Reverse Transcription-polymerase Chain Reaction Array Research Articles

Plasma-Based Scaffold Containing Bone-Marrow Mononuclear Cells Promotes Wound Healing in a Mouse Model of Pressure Injury.

Pressure injuries, or pressure ulcers, are a common problem that may lead to infections and major complications, besides being a social and economic burden due to the costs of treatment and hospitalization. While surgery is sometimes necessary, this also has complications such as recurrence or wound dehiscence. Among the newer methods of pressure injury treatment, advanced therapies are an interesting option. This study examines the healing properties of bone marrow mononuclear cells (BM-MNCs) embedded in a plasma-based scaffold in a mouse model. Pressure ulcers were created on the backs of mice (2 per mouse) using magnets and assigned to a group of ulcers that were left untreated (Control, n = 15), treated with plasma scaffold (Plasma, n = 15), or treated with plasma scaffold containing BM-MNC (Plasma + BM-MNC, n = 15). Each group was examined at three time points (3, 7, and 14 days) after the onset of treatment. At each time point, animals were subjected to biometric assessment, bioluminescence imaging, and tomography. Once treatment had finished, skin biopsies were processed for histological and wound healing reverse transcription polymerase chain reaction (RT-PCR) array studies. While wound closure percentages were higher in the Plasma and Plasma + BM-MNC groups, differences were not significant, and thus descriptive data are provided. In all individuals, the presence of donor cells was revealed by immunohistochemistry on posttreatment onset Days 3, 7, and 14. In the Plasma + BM-MNC group, less inflammation was observed by positron emission tomography-computed tomography (PET/CT) imaging of the mice at 7 days, and a complete morphometabolic response was produced at 14 days, in accordance with histological results. A much more pronounced inflammatory process was observed in controls than in the other two groups, and this persisted until Day 14 after treatment onset. RT-PCR array gene expression patterns were also found to vary significantly, with the greatest difference noted between both treatments at 14 days when 11 genes were differentially expressed.

Nonerythroid hemoglobin promotes human cumulus cell viability and the developmental capacity of the human oocyte

To determine the relationship between the levels of cumulus cell (CC) hemoglobin messenger ribonucleic acid (mRNA) and the developmental potential of the associated oocyte and whether hemoglobin protects the CCs from oxidative stress-induced apoptosis. Laboratory-based study. University laboratory and university-affiliated invitro fertilization center. Cumulus cells from the oocytes of patients who underwent invitro fertilization with intracytoplasmic sperm injection with and without preimplantation genetic testing between 2018 and2020. Studies on individual and pooled CCs collected at the time of oocyte retrieval or cultured under 20% or 5% O2. Quantitative polymerase chain reaction analysis of individual and pooled patient CC samples were used to monitor the hemoglobin mRNA levels. Reverse transcription-polymerase chain reaction arrays were used to assess genes that regulate oxidative stress in CCs associated with aneuploid and euploid blastocysts. Studies were conducted to assess the effect of oxidative stress on the rate of apoptosis, level of reactive oxygen species, and gene expression in CCs invitro. Compared with CCs associated with arrested and aneuploid blastocysts, the mRNA levels encoding the alpha and beta chains of hemoglobin increased by 2.9- and 2.3-fold in CCs associated with euploid blastocysts, respectively. The mRNA levels encoding the alpha and beta chains of hemoglobin also increased by 3.8- and 4.5-fold in CCs cultured under 5% O2 vs. 20% O2, respectively, and multiple regulators of oxidative stress were overexpressed in cells cultured under 20% O2 compared with those under 5% O2. However, the rate of apoptosis and amount of mitochondrial reactive oxidative species increased by 1.25-fold in CCs cultured under 20% O2 compared with those under 5% O2. Variable amounts of the alpha and beta chains of hemoglobin were also detected within the zona pellucida and oocytes. Higher levels of nonerythroid hemoglobin in CCs are associated with oocytes that result in euploid blastocysts. Hemoglobin may protect CCs from oxidative stress-induced apoptosis, which may enhance cumulus-oocyte interactions. Moreover, CC-derived hemoglobin may be transferred to the oocytes and protect it from the adverse effects of oxidative stress that occurs invivo and invitro.

