MicroRNA expression changes in Bone Marrow Stromal Cells from Myeloma Patients vs Normal Patients, and From Normal Patients cultured with or without Myeloma cells in 3D culture
ABSTRACT: The objectives of this study were to assess differences in Bone Marrow Derived Menenchymal Stromal Cells (MSCs) during co-culture with myeloma cells, and to assess differences in myeloma patient MSCs compared to normal donor MSCs. In the study presented here, a Bone Marrow Derived Menenchymal Stromal Cells (MSCs) were analyzed after FACS sorting from 2 week culture in osteogenic media lacking dexamethasone in 3D silk scaffold matrices either in co-culture with the multiple myeloma cell line GFP+Luc+MM1.S or Alone, as controls. Also, monocultures of MSCs grown in 2D, in MSC expansion media, from Normal Donor Controls (ND) or Multiple myeloma patients (MM) were analyzed. Analysis was done looking at microRNA expression in samples with the nanoString microRNA platform for 800 microRNAs.
Project description:miRNAs are known to be involved in PDAC tumorigenesis, but only a few biologically relevant gene targets have been identified. Here we show that three miRNAs (miR-21, miR-23a and miR-27a) act in concert for the cooperative suppression of several tumor suppressor genes of which we experimentally validated PDCD4, BTG2 and NEDD4L. The synergistic inhibition of this triple miRNA combination is capable of reducing PDAC growth in a mouse model greater than inhibition of oncomiR-21 alone. Patients samples of normal pancreas (n=9) or pancreatic ductal adenocarcinoma (PDAC; n=9) were retrieved during surgery and placed in RNA Later stabilization fluid and then kept at minus 80 until required.
Project description:Cutaneous squamous cell carcinoma (cSCC) is the second most commonly diagnosed cancer in the United States each year. Despite a generally good prognosis, metastatic cSCC results in over 3500 deaths annually. There are no specifically targeted therapies or biomarkers for metastatic cSCC. To determine whether aberrant microRNA expression occurs in metastatic cSCC which could provide novel targets for therapy or biomarkers for earlier diagnosis or prognosis, microRNA expression profiling was performed in 48 samples including normal skin, primary tumors and metastases. Multiple microRNAs showed differential expression; miR-4286, miR-200a-3p and miR-148-3p showed increased expression and miR-1915-3p, miR-205-5p, miR-4516 and miR-150-5p showed reduced expression in metastatic samples. Several microRNAs previously showing aberrant expressionshown to be aberrantly expressed in primary cSCCs were also observed in this study including miR-100, miR-135b, miR-145, miR-21, and miR-214. In summary, several microRNAs show differential expression between primary and metastatic cSCCs; these may be useful as biomarkers for metastasis or as targets for therapytherapeutic targets. RNA extracted from primary human tissues
Project description:Background: Molecular adaptations in the striatum mediated by dopamine (DA) denervation or Levodopa (L-dopa) treatment have been implicated with the motor deficits found in Parkinson’s disease (PD). Alterations in glutamatergic neurotransmission and anti-oxidant mechanisms are reported to play important roles in mediating these changes. However, the mechanisms mediating the molecular adaptations in the striatum are not well understood. In recent years, microRNAs (miRNAs) have been recognized as potent post-transcriptional regulators of gene expression with fundamental roles in numerous biological processes. miRNAs are known to influence the development and maintenance of striatal neurons. Therefore, we sought to determine the genome-wide expression levels of miRNAs in PD striatal tissues. Methods: Using a digital gene expression platform to quantify miRNA levels, we compared the expression of 800 miRNAs in human postmortem putamen tissues from PD patients and controls. Results: We detected the expression of approximately 250 miRNAs in postmortem human putamen samples collected from patients with PD and healthy controls. There was an abundance of a subset of 17 miRNAs (10 up- and 7 down-regulated) differing substantially between PD and the control tissues. Conclusions: We identified deregulated miRNAs most likely associated with altered striatal functions found in PD. This approach may provide insight into pathogenesis and additional therapeutic targets for the development novel treatment strategies for the disease. Human postmortem putamen tissues from 12 patients with PD symptoms and 12 neurologically normal controls. Samples were obtained from the Human Brain and Spinal Fluid Resource Center, Los Angeles, CA through NIH NeuroBioBank, and stored at -80°C until RNA isolation. Total RNA was extracted using the miRVana RNA isolation kit, following the manufacturer’s instructions (Ambion). Using 225ng total RNA, miRNA levels were assayed by direct digital detection using Nanostring miRNA assay kits (Nanostring Technologies).
Project description:Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1. Total RNA was hybridised to NanoString miRNA Human v2.1 probes, immobilized to NanoString cartridge and analysed on the NanoString Digital Analyzer. NanoString nSolver Analysis Software was utilised to check QC metrics and extract raw miRNA counts. Expression was normalised for input using the housekeeping genes. Contributor: The Australian Ovarian Cancer Study Group
Project description:miRNA profiling in multiple myeloma - microRNAs represent a class of noncoding regulators of gene expression implicated in several biological and pathophysiological processes, including cancer. We investigate here their role in multiple myeloma using miChip-arrays interrogating 559 miRNAs in 92 purified myeloma-, MGUS-, normal plasma cell- and myeloma cell line samples. Impact on gene expression is assessed by Affymetrix U133 2.0 DNA-microarrays in 741 samples including two cohorts of 332 and 345 myeloma patients; chromosomal aberrations are assessed by iFISH, survival for 247 and 345 patients undergoing up-front high-dose therapy and autologous stem cell transplantation. Compared to normal plasma cells, 67/559 (12%) miRNAs are differentially expressed with fold changes of 4.6 to -3.1 in myeloma-, 20 (3.6%) in MGUS-samples, and three (0.5%) between MGUS- and myeloma-samples. Expression of miRNAs is associated with biological and pathophysiological parameters, i.e. proliferation, chromosomal aberrations, e.g. t(4;14), tumor mass, and gene expression-based high-risk scores. This holds true for target-gene signatures of regulated mRNAs. miRNA-expression confers prognostic significance for event-free (72/559) and overall survival (69/559), as do respective target-gene signatures. In conclusion, the miRNome of myeloma confers a pattern of small changes of individual miRNAs compared to normal plasma cells impacting on gene expression, biological functions, and survival.
Project description:Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here we show that YAP, the downstream target of the tumor-suppressive Hippo signaling pathway regulates miRNA biogenesis in a cell density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding post-transcriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer. Two conditions (Low density and High density) were analyzed in duplicate.
Project description:Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis. The mechanisms responsible for this phenomenon remain poorly understood. Here we show that YAP, the downstream target of the tumor-suppressive Hippo signaling pathway regulates miRNA biogenesis in a cell density-dependent manner. At low cell density, nuclear YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA processing machinery. At high cell density, Hippo-mediated cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs. Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors and a corresponding post-transcriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer. Two conditions (siCtrl and siYAP) were analyzed in duplicate.
Project description:We generated H460 cells with acquired TRAIL resistance by exposing the parental sentisitve cells to subtoxic concentrations of TRAIL for 6 months. Then we compared the microRNA expression profile in the sensitive versus resistant cells. H460 cells were treated with subtoxic concentrations of TRAIL for 6 month. After confirmation of the resistance, the RNA was extracted and the microRNA expression profile was analyzed using the NanoString technology.