MicroRNA Dysregulation in Human Prostate Cancer Cell Model (M12 and P69)
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ABSTRACT: An isogenic prostate cancer cell model was used to profile miRNA changes that contribute to the tumorigenic state of prostate cancer. P69 cells are an SV40 large T antigen immortalized cell line that is poorly tumorigenic and non-metastatic. The M12 derivative was derived from an in vivo selection process using nude, athymic mice. Immortalized cells were cultured and used to extract RNA and probed against the human miRNA panel from Exiqon. There were a total of 186 miRNAs that were at least two fold differentially regulated . Two human prostate cancer cell lines were used to evaluate miRNA expression differences contributing to oncogenesis. Two replicates were performed and the data presented as the mean fold difference between P69 and M12.
Project description:An isogenic prostate cancer cell model was used to profile miRNA changes that contribute to the tumorigenic state of prostate cancer. P69 cells are an SV40 large T antigen immortalized cell line that is poorly tumorigenic and non-metastatic. The M12 derivative was derived from an in vivo selection process using nude, athymic mice. Immortalized cells were cultured and used to extract RNA and probed against the human miRNA panel from Exiqon. There were a total of 186 miRNAs that were at least two fold differentially regulated .
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 study was to determine the miRNA expression signature during human pancreas development. We identified 212 miRNAs that were expressed throughout different gestational ages. From those, 4 miRNAs increased (group I), 35 miRNAs decreased (group II), during pancreatic development. The expression of the remaining 173 miRNAs was relatively constant throughout all assessed gestational ages. The determination of the specific group of miRNAs expressed in the human developing pancreas may further the understanding of gene expression regulation during this important process. In this study we define expression profiles of a total of 352 miRNAs for different stages of the pancreas development. Each TLDA card contains 2 endogenous controls which are present in 8 replicates each. Analysis of these controls allows calculating the intra- and inter-assay variation. Quantitative values (RQ) were calculated measuring the dCt between the Ct values of each miRNA and the Ct value of the small nucleolar RNU48 RNA.
Project description:Factors responsible for radiographic severity of rheumatoid arthritis (RA) in African-Americans are poorly understood. We sought to examine genes whose expression in peripheral blood mononuclear cells (PBMCs) is associated with radiographic severity of RA. 20 control samples (from individuals without RA) were compared with 10 early mild, 10 early severe, 10 late mild, and 10 late severe RA samples. All samples were obtained from African-American individuals.
Project description:Factors responsible for radiographic severity of rheumatoid arthritis (RA) in African-Americans are poorly understood. We sought to examine genes whose expression in peripheral blood mononuclear cells (PBMCs) is associated with radiographic severity of RA. 20 control samples (from individuals without RA) were compared with 10 early mild, 10 early severe, 10 late mild, and 10 late severe RA samples. All samples were obtained from African-American individuals.
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:EGR3 expression is upregulated in human prostate cancer compared to normal prostate tissue and is associated with absence of relapse, while low EGR3 expression in tumors is predicitive of disease relapse (Pio et al., PLOS One 2013; 8(1):e54096). However the function of EGR3 in prostate cancer is unknown. Human prostate cancer cells M12 containing high levels of EGR3 were used for shRNA-mediated silencing of EGR3. Gene expression analysis of EGR3 knockdown cells reveals a role in inflammation and the existence of a crosstalk with the NFkB pathway. The M12 prostate cancer cell line is a tumorigenic derivative of the P69 SV40-T immortalized epithelial cells obtained through serial passages of P69 tumor xenografts in mice (Bae et al., Prostate 1998; 34:275-82). M12 cells were transfected with shRNA scramble plasmid (shSCR) or shEGR3 plasmid and selected with puromycin (1 μg/ml). Single-cell clones were isolated using standard methods. Stably transfected cells (shSCR-M12 and shEGR3-M12) were then maintained in growth medium supplemented with puromycin. Two separate clones (termed cl 2 and cl 3) were studied. Knockdown of EGR3 (50% efficacy on average) was stable over many cell culture passages. Biological duplicates of control cells (scramble shRNA) and of two separate clones of shEGR3 cells (clone 2 and clone 3) were used for the gene expression arrays (i.e. 6 samples).
Project description:Purpose: Next-generation sequencing (NGS) of miRNA expression in cancer cell lines provides a path for analysis of dysregulated pathways and potential treatment of dysregulation. The goals of this study are to compare NGS-derived miRNA expression data in a non-tumorigenic prostate cell line (P69) to its metastatic derivative cell lines M12 and M2182.
Project description:Osteoporosis is the consequence of altered bone metabolism resulting in the systemic reduction of bone strength and increased risk of fragility fractures. MicroRNAs (miRNAs) regulate gene expression on a post-transcriptional level are known to take part in the control of bone formation and bone resorption. Recently, targeted secretion of miRNAs from cells originating from various tissues has been described, which allows for their minimal-invasive detection in serum/plasma and use as biomarkers for presence and progression of pathological conditions. One pilot study has reported circulating miRNAs in serum and tissue of fracture patients. However, further studies are required to explore whether a dysbalance in bone homeostasis of fracture patients can reliably be reflected by specific circulating miRNAs, and whether these miRNAs might serve as drugable targets. Here, we report results from a comprehensive multiplex study of 175 miRNAs in serum samples obtained from 7 patients with osteoporotic fractures at the femoral neck, and 7 age-matched controls. Following elaborate quality control statistical analysis of this exploratory dataset identified 9 microRNAs with altered serum levels in response to fracture (adjusted p-value < 0.1). Of these, hsa-miR-10a/b gave excellent discrimination of both groups (AUC = 1.0), and clustering of samples based on the top10 miRNAs confirmed the high discriminatory power of circulating microRNAs for osteoporotic fractures. In the next step 3 miRNAs with unknown roles in osteogenic differentiation and 4 miRNA from a previous study were tested for their effects on osteogenic differentiation. Of these, 3 miRNAs showed robust effects on osteogenic differentiation. Overall, these data provide important insights into changes in serum miRNA in post-traumatic patients. Future studies will show, whether this knowledge can be used to improve current diagnostic methodologies to predict fracture risk and design novel treatment strategies for osteoporosis patients. Two groups with n=7 per group; one groups represents cases with osteoporotic fractures, the control group is age-matched without fractures
Project description:Recent studies have reported both mRNA and microRNA (miRNA) in saliva, but little information has been documented on the quality and yield of RNA collected. Therefore, the aim of the present study was to develop an improved RNA isolation method from saliva and to identify major miRNA species in human whole saliva. RNA samples were isolated from normal human saliva using a combined protocol based on the Oragene RNA collection kit and the mirVana miRNA isolation kit in tandem. RNA samples were analyzed for quality and subjected to miRNA array analysis. Twelve representing 6 males and 6 females, were selected for miRNA profiling using the TaqManM-BM-. Low Density Array Card (TLDA) Human miRNA Panel v2.0 (Applied Biosystems). The analysis of expression of the >700 miRNAs was performed by the DNA Core at the Interdisciplinary Center for Biotechnology Research Center at the University of Florida, according to the manufacturerM-bM-^@M-^Ys protocol except the pre-amplification step was omitted. The NormFinder algorithm was used to identify the optimal normalization of miRNA among the 25 most abundantly expressed miRNAs detected.