Cognitive effects of second-generation antipsychotic polypharmacy in stabilized patients with schizophrenia

To assess the utility of circulating serum microRNAs (c-miRNAs) to predict response to high-dose radiation therapy for locally advanced non-small cell lung cancer (NSCLC).Data from 80 patients treated from 2004 to 2013 with definitive standard- or high-dose radiation therapy for stages II-III NSCLC as part of 4 prospective institutional clinical trials were evaluated. Pretreatment serum levels of 62 miRNAs were measured by quantitative reverse transcription–polymerase chain reaction array. We combined miRNA data and clinical factors to generate a dose–response score (DRS) for predicting overall survival (OS) after high-dose versus standard-dose radiation therapy. Elastic net Cox regression was used for variable selection and parameter estimation. Model assessment and tuning parameter selection were performed through full cross-validation. The DRS was also correlated with local progression, distant metastasis, and grade 3 or higher cardiac toxicity using Cox regression, and grade 2 or higher esophageal and pulmonary toxicity using logistic regression.Eleven predictive miRNAs were combined with clinical factors to generate a DRS for each patient. In patients with low DRS, high-dose radiation therapy was associated with significantly improved OS compared to treatment with standard-dose radiation therapy (hazard ratio 0.22). In these patients, high-dose radiation also conferred lower risk of distant metastasis and local progression, although the latter association was not statistically significant. Patients with high DRS exhibited similar rates of OS regardless of dose (hazard ratio 0.78). The DRS did not correlate with treatment-related toxicity.Using c-miRNA signature and clinical factors, we developed a DRS that identified a subset of patients with locally advanced NSCLC who derive an OS benefit from high-dose radiation therapy. This DRS may guide dose escalation in a patient-specific manner.

Maternal undernutrition during pregnancy alters the epigenetic landscape and the expression of endothelial function genes in male progeny

Recent studies point to the important role of in utero malnutrition in gene programming and in the development of vascular diseases. We hypothesize that maternal undernutrition affects vascular function in the offspring by promoting epigenetic changes that drive the differential expression of genes involved in endothelial function. To test this, we exposed mice to nutrient deprivation in utero and analyzed its effect on global DNA methylation and expression of endothelium-specific genes in the pulmonary endothelium of the adult progeny. Mice were kept either on ad libitum (AL) or energy-restricted (ER) diet during the second and third trimesters of gestation. Mice in the ER group received 65% of energy compared to mice in the AL diet group. Pulmonary endothelial cells were isolated from 6-week-old male offspring mice (AL-F1 and ER-F1). The expression of genes in the pulmonary endothelium was analyzed using quantitative reverse-transcription polymerase chain reaction array and confirmed by qRT-PCR. Several genes including fibronectin 1 and plasminogen activator inhibitor 1 were upregulated in the endothelium of male ER-F1 mice, whereas the expression of genes involved in regulation of histone acetylation was significantly attenuated. At the same time, the global DNA methylation did not change in pulmonary endothelial cells of ER-F1 mice compared to AL-F1 mice. Overall, we found that maternal undernutrition during pregnancy affects the expression of genes involved in regulation of endothelial cell function in the pulmonary vasculature of male progeny, which could potentially promote pulmonary vascular remodeling.

Celiac disease gene expression data can be used to classify biopsies along the Marsh score severity scale.

The diagnosis of celiac disease autoimmune pathology relies on the subjective histological assignment of biopsies into Marsh score categories. It is hypothesized that Marsh score categories have unique gene expression signatures. The aims were as follows: first, to develop a celiac disease quantitative reverse transcription-polymerase chain reaction (RT-PCR) array; second, define gene expression signatures associated with Marsh score categories; and third, develop equations that classify biopsies into Marsh score categories and to monitor the efficacy of patient treatment. Gene targets for inclusion in the celiac RT-PCR (qRT-PCR) array were identified using systematic analysis of published celiac transcriptomic data. The array was used to assess the gene expression associated with histological changes in duodenal biopsies obtained from adult patients. Finally, Marsh score classification equations were defined using discriminant analysis. The array contained 87 genes. The expression of 26 genes were significantly (p<0.06) associated with the discrete Marsh score categories. As the Marsh score pathology of biopsies increased, there was a progression of innate immune gene expression through adaptive Th1-specific gene expression with a concurrent decrease in intestinal structural gene expression in high Marsh score samples. These 26 genes were used to define classification equations that accounted for 99% of the observed experimental variation and which could classify biopsies into Marsh score categories and monitor patient treatment progression. This proof-of-concept study successfully developed a celiac RT-PCR array and has provided evidence that discriminant equations defined using gene expression data can objectively and accurately classify duodenal biopsies into Marsh score categories.

