ABSTRACT: To identify microRNA changes during plasmacytoid dendritic cell (PDC) activation, we stimulated human primary PDCs with 10ug/ml R837 (Invivogen, San Diego, CA, USA) for 4 hours. Purified human pDCs were divided into two parts: one was cultured with medium alone, another was cultured with R837. 4 hours later, cells were collected and total RNA was extracted for the TaqMan® Human MicroRNA Arrays. The experiment was duplicated (sample1 and sample2).
Project description:Hematopoietic stem cells (HSCs) can regenerate the entire hematopoietic system in vivo, providing the most relevant criteria to measure candidate HSCs derived from human embryonic stem cell (hESC) or induced pluripotent stem cell (hiPSC) sources. Here, we show that unlike primitive hematopoietic cells derived from hESCs, phenotypically identical cells derived from hiPSC are more permissive to graft the bone marrow of xenotransplantation recipients. Despite establishment of bone marrow graft, hiPSC-derived cells fail to demonstrate hematopoietic differentiation in vivo. However, once removed from recipient bone marrow, hiPSC-derived grafts were capable of in vitro multilineage hematopoietic differentiation, indicating that xenograft imparts a restriction to in vivo hematopoietic progression. This failure to regenerate multilineage hematopoiesis in vivo was attributed to the inability to downregulate key microRNAs involved in hematopoiesis. Based on these analyses, our study indicates that hiPSCs provide a beneficial source of pluripotent stem cell-derived hematopoietic cells for transplantation compared with hESCs. Since use of the human-mouse xenograft models prevents detection of putative hiPSC-derived HSCs, we suggest that new preclinical models should be explored to fully evaluate cells generated from hiPSC sources. Human pluripotent stem cell-derived hematopoietic cells were isolated and qPCR-based microRNA profiling was performed.
Project description:In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a 4-stage differentiation protocol involving: embryoid body (EB) formation (Stage-1); EB culture with hematopoietic cytokines (Stage-2); HSPC expansion (Stage-3); and neutrophil maturation (Stage-4). CD34+CD45- putative hemogenic endothelial cells were observed in Stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34+CD45- cells generated CD34+CD45+ HSPCs that produced hematopoietic CFUs. Mid-Stage-3 CD34+CD45+ HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34-CD45+ cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBPε expression were observed, with 25-65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. miRNA expression profiles were analyzed in iNC-01-3 and iNC-01-4 induced pluripotent stem cell lines at day 0 (undifferentiated iPSCs), day 18 of differentiation (embryoid bodies), and in 3 FACS-sorted cell populations at day 22 of differentiation (CD34+CD45- cells, CD34+CD45+ cells, and CD34-CD45+ cells). Comparative CT analysis was normalized to U6 snRNA and RNU48 control RNAs relative to expression in undifferentiated iPSCs. Data includes 2 files for each sample, one for the card A array and one for the card B array. This includes a total of 32 data files consisting of: 5 replicates for undifferentiated iPSCs (10 files), 2 replicates for EB (4 files), 3 replicates for CD34+CD45- (6 files), 3 replicates for CD34+CD45+ (6 files), and 3 replicates for CD34-CD45+ (6 files).
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 (9-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 manufacturer’s 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 (Δ9-THC) administration to SIV-infected rhesus macaques. Chronic Δ9-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 Δ9-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 Δ9-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 manufacturer’s recommendation. microRNA expression profiling was performed using TaqMan ®OpenArray® Human microRNA panels. Data analysis was performed using ExpressionSuite® 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: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 TaqMan® 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:MicroRNA (miRNA) expression profiling identified miR-638 as one of the most significantly overexpressed miRNAs in metastatic lesions compared with primary melanomas. miR-638 enhanced the tumourigenic properties of melanoma cells in vitro and lung colonization in vivo. mRNA expression profiling of miR-638 and antagomir-transduced cells identified new candidate genes as miR-638 targets, the majority of which is involved in p53-mediated apoptosis regulation. miR-638 depletion stimulated expression of p53 and its downstream target genes and induced apoptosis and autophagy in melanoma cells. miR-638 promoter analysis revealed transcription factor associated protein 2-α (TFAP2A) as a direct negative regulator of miR-638. Further analyses provided strong evidence for a double negative regulatory feedback loop between miR-638 and TFAP2A. Taken together, miR-638 may support melanoma progression by suppressing p53-mediated apoptosis pathways and by targeting the transcriptional repressor TFAP2A. TaqMan® low-density arrays (TLDA; human microRNA Cards A v2.1 & B v2.0, Applied Biosystems, Darmstadt, Germany) were used for measuring the expression of 667 human miRNAs in primary melanomas (PM, n=8), lymph node metastases (LNM, n=9) or distant cutaneous metastases (DCM, n=10).
