is one of the most common microvascular complications in patients with diabetes. MicroRNA (miRNA, miR) is closely related to the formation, development and pathophysiology of diabetic nephropathy. We aimed to investigate whether miR-193a-3p could be used as a potential biomarker for the diagnosis of diabetic nephropathy. Plasma samples were collected from all the participants. TaqMan Low Density Array analysis was employed to obtain the miRNA profiles of plasma samples, and qRT-PCR was used to confirm the result. Receiver operating characteristic curves were employed to evaluate the specificity and sensitivity of miR-193a-3p for predicting diabetic nephropathy. The expression of miR-193a-3p and miR-320c was elevated and miR-27a-3p was decreased in diabetic nephropathy patients compared to patients with type 2 diabetes and healthy controls. We found that, in diabetic nephropathy patients, the elevated miR-193a-3p expression had a negative correlation with the level of evaluate glomerular filtration rate, while a positive correlation with the level of proteinuria. We further demonstrated that miR-193a-3p could be employed to distinguish patients with diabetic nephropathy. The Kaplan-Meier analysis showed that the high expression of miR-193a-3p significantly shortened the dialysis-free survival of diabetic nephropathy patients. In conclusion, miR-193a-3p is involved in diabetic nephropathy pathogenesis and may serve as a potentially novel diagnostic biomarker for diabetic nephropathy.

MicroRNA Profile of Cardiovascular Risk in Patients with Obstructive Sleep Apnea

Background: Obstructive sleep apnea (OSA) is a common disease caused by repeated episodes of collapse of the upper airway during sleep and is associated with the development of cardiovascular disease (CVD). However, there is high heterogeneity in the impact of OSA on patients. Until now, the profile of OSA patients at risk of developing CVD has not been defined, including the measurable variables that could be used to predict the CVD risk of a patient with OSA. Objective: The aim of this study was to identify the microRNA (mi­RNA) profile associated with CVD in patients with OSA. Method: This is an observational, cross-sectional study that included 132 male patients. Three groups were defined as OSA patients, OSA patients with hypertension, and OSA patients who developed a major cardiovascular event. Polysomnography and ambulatory blood pressure measurements were performed. The expression profiling of 188 miRNAs in plasma was performed in 21 subjects (matched by BMI and age) by the TaqMan low density array (TLDA). miRNAs differentially expressed in the different subgroups of patients and miRNAs that correlated with the cardiovascular risk SCORE were selected for validation by RT-qPCR in the 111 remaining patients. Results: From the TLDA analysis, 7 miRNAs were selected for validation. Differential expression was not confirmed in any of the miRNAs. miR-143 was associated with nocturnal systolic blood pressure. miR-107 correlated with 24-h blood pressure parameters and with nocturnal hypertension. miR-486 was associated with the cardiovascular risk SCORE. Conclusions: The circulating profile of miRNAs does not seem to be different in any of the subgroups of patients with OSA and different cardiovascular risk factors. Nevertheless, miR-107 and miR-143 are associated with specific blood pressure parameters in patients with OSA and miR-486 is associated with the cardiovascular risk SCORE.

169 PITX2C deficiency augments the anti-arrhythmic properties of flecainide: results in a mouse model and validation in a human atrium simulation study