Serum MicroRNA Signature Predicts Response to High-Dose Radiation Therapy in Locally Advanced Non-Small Cell Lung Cancer

Toassess the utility of circulating serum microRNAs (c-miRNAs) to predict response to high-dose radiation therapy for locally advanced non-small cell lung cancer(NSCLC). Data from 80 patients treated from 2004 to 2013 with definitive standard- or high-dose radiation therapy for stages II-III NSCLC as part of 4 prospective institutional clinical trials were evaluated. Pretreatment serum levels of 62 miRNAs were measured by quantitative reverse transcription-polymerase chain reaction array. We combined miRNA data and clinical factors to generate a dose-response score (DRS) for predicting overall survival (OS) after high-dose versus standard-dose radiation therapy. Elastic net Cox regression was used for variable selection and parameter estimation. Model assessment and tuning parameter selection were performed through full cross-validation. The DRS was also correlated with local progression, distant metastasis, and grade 3 or higher cardiac toxicity using Cox regression, and grade 2 or higher esophageal and pulmonary toxicity using logistic regression. Eleven predictive miRNAs were combined with clinical factors to generate a DRS for each patient. In patients with low DRS, high-dose radiation therapy was associated with significantly improved OS compared to treatment with standard-dose radiation therapy (hazard ratio 0.22). In these patients, high-dose radiation also conferred lower risk of distant metastasis and local progression, although the latter association was not statistically significant. Patients with high DRS exhibited similar rates of OS regardless of dose (hazard ratio 0.78). The DRS did not correlate with treatment-related toxicity. Using c-miRNA signature and clinical factors, we developed a DRS that identified a subset of patients with locally advanced NSCLC who derive an OS benefit from high-dose radiation therapy. This DRS may guide dose escalation in a patient-specific manner.

MicroRNA-mediated target mRNA cleavage and 3′-uridylation in human cells

MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5′-fragments were 3′-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5′-3′ degradation or other molecular fates. Some 3′-5′ decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

Porphyromonas gingivalis Lipopolysaccharide Stimulation of Vascular Smooth Muscle Cells Activates Proliferation and Calcification.

Porphyromonas gingivalis (Pg) is a major etiologic agent of periodontitis, whose virulence has been attributed to different factors, including lipopolysaccharide (LPS). Vascular ectopic calcification as a well-known major risk factor for adverse cardiovascular diseases is a highly prevalent vascular pathophenotype, and vascular smooth muscle cells (VSMCs) play an important role in mediating vascular calcification. It was hypothesized that Pg-LPS may stimulate vascular calcification through a direct effect on VSMC function. To test this hypothesis, the effect of Pg-LPS on VSMC calcification was determined. Primary cultures of VSMCs were obtained and identified by immunochemistry in vitro. The proliferation and alkaline phosphatase (ALP) activity of VSMCs were measured using a cell counting kit and an ALP activity test. Mineral deposition was examined using alizarin red staining. Gene (e.g. ALP, core binding factor α1 [Cbfα1], bone sialoprotein [BSP], and osteopontin [OPN]) expression levels altered by Pg-LPS were determined by reverse transcription-polymerase chain reaction array. Pg-LPS could increase the proliferation of VSMCs at different times and enhance ALP activity of VSMCs after 1 day. Alizarin red staining and quantification showed that, with Pg-LPS treatment, VSMCs displayed more obvious calcification nodules. When stimulated with Pg-LPS, the expression of specific osteogenic genes (e.g., ALP, Cbfα1, BSP, and OPN) was significantly promoted in the presence or absence of mineralization-inducing medium, whereas the expression of the OPN gene was inhibited in the mineralization induction group at day 7. Pg-LPS can stimulate VSMC calcification, which results in vascular calcification, further proving the precise relationship between periodontitis and vascular calcification.

