MiRNA profiling of Macaca mulatta intact colon samples during acute SIV infection
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ABSTRACT: This study describes differential miRNA expression in intact colon tissue during acute SIV infection of rhesus macaques. Nine miRNAs were found to be significantly affected by infection, with 5 down-regulated and 4 up-regulated miRNAs. The expression of one upregulated miRNA was further characterized and found to be significantly elevated specifically in response to SIV replication and not immune activation/inflammation accompanying SIV infection. We performed TaqMan Low Density Array based high throughput miRNA analysis on intact colon tissue from 10 acutely SIV-infected and 5 uninfected control macaques. All SIV-infected animals were inoculated intravenously with 100TCID50 of SIV. Out of the ten, one animal each was at 7, 8 and 10DPI (days post infection), 3 each at 13 and 21DPI, and 1 at 29DPI. microRNA reverse transcription and preamplification was performed according to the manufacturerM-bM-^@M-^Ys recommendation. Data analysis was performed using RQ Manager 1.2.2 and DataAssist v3.01 software. Data was normalized using Global normalization method and multiple comparisons correction was performed using Benjamini-Hochberg method.
Project description:This study describes differential miRNA expression in small intestinal lamina propria leukocyte samples longitudinally during the course of SIV infection of rhesus macaques. Notably, the T-cell activation associated miR-15b, miR-142-3p, miR-142-5p and miR-150 expression was significantly downregulated at 90 and 180DPI. Further, reporter and overexpression assays validated IRAK1 as a direct miR-150 target. Furthermore, IRAK1 protein levels were markedly elevated in intestinal LPLs and epithelium. Finally, blockade of CD8+ T-cell activation/proliferation with delta-9 tetrahydrocannabinol (ï9-THC) significantly prevented miR-150 downregulation and IRAK1 upregulation. Our findings suggest that miR-150 downregulation during T-cell activation may disrupt the translational control of IRAK1 facilitating persistent GI inflammation. We performed TaqMan Low Density Array based high throughput miRNA analysis on small intestine tissue from 12 chronically SIV-infected and 4 uninfected control macaques. All SIV-infected animals were inoculated intravenously with 100TCID50 of SIV. Out of the ten, one animal each was at 7, 8 and 10DPI (days post infection), 3 each at 13 and 21DPI, and 1 at 29DPI. microRNA reverse transcription and preamplification was performed according to the manufacturerâs recommendation. Data analysis was performed using RQ Manager 1.2.2 and DataAssist v3.01 software. Data was normalized using Global normalization method and multiple comparisons correction was performed using Benjamini-Hochberg method.
Project description:The study describes miRNA expression in intact duodenum following chronic delta 9 tetrahydrocannabinol (M-NM-^T9-THC) administration to SIV-infected rhesus macaques. Chronic M-NM-^T9-THC administration to uninfected macaques significantly and positively modulated intestinal miRNA expression by increasing the total number of differentially expressed miRNAs from 14 to 60 days post infection (DPI). At 60DPI, ~28% of miRNAs showed decreased expression in VEH/SIV compared to none in the THC/SIV group. Furthermore, compared to the VEH/SIV group, THC selectively upregulated the expression of miR-10a, miR-24, miR-99b, miR-145, miR-149 and miR-187 previously shown to target proinflammatory molecules. NOX4, a potent reactive oxygen species generator was confirmed as a direct miR-99b target. A significant increase in NOX4+ crypt epithelial cells was detected in VEH/SIV compared to the THC/SIV group. We speculate that miR-99b-mediated NOX4 downregulation may protect the intestinal epithelium from oxidative stress-induced damage. Twelve age and weight matched male Indian rhesus macaques were randomly divided into 4 groups. Group 1 (n=1) received vehicle (1:1:18 of emulphor : alcohol : saline) and no infection. Group 2 (THC only, n=3) animals received twice daily intramuscular injections of M-NM-^T9-THC and no infection. Group-3 THC/SIV, (n=4) animals received twice daily injections of vehicle and were infected intravenously with 100TCID50 of SIVmac251. Group-4 (VEH/SIV, n=4) animals received twice daily injections of M-NM-^T9-THC similar to group 1 for four weeks prior to SIV infection. Duodenal pinch biopsies were collected before infection and thereafter at 14 and 30 days post infection. All animals were necropsied at 60 days post SIV infection. ~100 ng of total RNA was first reverse transcribed and preamplified according to the manufacturerM-bM-^@M-^Ys recommendation. microRNA expression profiling was performed using TaqMan M-BM-.OpenArrayM-BM-. Human microRNA panels. Data analysis was performed using ExpressionSuiteM-BM-. software. Data was normalized to three endogenous controls (RNU44, RNU48 and snoU6). Delta CT values were calculated by subtracting individual miRNA CT values from an average of all three endogenous controls. Comparisons were made between preinfection and all three treatment groups at 14, 30 and 60 DPI. To determine the effect of chronic THC treatment during SIV infection, comparisons were also made between VEH/SIV and THC/SIV at all three time points.