Background and objectives Polymorphisms close to the PITX2 gene on chromosome 4q25 are associated with incident and recurrent atrial fibrillation (AF). Carriers of variant rs10033464 respond relatively well to antiarrhythmic drug therapy with sodium channel blockers. To test whether altered atrial PITX2 mRNA expression alters the electrophysiological effect of sodium channel blockers, we studied the effect of flecainide (Flec) in hearts of mice with heterozygous Pitx2c gene deletion, a model for reduced Pitx2 mRNA expression and AF susceptibility. Methods We assessed the effect of 1µM Flec on left atrial (LA) monophasic, transmembrane and optical action potentials in Pitx2c +/– mice and their wild type (WT) littermates. We measured conduction velocity (CV), action potential duration (APD), AP amplitude (APA), maximum upstroke velocity (dV/dt max ) and resting membrane potential (RMP) during atrial pacing at 80–120 ms fixed-rate cycle lengths (CL); and effective refractory periods (ERP) with arrhythmia inducibility during programmed (S2) stimulation. Post-repolarisation refractoriness (PRR) was defined as the difference between ERP and APD90. We assessed whether the electrophysiological changes associated with reduced Pitx2c mRNA expression alters the response to sodium current (I Na ) block using the Courtemanche et al . human atrial model. Taqman low density array analysis was also used to measure changes in 20 ion channel, and related, genes of interest. Results Flec abolished arrhythmias in Pitx2c +/– (6/18 base vs 0/15 Flec, p +/– atria compared with WT (Pitx2c +/– 14 ± 2 ms, n = 13 vs WT 7 ± 2 ms, n = 10; p +/– 21 ± 3 ms, n = 6 vs WT 6 ± 4 ms, n = 6, p +/– atria had a slightly more positive resting membrane potential (WT –70 ± 0.7 mV, n = 30 cells vs PITX2c +/– –68 ± 0.7 mV, n = 32 cells, p +/– 73 ± 1.3 mV, n = 32). Flec had no effect on the RMP but reduced CV, APA and dV/dt max in both genotypes to a similar extent. Hearts with PRR were not susceptible to arrhythmias (0/17). As with the isolated hearts, I Na block prolonged PRR in the Pitx2c deficiency simulation (500 ms CL: reference model 30ms; Pitx2c deficiency model 42 ms) and had no differential effects of I Na on RMP, CV, APD, or dV/dt max . The human atrial model thus qualitatively reproduced the experimental findings. mRNA expression of Kcnk5 and Kcna6 was altered in Pitx2c +/– left atria. Conclusion Flecainide suppresses atrial arrhythmias in mice with reduced Pitx2 mRNA expression. We propose that this genotype-specific antiarrhythmic effect can be explained by a more positive resting membrane potential and higher post-repolarisation refractoriness. These data suggest that flecainide may be effective for rhythm control in carriers of genetic variants on chromosome 4q25.

Influence of neonatal sevoflurane exposure on nerve development-related microRNAs and behavior of rats.

Commonly used anesthetics adversely affect the developing brain, but the mechanisms remain unknown. We previously showed that the expressions of microRNAs (miRNAs) in major organs are affected by anesthetics. Therefore, we used TaqMan low-density array (TLDA) to analyze gene expression in the hippocampus of neonatal rats exposed to sevoflurane and performed behavioral tests after they reached adulthood to evaluate cognitive and memory function. Rat male pups at postnatal day 7 were exposed to 1.9% sevoflurane for 3 h, and the hippocampus-miRNA expression profile on postnatal day 8 was determined. Open field and fear conditioning tests conducted during postnatal weeks 7 and 8 indicated that sevoflurane-exposed rats, but not controls, exhibited anxiety-like disorders. TLDA analysis identified 20 differentially expressed miRNAs, which were not shared between postnatally and maturely sevoflurane-exposed rats. The level of rno-miR-632, which targets brain-derived neurotrophic factor and calcium channel, voltage-dependent, alpha 2/delta subunit 2, increased by 10-fold, indicating that exposure to sevoflurane during early neural development alters hippocampus-miRNA expression and may induce subsequent behavioral disorders.

TRAIL-based tumor sensitizing galactoxyloglucan, a novel entity for targeting apoptotic machinery

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an attractive target for cancer therapy due to its ability to selectively induce apoptosis in cancer cells, without causing significant toxicity in normal tissues. We previously reported that galactoxyloglucan (PST001) possesses significant antitumor and immunomodulatory properties. However, the exact mechanism in mediating this anticancer effect is unknown. This study, for the first time, indicated that PST001 sensitizes non-small cell lung cancer (A549) and nasopharyngeal (KB) cells to TRAIL-mediated apoptosis. In vitro studies suggested that PST001 induced apoptosis primarily via death receptors and predominantly activated caspases belonging to the extrinsic apoptotic cascade. Microarray profiling of PST001 treated A549 and KB cells showed the suppression of survivin (BIRC5) and anti-apoptotic Bcl-2, as well as increased cytochrome C. TaqMan low density array analysis of A549 cells also confirmed that the induction of apoptosis by the polysaccharide occurred through the TRAIL-DR4/DR5 pathways. This was finally confirmed by in silico analysis, which revealed that PST001 binds to TRAIL-DR4/DR5 complexes more strongly than TNF and Fas ligand–receptor complexes. In summary, our results suggest the potential of PST001 to be developed as an anticancer agent that not only preserves innate biological activity of TRAIL, but also sensitizes cancer cells to TRAIL-mediated apoptosis.