Combination of methamphetamine and HIV-1 gp120 causes distinct long-term alterations of behavior, gene expression, and injury in the central nervous system

Methamphetamine (METH) abuse is frequent in individuals infected with human immunodeficiency virus type-1 (HIV-1) and is suspected to aggravate HIV-associated neurocognitive disorders (HAND). METH is a psychostimulant that compromises several neurotransmitter systems and HIV proteins trigger neuronal injury but the combined effects of viral infection and METH abuse are incompletely understood. In this study we treated transgenic mice expressing the HIV envelope protein gp120 in the brain (HIV-1 gp120tg) at 3–4months of age with an escalating-dose, multiple-binge METH regimen. The long-term effects were analyzed after 6–7months of drug abstinence employing behavioral tests and analysis of neuropathology, electrophysiology and gene expression. Behavioral testing showed that both HIV-1 gp120tg and WT animals treated with METH displayed impaired learning and memory. Neuropathological analysis revealed that METH similar to HIV-1 gp120 caused a significant loss of neuronal dendrites and pre-synaptic terminals in hippocampus and cerebral cortex of WT animals. Electrophysiological studies in hippocampal slices showed that METH exposed HIV-1 gp120tg animals displayed reduced post-tetanic potentiation, whereas both gp120 expression and METH lead to reduced long-term potentiation. A quantitative reverse transcription-polymerase chain reaction array showed that gp120 expression, METH and their combination each caused a significant dysregulation of specific components of GABAergic and glutamatergic neurotransmission systems, providing a possible mechanism for synaptic dysfunction and behavioral impairment. In conclusion, both HIV-1 gp120 and METH caused lasting behavioral impairment in association with neuropathology and altered gene expression. However, combined METH exposure and HIV-1 gp120 expression resulted in the most pronounced, long lasting pre- and post-synaptic alterations coinciding with impaired learning and memory.

A Site-Specific Integrated Col2.3GFP Reporter Identifies Osteoblasts Within Mineralized Tissue Formed In Vivo by Human Embryonic Stem Cells.

The use of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) for study and treatment of bone diseases or traumatic bone injuries requires efficient protocols to differentiate hESCs/iPSCs into cells with osteogenic potential and the ability to isolate differentiated osteoblasts for analysis. We have used zinc finger nuclease technology to deliver a construct containing the Col2.3 promoter driving GFPemerald to the AAVS1 site (referred to as a "safe harbor" site), in human embryonic stem cells (H9Zn2.3GFP), with the goal of marking the cells that have become differentiated osteoblasts. In teratomas formed using these cells, we identified green fluorescent protein (GFP)-positive cells specifically associated with in vivo bone formation. We also differentiated the cells into a mesenchymal stem cell population with osteogenic potential and implanted them into a mouse calvarial defect model. We observed GFP-positive cells associated with alizarin complexone-labeled newly formed bone surfaces. The cells were alkaline phosphatase-positive, and immunohistochemistry with human specific bone sialoprotein (BSP) antibody indicates that the GFP-positive cells are also associated with the human BSP-containing matrix, demonstrating that the Col2.3GFP construct marks cells in the osteoblast lineage. Single-cell cloning generated a 100% Col2.3GFP-positive cell population, as demonstrated by fluorescence in situ hybridization using a GFP probe. The karyotype was normal, and pluripotency was demonstrated by Tra1-60 immunostaining, pluripotent low density reverse transcription-polymerase chain reaction array and embryoid body formation. These cells will be useful to develop optimal osteogenic differentiation protocols and to isolate osteoblasts from normal and diseased iPSCs for analysis.