Project description:Assessment of the extent to which the altered profiles of miRNA expression influence viral replication and latency, as well as the efficiency of host defenses, may be useful for understanding the basis of the HIV-1-related alterations in cellular physiology and immunologic control. To this end, three patient groups were enrolled. One group consisted of subjects who were classified as M-CM-)lite control long-term non-progressors (M-CM-)LTNP). A second study group was HIV-1-positive subjects, who were antiretroviral therapy naive. A third group was multiply exposed to HIV-1, but uninfected (MEU). This study allowed us to investigate the existence of a CD4+T- lymphocytes miRNAs signature able to discriminate among different stages of HIV-1 infection, and to evaluate whether the exposure to HIV-1 antigen is sufficient to change the miRNA profile. MicroRNAs inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4+ T cells from HIV-1 M-CM-)lite LTNP (M-CM-)LTNP), naive and multiply exposed uninfected individuals (MEU) and we observed that the M-CM-)LTNP patients clustered with naive, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated M-CM-)LTNP from MEU and 23 miRNAs distinguished naive from MEU, while only 1 (miR-155) discriminated M-CM-)LTNP from naive. We proposed that miRNA expression may discriminate between HIV-1 infected and exposed but negative individuals. Analysis of miRNAs expression after exposure of healthy CD4+T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection, but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro conditions revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway. We have compared miRNA profiles of CD4+ T-lymphocytes from 18 HIV-1-exposed subjects with healthy CD4+ T-lymphocytes following exposure to gp120 using a RT-qPCR assay.
Project description:This study describes a circulating miRNA signature of acute retroviral infection, which may include candidate diagnostic or prognostic miRNAs for lentivirus-associated neurologic conditions. Plasma samples were taken from six macaques prior to infection and at ten days post-infection. Three macaques each went on to develop no or severe encephalitis. miRNA profiles were generated for each animal before and 10 days after SIV infection.
Project description:The study sought to determine the global miRNA profiles of mouse pancreatic cell lines aTC1-6 and bTC1 at steady state and after treatment with a cocktail of pro-inflammatory cytokines (IL1b; IFNg and TNFa) for a time course of 24 and 48 hours. Inflammatory cocktail contains IL1b 50 IU/ml; IFNg 1000 IU/ml; TNFa 1000 IU/ml. MicroRNA expression profiles in both cell lines during the different experimental conditions were determined using TaqManM-BM-. Rodent miRNA A+B Cards Set v2.0 (TLDA, Life Technologies). MicroRNA profiles were measured in 3 independent biological replicates. qRT-PCR analysis of microRNAs of aTC1-6 treated with cytokines for 24 and 48 h and of their respective not-treated controls as well as of M-NM-2TC1 cells not treated with cytokines. 180 ng RNA of each sample was reverse transcribed through MegaplexM-bM-^DM-" RT Rodent Primer Pool sets A and B, and preamplified through MegaplexM-bM-^DM-" PreAmp Rodent Primer Pool sets (LifetechnologiesM-bM-^DM-"). Complementary DNA (cDNA) was amplified usingTaqManM-BM-. Rodent miRNA A+B Cards Set v2.0 (Life TechnologiesM-bM-^DM-") with TaqMan Universal PCR Master Mix on an ABI 7900HT Sequence Detection System.