Project description:Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin’s 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-2’-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-β) 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-β 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-β, 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 TaqMan® MicroRNA Reverse Transcription Kit. cDNA was pre-amplified using MegaplexTM PreAmp Primer and loaded onto 384-well format Taqman® 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 ΔΔCt using endogenous controls and untreated controls using SDS 2.4 and RQ manager 1.2. All experimental procedures and analyses were performed according to manufacturer’s instruction, using reagents, system and softwares acquired from Applied Biosystems (Foster City, USA).
Project description:MicroRNAs (miRNAs) are non-coding RNAs that play a fundamental role in regulation of gene expression affecting differentiation and development. In particular, miRNAs have been described to regulate genes important for pancreatic development and islet function. The aim of this work was to determine the miRNA expression signature in human pancreatic alpha and beta cells. miRNA stability to fixation allowed the study of microRNA in pure populations of human alpha and beta cells sorted by FACS after intracellular staining with glucagon and insulin, respectively. The determination of the specific group of miRNAs expressed in the human pancreatic alpha and beta cells may further the understanding of gene expression regulation of the islet differentiation process. The alpha and beta cells come from 6 different preparations of human pancreatic islets from donors. In this study we define expression profiles of a total of 665 miRNAs for pancreatic alpha and beta cells. For this purpose, cells were fixed with paraformaldehyde, 7AAD was applied to exclude dead cells. Then, cells were sorted after intracellular staining with C peptide to detect beta cells and glucagon to detect alpha cells. After sorting, we confirmed enriched beta cells have a purity of on average over 98%. Enriched alpha cells have a purity of on average over 98%. To determine the miRNA expression profiles, we used human miRNA TLDAs version 2. For each sample card A and card B were run after cDNA synthesis and 12 cycles of preamplification according to the manufacturer protocol. Each TLDA card A contains 1 probe for the endogenous control RNU48 while each TLDA card B contains 4 replicates of the RNU48 probe. Analysis of these controls allows calculating the intra- and inter-assay variation. Quantitative values (RQ) were calculated measuring the ddCt between the Ct values of each miRNA and the Ct value of the small nucleolar RNU48 RNA comparing the target sample and the control sample.
Project description:Signal transducer and activator of transcription 3 (STAT3) is a critical transcription factor in cancer. However, while the protein-coding target genes of STAT3 have been extensively studied, the microRNA target genes of STAT3 are less understood. MicroRNAs are short, non-coding RNAs that regulate messenger RNAs through translational inhibition and transcript degradation. They have been found to be involved in all aspects of cancer biology. Given the roles of both STAT3 and miRNAs in cancer, the function of STAT3 as a transcription factor, and the dearth of known STAT3 miRNA targets, our goal was to identify novel STAT3 miRNA targets relevant to cancer. To do so, we engineered MCF-10A cells with doxycycline-inducible expression of STAT3C. STAT3C is a constitutively-active mutant version of STAT3. Although STAT3 can be activated by various growth factors and kinases, other pathways can be activated as well, which would confound analysis of the results. Thus, the advantage of STAT3C is that it allowed specific and focused activation of STAT3 alone, and this screen represents the first genome-wide survey of miRNA expression changes associated specifically with STAT3 activity. MCF-10A, a non-transformed breast epithelial cell line, was chosen because STAT3C has been reported to be sufficient to cause their neoplastic transformation. Therefore, we reasoned that analysis of STAT3C’s effects in MCF-10A cells would be especially informative for STAT3-regulated miRNAs relevant to cancer. As a result of our study, we identified previously-known as well as novel miRNA targets of STAT3. Doxycycline-inducible MCF-10A cells were seeded, and then untreated (grown in standard growth media alone) or treated with 2μg/ml doxycycline for 48hr. Each condition was performed in biological triplicate (labeled A, B, and C), for a total of 6 samples. Total RNA was harvested and submitted to the Dana-Farber Cancer Institute Molecular Diagnostics Laboratory. MicroRNA expression profiling was performed using TaqMan Low-Density Arrays (TLDA), human miRNA version 2.0A and version 3.0B cards (Applied Biosystems).
Project description:In this study, we performed a miRNA global profiling in human lung epithelial cells (A549) infected by two different subtypes of human influenza A viruses (H1N1 and H3N2). A549 cells were either mock-infected or infected at a multiplicity of infection (MOI) of 1 with H1N1 or H3N2 viruses, and total RNAs were isolated at 24 hours post-infection (hpi). An MOI of 1 was performed to ensure that 100% of the cells were infected at 24 hpi, a strategy that we have previously validated and used for a transcriptional profiling study of infected cells (Josset et al. , 2010). The purified RNAs were subjected to reverse transcription using a pool of miRNA RT primers (Human pool A v2.1, Applied Biosystems) and subsequently amplified and quantified by RT-qPCR in a TaqMan array MicroRNA card (Applied biosystems).