Comparative Analysis of Mouse-Induced Pluripotent Stem Cells and Mesenchymal Stem Cells During Osteogenic Differentiation In Vitro

Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and are, therefore, expected to be useful for bone regenerative medicine; however, the characteristics of iPSC-derived osteogenic cells remain unclear. Here, we provide a direct in vitro comparison of the osteogenic differentiation process in mesenchymal stem cells (MSCs) and iPSCs from adult C57BL/6J mice. After 30 days of culture in osteogenic medium, both MSCs and iPSCs produced robustly mineralized bone nodules that contained abundant calcium phosphate with hydroxyapatite crystal formation. Mineral deposition was significantly higher in iPSC cultures than in MSC cultures. Scanning electron microscopy revealed budding matrix vesicles in early osteogenic iPSCs; subsequently, the vesicles propagated to exhibit robust mineralization without rich fibrous structures. Early osteogenic MSCs showed deposition of many matrix vesicles in abundant collagen fibrils that became solid mineralized structures. Both cell types demonstrated increased expression of osteogenic marker genes, such as runx2, osterix, dlx5, bone sialoprotein (BSP), and osteocalcin, during osteogenesis; however, real-time reverse transcription-polymerase chain reaction array analysis revealed that osteogenesis-related genes encoding mineralization-associated molecules, bone morphogenetic proteins, and extracellular matrix collagens were differentially expressed between iPSCs and MSCs. These data suggest that iPSCs are capable of differentiation into mature osteoblasts whose associated hydroxyapatite has a crystal structure similar to that of MSC-associated hydroxyapatite; however, the transcriptional differences between iPSCs and MSCs could result in differences in the mineral and matrix environments of the bone nodules. Determining the biological mechanisms underlying cell-specific differences in mineralization during in vitro iPSC osteogenesis may facilitate the development of clinically effective engineered bone.

Negative regulation of lncRNA GAS5 by miR-21

In addition to protein-coding genes, the human genome makes a large amount of noncoding RNAs, including microRNAs and long noncoding RNAs (lncRNAs). Both microRNAs and lncRNAs have been shown to have a critical role in the regulation of cellular processes such as cell growth and apoptosis, as well as cancer progression and metastasis. Although it is well known that microRNAs can target a large number of protein-coding genes, little is known whether microRNAs can also target lncRNAs. In the present study, we determine whether miR-21 can regulate lncRNA expression. Using the lncRNA RT-PCR (reverse transcription-polymerase chain reaction) array carrying 83 human disease-related lncRNAs, we show that miR-21 is capable of suppressing the lncRNA growth arrest-specific 5 (GAS5). This negative correlation between miR-21 and GAS5 is also seen in breast tumor specimens. Of interest, GAS5 can also repress miR-21 expression. Whereas ectopic expression of GAS5 suppresses, GAS5-siRNA increases miR-21 expression. Importantly, there is a putative miR-21-binding site in exon 4 of GAS5; deletion of the miR-21-binding site abolishes this activity. Experiments with in vitro cell culture and xenograft mouse model suggest that GAS5 functions as a tumor suppressor. We further show that the biotin-labeled GAS5-RNA probe is able to pull down the key component (AGO2) of the RNA-induced silencing complex (RISC) and we subsequently identify miR-21 in this GAS5-RISC complex, implying that miR-21 and GAS5 may regulate each other in a way similar to the microRNA-mediated silencing of target mRNAs. Together, these results suggest that miR-21 targets not only tumor-suppressive protein-coding genes but also lncRNA GAS5.

Angiotensin II upregulated the expression of microRNA-224 but not microRNA-21 in adult rat cardiac fibroblasts

The role of microRNA-21 (miRNA-21, miR-21) in cardiac fibrosis remains controversial, while the role of microRNA-224 (miRNA-224, miR-224) in cardiac fibroblasts has not been reported. Angiotensin II (Ang II) is known to play a pivotal role in the pathogenesis of cardiac fibrosis. The aim of this study was to confirm whether the expression of miR-21 and miR-224 is regulated by Ang II in adult rat cardiac fibroblasts. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR) were performed to measure the levels of miR-21 and miR-224 in Ang II-treated or untreated adult rat cardiac fibroblasts. The RT-PCR, qPCR and previous miRNA array results demonstrated that treatment with Ang II (100 nM) for 24 h did not induce the increase of miR-21 in cardiac fibroblasts, although the level of miR-21 in cardiac fibroblasts was not considered as low. The results of the present study also demonstrated that Ang II significantly upregulated the expression of miR-224 in adult rat cardiac fibroblasts. Bioinformatic analysis revealed that the potential target genes of miR-224 included SMAD4, SMAD5, cyclin-dependent kinase 9 and early growth response 1/2. In previous studies, it was reported that miR-224 was upregulated in tumors by promoting cell proliferation and targeting SMAD4. Those results indicated the potential roles of miR-224 in cardiac fibroblasts and cardiac fibrosis. In conclusion, results of the present study demonstrated that miR-21 was not induced by Ang II, whereas Ang II upregulated miR-224 expression in adult rat cardiac fibroblasts, a finding that may provide a starting point for the investigation of the potential role of miR-224 in cardiac fibrosis.