Project description:This study describes a circulating miRNA signature of unstable angina (UA), which may be used as a novel biomarker for unstable coronary artery disease (CAD). The Taqman low-density miRNA array were used to identify distinct miRNA expression profiles in the plasma of patients with typical UA and angiographically documented CAD (UA group, n = 13) compared to individuals with non-cardiac chest pain (control group, n = 13). EDTA-plasma samples were obtained before the cardiac catheterization procedure.The study included 2 groups that were classified according to angiographic evidence and clinical evaluation of chest pain. Patients with chest pain or discomfort but with angiographic exclusion of coronary atherosclerosis were enrolled in the control group (n = 13). Chest discomfort referred to the following complaints: chest pain, pressure, tightness, or heaviness; pain that radiated to the neck, jaw, shoulders, back, or one or both arms; and persistent shortness of breath. Patients with typical unstable angina (UA) and angiographically documented CAD were enrolled in the UA group (n = 13).
Project description:microRNA expression signatures can differentiate normal and breast cancer tissues and can define specific clinico-pathological phenotypes in breast tumors. In order to further evaluate the microRNA expression profile in breast cancer, we analyzed the expression of 667 microRNAs in 29 tumors and 21 adjacent normal tissues using TaqMan Low-density arrays. 130 miRNAs showed significant differential expression (adjusted P value=0.05, Fold Change=2) in breast tumors compared to the normal adjacent tissue. Importantly, the role of 43 of these microRNAs has not been previously reported in breast cancer, including several evolutionary conserved microRNA*, showing similar expression rates to that of their corresponding leading strand. The procedure begins with the retro-transcription of 70ng of total RNA with stem-loop primers to obtain a cDNA template. A pre-amplification step was included in order to increase the concentration of the original material and to detect microRNAs that are expressed at low levels. The pre-amplified product was loaded into the TaqManM-BM-. Low Density Arrays and amplification signal detection was carried out using the 7900 FAST real time thermal cycler (ABI). A total of 29 tumor and 21 normal samples (two pools: one containing five samples, other containing 12 samples, plus 4 independent normal samples) were analyzed. 23 tumors and the two normal pools were processed by triplicate, representing 82% of the total samples.
Project description:Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA expression signature of this response is different from that of a PAMP-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human PBMCs exposed to DAMPcontaining freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6x10-4 and p<3.7x10-3), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from HMGB1+/+ mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1-/- MEFs. miR-155 expression in these cultures was negligible, but was significantly higher in PBMCs stimulated with LPS or most other TLR ligands, making it the prototypic PAMPmiR. Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKgamma mRNA, a putative target of miR-34c, increased, while protein levels of IKKgamma in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1beta and TNFalpha) decreased when PBMC cultures were briefly pre-incubated with the K+ channel (inflammasome) inhibitor, glybenclamide, suggesting that miR-34c is involved in the inflammasome pathway in response to DAMPs. Our findings suggest that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders. Human PBMC (peripheral blood mononuclear cells) were exposed to 4 conditions for 48 hours. In the first condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1-/- MEF cells (mouse embryonic fibroblast cells). In the second condition, PBMCs were exposed to conditioned media from serum-starved and glucose-deprived and heat shocked HMGB1+/+ MEF cells (mouse embryonic fibroblast cells). In the third condition, PBMCs were exposed to LPS (Lipopolysaccharide). In the fourth condition, the PBMCs where left untreated. Four biological repeats were done for each condition for a total of 16 samples.