Circulating microRNAs as biomarkers for evaluating the severity of acute spinal cord injury

An in vivo study in mouse models of spinal cord contusion. To develop a novel indicator to anticipate the severity of spinal cord injury (SCI) during the acute phase and for the assessment of the efficacy of novel therapies. MicroRNAs (miRNAs) circulating in the peripheral blood are reported to modulate signaling between cells, and to be diagnostic markers for cancers. The purpose of this study was to identify circulating miRNAs for predicting the severity of SCI in the acute phase. Department of Orthopaedic Surgery, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Mouse SCI models were made using Infinite Horizon impactor with 50 or 70 kdyn compressing power following thoracic laminectomy. The mice were then divided into four groups: normal (without surgery), sham (laminectomy only), mild (50 kdyn), and severe (70 kdyn). TaqMan low-density array analysis and real-time PCR were performed to identify candidate miRNAs that were increased in the serum relative to the severity of SCI. The expression levels of miR-9*, miR-219 and miR-384-5p in the serum were significantly increased relative to the severity of SCI 12 h after injury. The expression of miR-9* was also significantly increased relative to injury severity at 3 and 24 h after injury. Serum miR-9*, miR-219 and miR-384-5p might be promising biomarkers for predicting the severity of SCI.

The miRNA and mRNA Changes in Rat Hippocampi after Chronic Constriction Injury

We elaborated the rat hippocampi in order to assess for central nervous system changes following a peripheral neuropathic injury. We examined the gene changes in the hippocampi of chronic constriction injury (CCI) rats with TaqMan® low-density array analysis (TLDA) and quantitative real-time polymerase chain reaction (qRT-PCR) of miR-125b, -132, and messenger RNAs (mRNAs) of neuropeptide Y, brain-derived neural factor, N-methyl-D-aspartate glutamate 2A receptor, gamma-aminobutyric acid A a1 receptor, gamma-aminobutyric acid A b1 receptor, gamma-aminobutyric acid B b2 receptor, serotonin 1A receptor, serotonin 2A receptor, serotonin 2C receptor, and serotonin 3A receptor on days 0, 7, and 15 after surgery. None. Two behavioral tests (thermal and mechanical stimulation tests) were performed three times at 5-minute intervals to assess pain thresholds. MicroRNA (miRNA) changes were examined by TLDA. mRNA changes were examined by qRT-PCR. Statistical significance was determined by Tukey-Kramer's method and paired t-test. All rats showed mechanical and thermal hypersensitivity on the ipsilateral side. Out of 373 miRNAs analyzed, 237 were expressed, and 51 changed their expressions after CCI. By TLDA, cluster analysis found obvious miRNA changes on day 7 that tended to recover by day 15. For miR-125b, the relative expression decreased to 0.70 ± 0.30 at day 7 and recovered to 1.65 ± 0.19 at day 15. The miR-132 relative expressions were 0.69 ± 0.30 and 0.70 ± 0.15, respectively. The mRNA changes followed the miRNA changes. Our results showed that the peripheral nerve injury altered rat hippocampal miRNA.

Research Resource: Nuclear Receptors as Transcriptome: Discriminant and Prognostic Value in Breast Cancer

To identify biologically relevant groupings or clusters of nuclear receptors (NR) that are associated with breast neoplasia, with potentially diagnostic, discriminant or prognostic value, we quantitated mRNA expression levels of all 48 members of the human NR superfamily by TaqMan low-density array analysis in 116 curated breast tissue samples, including pre- and postmenopausal normal breast and both ERα(+) and ERα(-) tumor tissue. In addition, we have determined NR levels in independent cohorts of tamoxifen-treated ERα(+) and ERα(-) tissue samples. There were differences in relative NR mRNA expression between neoplastic and normal breast, and between ER(+) and ER(-) tumors. First, there is overexpression of the NUR77 subgroup and EAR2 in neoplastic breast. Second, we identify a signature of five NR (ERα, EAR2, NUR77, TRα, and RARγ) that classifies breast samples with more than 97% cross-validated accuracy into normal or cancer classes. Third, we find a novel negative association between five NR (TRβ, NUR77, RORγ, COUP-TFII, and LRH1) and histological grade. Finally, four NR (COUP-TFII, TRβ, PPARγ, and MR) are significant predictors of metastasis-free survival in tamoxifen-treated breast cancers, independent of ER expression. The present study highlights the discriminant and prognostic value of NR in breast cancer; identifies novel, clinically relevant, NR signatures; and highlights NR signaling pathways with potential roles in breast cancer pathophysiology and as new therapeutic targets.