Microarray determination of Bcl-2 family protein inhibition sensitivity in breast cancer cells

This study tests the hypothesis that reverse transcription polymerase chain reaction (RT-PCR) microarrays can be used to predict the relative sensitivity to induction of apoptosis in breast cancer cells exposed to inhibitors of antiapoptotic Bcl-2 family proteins. Four cell lines, MDA-MB-231 (MDA-231) and MDA-MB-468 (MDA-468), BT-20 and T47-D were screened for relative expression of Bcl-2 family members A1, Mcl-1, Bcl-2, Bcl-xL and Bcl-w mRNA by RT-PCR microarrays and Western analysis. The four cell lines were treated with 1 μmol/L obatoclax (GX15-070) and/or 2 Gy radiation (RT) and monitored for apoptosis after 48 h. Cell lines showing the highest total fold-increase of Bcl-2 family member mRNA, MDA-231 and MDA-468, also showed the highest levels of apoptosis induction (approximately 70% with obatoclax alone and 82% with obatoclax plus RT). Cell lines with little or no increase in Bcl-2 family mRNA (BT-20 and T47-D) showed less apoptosis (30% following treatment with obatoclax and 42% with obatoclax plus RT). RT-PCR arrays can predict the relative apoptosis response of breast cancer cells to the pan Bcl-2 inhibitor obatoclax alone or when combined with radiation.

Extensive Sex- and/or Hormone-Dependent Expression of Rat Housekeeping Genes

Aim. Identify sex- and hormone - independent housekeeping genes in rat liver by using a commercially available quantitative reverse transcription-polymerase chain reaction array designed to measure the expression of 32 rat housekeeping genes. Results. We found that the levels of five of the genes were sexually dimorphic, 22 genes were overexpressed, and one was underexpressed in multi-hormone-deficient hypophysectomized rats of both sexes. Only three genes fulfilled the stability criteria determined by geNorm and NormFinder as suitable housekeeping genes. Normalizing quantitative reverse transcription-polymerase chain reaction data with either of these three genes alone, the geometric means of any two of the genes, or even the geometric mean of all the three genes, produced similar results. In contrast, application of unproven housekeeping genes could lead to erroneous conclusions, having found that insulin-like growth factor 1 messenger RNA levels could be calculated dramatically either as male or as female predominant depending on the choice of housekeeping gene. Conclusion. It is essential to validate the constancy of housekeeping genes under every experimental condition. (This research protocol was approved by the university's Institutional Animal Care and Use Committee.)

Gene Expression Profiling of Rat Fetuses Exposed to 2-Dimensional Ultrasound

This study evaluated the possible effects of ultrasound (US) on gene expression in brain tissue of rat embryos. Four groups (n = 5 each) of pregnant Wistar Han rats were exposed to US for different durations (55, 100, 145, and 195 seconds) via a multifrequency transducer in the 2-dimensional imaging mode with a pulse duration of 1.29 microseconds, a pulse repetition frequency of 1 kHz, and a derated spatial-peak pulse-average intensity of 222.4 W/cm(2) on day 5, 9, 7, or 13 of gestation. Gene expression profiling was performed in fetal brain tissue (n = 5 per group) by quantitative reverse transcription-polymerase chain reaction arrays. The results indicated substantial alterations in gene expression. The most differentially expressed genes were Adamts5, Gadd45a, Npy2r, and Chrna1, which are implicated in important developmental signaling pathways. On the basis of our findings, routine short US examinations for monitoring fetal development are not contraindicated, but prolonged exposures should be used only when needed to obtain important diagnostic information.