Project description:Purpose: To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell carcinoma (MRCC) and to validate them in a cellular model. Selected microRNAs were studied in serum from MRCC patients and healthy individuals. Methods: We screened 673 microRNAs using TaqMan Low-density Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n=41). Differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenoytpes selected from an independent cohort (n=117). Results were validated in a cellular model of sunitinib resistance and studied in serum from healthy individuals and MRCC patients. Results: TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p=0.0074). High expression of miR-942, miR-133a, miR484, and miR-628-5p was significantly associated with decreased time-to-progression and overall survival. These microRNAs were overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive parental cell line. Serum levels of miR-942, miR-133a, miR-484, miR-146a-5p, miR-374a and miR-486-5p were significantly reduced in MRCC patients compared to healthy controls. Conclusions: Our strategy identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity and resistance to sunitinib. Mir-942 was the best predictor of efficacy. Results were confirmed in a cellular model of sunitinib resistance. We also identified exosome derived serum microRNAs differentially expressed in MRCC patients and healthy individuals. Taqman Low Density Array for 6 FFPE tissues obtained from extreme phenotype MRCC patients, (n=3 marked resistance to sunitinib treatment patients and n=3 marked sensitivity to sunitinib treatment patients), was performanced to screen 667 microRNAs.
Project description:Mantle cell lymphoma (MCL) is an aggressive B-cell non-HodgkinM-bM-^@M-^Ys lymphoma (NHL). In cancers, tumour suppressive microRNAs may be silenced by DNA hypermethylation. By microRNA profiling, miR-155-3p was significantly upregulated upon demethylation treatment of MCL cell lines with 5-aza-2M-bM-^@M-^Y-deoxycytidine (5-azadC). Methylation-specific PCR, verified by pyrosequencing, showed complete methylation of miR-155-3p in one MCL cell line (REC-1). 5-azadC treatment of REC-1 led to demethylation and re-expression of miR-155-3p. Over-expression of miR-155-3p led to increased sub-G1 apoptotic cells and reduced cellular viability, demonstrating its tumour suppressive properties. By luciferase assay, lymphotoxin-beta (LT-M-NM-2) was validated as a miR-155-3p target. In 31 primary MCL, miR-155-3p was found hypermethylated in 6(19%) cases. To test if methylation of miR-155-3p was MCL-specific, miR-155-3p methylation was tested in an additional 191 B-cell, T-cell and NK-cell NHLs, yielding miR-155-3p methylation in 66(34.6%) including 36(27%) non-MCL B-cell, 24(53%) T-cell and 6(46%) of NK-cell lymphoma. Moreover, in 72 primary NHL samples with RNA, miR-155-3p methylation correlated with miR-155-3p downregulation (p=0.030), and LT-M-NM-2 upregulation (p=0.004). Collectively, miR-155-3p is tumour suppressive microRNA hypermethylated in MCL and other NHL subtypes. As miR-155-3p targets LT-M-NM-2, which is an upstream activator of the non-canonical NF-kB signalling, miR-155-3p methylation is potentially important in lymphomagenesis Total RNA isolated from MINO and JEKO-1 before and after 5-azadC treatment were converted into cDNA by MegaplexTM RT Primers and TaqManM-BM-. MicroRNA Reverse Transcription Kit. cDNA was pre-amplified using MegaplexTM PreAmp Primer and loaded onto 384-well format TaqmanM-BM-. human microRNA array A V2.0 & B V3.0. Real-time PCR was performed on 7900HT Real-Time PCR system and raw data were analyzed normalizing to mean of three endogenous controls (U6snRNA, RNU44 and RNU48). Relative microRNA levels were determined by M-NM-^TM-NM-^TCt using endogenous controls and untreated controls using SDS 2.4 and RQ manager 1.2. All experimental procedures and analyses were performed according to manufacturerM-bM-^@M-^Ys instruction, using reagents, system and softwares acquired from Applied Biosystems (Foster City, USA).