Enoxaparin Improves the Course of Dextran Sodium Sulfate-Induced Colitis in Syndecan-1-Deficient Mice

Syndecan-1 (Sdc1) plays a major role in wound healing and modulates inflammatory responses. Sdc1 expression is reduced in lesions of patients with ulcerative colitis. The aim of this study was to investigate the role of Sdc1 in murine dextran sodium sulfate (DSS)-induced colitis. DSS colitis was induced in Sdc1-deficient (knockout (KO)) and wild-type mice by oral administration of 3% DSS. KO mice exhibited a significantly increased lethality as compared with wild-type controls (61 versus 5%, P < 0.05). Impaired mucosal healing and prolonged recruitment of inflammatory cells in KO mice were accompanied by significant up-regulation of tumor necrosis factor-alpha, CC chemokine ligand 3/macrophage inflammatory protein-1alpha, and vascular cell adhesion molecule-1, as determined by histological correlation between 0 and 15 days after colitis induction, TaqMan low-density array analysis, and quantitative real-time PCR. Treatment from days 7 through 14 with enoxaparin, a functional analogue of the Sdc1 heparan sulfate chains, significantly reduced lethality of KO mice due to DSS-induced colitis, which was correlated with improved mucosal healing. In vitro, Sdc1-deficient polymorphonuclear cells displayed increased adhesion to endothelial cells and intercellular adhesion molecule-1, and enoxaparin reverted adhesion to wild-type levels. Small interfering RNA-mediated knockdown of Sdc1 expression resulted in reduced basic fibroblast growth factor-mediated mitogen-activated protein kinase signaling and reduced Caco-2 cell proliferation. We conclude that Sdc1 has a protective effect during experimental colitis. The modification of missing Sdc1 function by heparin analogues may emerge as a promising anti-inflammatory approach.

Analyzing miRNAs in Ductal Adenocarcinomas of the Pancreas

MicroRNAs (miRNAs) have gained attention as an epigenetic component involved in the development of pancreatic ductal adenocarcinoma (PDAC). Several methods for miRNA profiling are in common use, but the validity of these methods is not defined. The aim of this study was to define the optimal method for miRNA detection in PDAC. miRNA expression was determined using different and partially redundant methods (miRNA microarray, TaqMan low density array (TLDA), single tube quantitative RT-PCR). The data from different methods were statistically evaluated and tested for intermethodic consistency and reliability of the results. Finally, the miRNA expression status and the cell lines' ability to metastasize were correlated. Comparing low and high metastatic cells, miRNA-microarrays identified fewer differentially expressed and only upregulated miRNAs (n=27; 27 up-regulated) compared with TLDAs (n=54; 19 up- and 35 down-regulated). Evaluating miRNAs that target tumor suppressor genes, expression of all single tube quantitative real-time reverse transcriptase PCR (qRT-PCR) validated miRNAs was detected to be significantly altered in TLDA analysis (100%). MiRNA microarrays detected only 25% of qRT-PCR validated miRNAs. Furthermore, results from TLDA analysis correlated well with data from qRT-PCR and presented ΔΔCt values from 3.5±1.86 (range 0.8-5.62) compared with 3.74±1.86 (range 0.78-5.95) in qRT-PCR. Notable differences comparing data obtained from different screening methods were found. While TLDA and qRT-PCR correlated well in quantity and quality of the measured miRNAs, several tumor suppressor gene targeting and down-regulated miRNAs were not detected by miRNA-microarrays. This heterogeneity shows that care must be exercised when comparing results from different methods in PDAC.