Abstract LB-464: Gene silencing by the mammalian microRNA-mediated and the mRNA-structure-dependent target mRNA cleavage, uridylation, and degradation

Abstract Argonaute (Ago) family proteins and GW182 are key components in microRNA(miRNA)-induced silencing complex (miRISC) and have recently been shown to play an important role in miRNA-mediated target gene expression interference by facilitating mRNA deadenylation, destabilization, and translational repression. However, the precise mechanism in mammalian miRNA-mediated mRNA degradation remains unknown. In this study, we used a stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) to specifically and efficiently detect the 3′ termini of cleaved mRNA fragments targeted by endogenous miR-98 in 3′-untranslated region (3′UTR) of a tumor suppressor gene TUSC2 mRNA. We detected the accumulation of miR-98 cleaved 5′ fragments of TUSC2 mRNA in its native forms by native SLA-RT-PCR and 3′-uridinylated forms by U-tract-specific SLA-RT-PCR, respectively, around miR-98 target sites. Significantly elevated bands with U-tract additions compared to the native ones of the cleaved target mRNA 5′ fragments were detected in mRNA sequences corresponding to the miR-98 seed region, 3′-complimentary pairing region, and as well as the regions immediately adjacent to the 3′- and 5′- ends of miR-98 sites, but no accumulation was detected in the central budge region of miR-98 in total RNAs isolated from human lung cancer H1299 cells. Knocking down terminal uridyltransferase 3 (TUTase-3) and TUTase-2 by gene-specific siRNAs respectively diminished and significantly reduced the accumulation of U-tract added fragments, confirming the miR-98-mediated target mRNA cleavage and 3-uridylation. We constructed an EGFP reporter plasmid vector by inserting two identical copies of TUSC2 miR-98 target sequences with 200 bp apart at the 3′ UTR of EGFP reporter gene. We transiently transfected this plasmid into human lung cancer H1299 cells with a high endogenous miR-98 expression or co-transfected with a miR-98-expressing plasmid into mouse F9 cells in total RNAs isolated at designed time points to analyze the potential effects of secondary or 3-dimensional (3D) structures of target mRNA on the precise positions and efficiency of the dynamic miR-98-mediated target mRNA cleavage and 3-uridylation in living cells. We found that the patterns, positions, and effectiveness of miRNA-mediated target mRNA cleavage and 3′-uridylation are significantly influenced by the unique secondary or 3D structures of target mRNAs. Our findings provided new insight into molecular mechanism in mammalian miRNA-mediated gene silencing by a cooperative action in miRISC through the miRNA-mediated and mRNA-structure-dependent target mRNA recognition, cleavage, uridylation, and degradation in mammalian cells. (Supported by grants P50CA70907, RO1 CA-116322, and W81XWH0920139). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-464. doi:1538-7445.AM2012-LB-464

Abstract LB-463: Target mRNA cleavage and degradation mediated by endogenous mammalian microRNAs

Abstract Recent studies suggest that the mammalian microRNAs (miRNAs) predominately act to decrease target mRNA levels, leading to reduced protein output. However, no direct evidence for miRNA-mediated target mRNA cleavage and degradation has been demonstrated in mammalian cells, in which such efforts are hampered by lacks of tools for accurately and reliably detecting the miRNA target cleavage sites and the cleaved mRNA products. In this study, we developed a sensitive, efficient, and the cleavage site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) technique and used the established miR-98-targeted high mobility group A2 (HMGA2) mRNA as a model system to determine the potential miRNA-directed mRNA target recognition, cleavage, and degradation in context of UTRs in mammalian cells under physiological conditions. We detected cleavage activities among the eight computer-predicted potential miR-98 target sites in UTRs of HMGA2 mRNA with varied efficiency, as demonstrated by the unique and specific SLA-RT-PCR products and patterns by agarose gel electrophoresis and confirmed by DNA sequencing. The first predicted miR-98 target site in HMGA2 3′ UTR is within 15 nt from the stop codon and showed almost no cleavage activity. The last two miR-98 target sites near the 3′ end of HMGA2 mRNA showed a significantly less cleavage efficiency than those in their up-stream 3′UTR target sites. These results suggest that the microRNA induced silencing complex (miRISC) might utilize a 5′ to 3′ mRNA scanning model similar to the translation machinery for processing mRNA target site recognition and cleavage and that the miRNA-directed target cleavage activity and efficiency are influenced by the positional and the structural contexts in the 3′UTR of the target mRNA. We also found that upon miRNA/Argonaute 2 (Ago 2) -mediated target mRNA cleavage a tract of uridine bases (U-tract) was added onto the exposed 3′ end of the resulting 5′ fragment, as demonstrated by an increased intensity of the cleaved fragments by SLA-RT-PCR with U-tract-specific detecting primers. The 3′-uridylation of the cleaved mRNA may lead to a 5′ to 3′ directional exonucleolytic decay, as evidenced by the undetectable 3′ to 5′ decay fragments in all miR-98 target sites on HMGA2 mRNA by SLA-RT-PCR. Furthermore, we also detected the miR-98-mediated cleavage at a 5′UTR target site of HMGA2 mRNA although the cleavage activity and efficiency are weaker and lower than those observed in 3′ UTR, providing an additional mechanism to the conventional model that restricts miRNA targets to the 3′UTR. Our findings provide direct evidence and new insights into molecular mechanism on mammalian miRNA-mediated gene expression repression by targeting mRNA cleavage and degradation. (Supported by grants P50CA70907, RO1 CA-116322, and W81XWH0920139). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-463. doi:1538-7445.AM2012-LB-463

MicroRNA-21 Regulates Vascular Smooth Muscle Cell Function via Targeting Tropomyosin 1 in Arteriosclerosis Obliterans of Lower Extremities

The goal of this study was to determine the expression signature and the potential role of microRNAs in human arteries with arteriosclerosis obliterans (ASO). The expression profiles of microRNAs in human arteries with ASO and in normal control arteries were determined by quantitative reverse transcription-polymerase chain reaction array. Among the 617 detected microRNAs, multiple microRNAs were aberrantly expressed in arteries with ASO. Some of these dysregulated microRNAs were further verified by quantitative reverse transcription-polymerase chain reaction. Among them, microRNA-21 (miR-21) was mainly located in arterial smooth muscle cells (ASMCs) and was increased by more than 7-fold in ASO that was related to hypoxia inducible factor 1-α. In cultured human ASMCs, cell proliferation and migration were significantly decreased by inhibition of miR-21. 3'-Untranslated region luciferase assay confirmed that tropomyosin 1 was a target of miR-21 that was involved in miR-21-mediated cellular effects, such as cell shape modulation. The results suggest that miR-21 is able to regulate ASMC function by targeting tropomyosin 1. The hypoxia inducible factor-1 α/miR-21/tropomyosin 1 pathway may play a critical role in the pathogenesis of ASO. These findings might provide a new therapeutic target for human ASO.

Circulating MicroRNA Is a Biomarker of Pediatric Crohn Disease

The gold standard for the diagnosis and evaluation of Crohn disease (CD) is endoscopy/colonoscopy, although this is invasive, costly, and associated with risks to the patient. Recently, circulating microRNAs (miRNAs) have emerged as promising noninvasive biomarkers. Here, we examined the utility of serum miRNAs as biomarkers of CD in children. Studies were conducted using sera samples from patients with pediatric CD, healthy controls, and a comparison group of patients with pediatric celiac disease. Serum miRNA levels were explored initially using a microfluidic quantitative reverse transcription-polymerase chain reaction array platform. Findings were subsequently validated using quantitative reverse transcription-polymerase chain reaction in larger validation sample sets. The diagnostic utility of CD-associated serum miRNA was examined using receiver operating characteristic analysis. A survey of miRNA levels in the sera of control and patients with CD detected significant elevation of 24 miRNAs, 11 of which were chosen for further validation. All of the candidate biomarker miRNAs were confirmed in an independent CD sample set (n = 46). To explore the specificity of the CD-associated miRNAs, they were measured in the sera of patients with celiac disease (n = 12); none were changed compared with healthy controls. Receiver operating characteristic analyses revealed that serum miRNAs have promising diagnostic utility, with sensitivities for CD above 80%. Significant decreases in serum miRNAs were observed in 24 incident patients with pediatric CD after 6 months of treatment. The present study identifies 11 CD-associated serum miRNA with encouraging diagnostic potential. Our findings suggest serum miRNAs may prove useful as noninvasive biomarkers in CD.