Project description:MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post-transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack ability to identify novel miRNAs and accurately determine expression at a range of concentration. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts. The results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 (chronic myelogenous leukemia) and HL60 (acute promyelogenous leukemia) are presented. Custom computation pipelines were used to generate expression profiles of known and for identification of novel miRNAs. Some of the highly expressed miRNAs in the leukocytes include several members of the let 7 family, mir-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 cells or HL60 revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by realtime RT-PCR and or RNAase protection assay. The small RNA population from four samples - Two Normal Peripheral blood mononuclear cells (PBMC) samples, K562 cell line (This cell line is used as a model to study Chronic Myelogenous Leukemia), HL60 (This cell line is used to study acute promyelogenous leukemia) was sequenced using Solexa technology.
Project description:Its a mathematical model depicting CML (chronic myelogenous leukemia) interaction with T cells and impact of T cell activations on CML progression over time.
Project description:Comparison of gene expression profiles of CD34+ hematopoietic stem and progenitor cells from bone marrow of patients with untreated chronic myelogenous leukemia (CML) in chronic phase with those from bone marrow of healthy volunteers. Chronic myelogenous leukaemia (CML) is a malignant disorder of the hematopoietic stem cell, which is characterized by the reciprocal translocation between chromosomes 9 and 22 (t(9;22)(q34;q11)) The translocation results in the formation of the BCR-ABL fusion oncogene encoding a protein with constitutive activated tyrosine kinase activity which plays a central role in the pathogenesis of the disease. There are still several open questions with respect to BCR-ABL-induced malignant transformation. A large limitation of the existing data about BCR-ABL effects is that they are derived to a great proportion from human hematopoietic cell lines, BCR-ABL-transformed murine cell lines or fibroblasts and mouse models, which might not be representative for chronic phase CML. A suitable cell population for studies on CML biology are primary hematopoietic stem and progenitor cells from patients with CML. Therefore, we provide in this study a genome-wide expression signature of highly enriched CD34+ cells from bone marrow (BM) of untreated patients with CML in chronic phase. Gene expression profiles of immunomagnetically enriched BM CML CD34+ cells (n=9) were compared with those of normal BM CD34+ cells (n=8) using microarrays covering 8.746 genes. Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays. Following quality control and normalization differentially expressed genes were identified by significance analysis of microarrays (SAM). Comparing both groups 918 genes were significantly differentially expressed (q value <0.1%; fold change > 1.3). Several of the BCR-ABL-induced effects described in cell lines and BCR-ABL-transduced cells could also be found in primary CML progenitor cells as for example the transcriptional activation of the classical MAPK pathway and the PI3 kinase/AKT pathway and the down-regulation of the pro-apoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include an up-regulation of components of the TGFb signalling pathway and the non-canonical Wnt/Ca2+ pathway, a transcriptional activation of fetal haemoglobin genes and genes associated with early hematopoietic stem cells (HSC) such as HoxA9 and MEIS1 and up-regulation of genes involved in fatty acid metabolism, of the thrombin receptor PAR1 and the neuroepithelial cell transforming gene 1. Differential expression of differentiation-associated genes suggested an alteration of the composition of the CD34+ cell population in CML. This was confirmed by immunophenotypical subset analyses of chronic phase CML CD34+ cells showing an increase of erythroid progenitors and a decrease of granulocyte-macrophage progenitor cells while the proportion of HSC was similar in normal and CML CD34+ cells. In conclusion, our results give novel insights into the biology of CML hematopoietic stem and progenitor cells and could be the basis for identification of new targets for therapy.
Project description:Imatinib has become the current standard therapy for patients with chronic myelogenous leukaemia (CML). For a better understanding of the Imatinib-related molecular effects in vivo, we assessed gene expression profiles of Philadelphia Chromosome positive (Ph+) CD34+ cells from peripheral blood of 6 patients with de novo CML in chronic phase. After 7 days of treatment with Imatinib the Ph+ CD34+ cells were reassessed to look for changes in the transcriptome. The expression level of 303 genes was significantly different comparing the transcriptome of the Ph+ CD34+ cells before and after 7 days of Imatinib therapy (183 down-regulated, 120 up-regulated, lower bound â¥1.2-fold). For a substantial number of genes governing cell cycle and DNA replication, the level of expression significantly decreased (CDC2, RRM2, PCNA, MCM4). On the other hand, therapy with Imatinib was associated with an increase of genes related to adhesive interactions, such as L-selectin or CD44. A group of 8 genes with differential expression levels were confirmed using a gene specific quantitative real-time PCR. Thus, during the first week of treatment, Imatinib is preferentially counteracting the bcr-abl induced effects related to a disturbed cell cycle and defective adhesion of leukemic Ph+ CD34+ cells. Experiment Overall Design: In total 6 patients with new diagnosis CML (Chronic Myelogenous Leukemia) in chronic phase are inculded in the study. The gene expression profiles of the CD34+ hematopoietic stem and progenitor cells from the patients before first treatment with Glivec (Imatinib) are compared to the gene expression profiles of the CD34+ hematopoietic stem and progenitor cells of the same patients after 7 days of treatment with 400 mg Glivec / day.
Project description:The effects of 7.5 micromolar parthenolide (PTL) were assessed on primary CD34+ acute myelogenous leukemia specimens obtained from 12 patients. Experiment Overall Design: Acute myelogenous leukemia (AML) specimens were obtained from 12 patients and CD34+ cells were isolated. For each patient, cells were cultured in vitro and exposed to either 7.5 micromolar parthenolide (PTL) or left untreated (UT) for 6 h. Total RNA was then harvested for global gene expression analysis.
Project description:MicroRNAs are a class of small non-coding RNAs that regulate mRNA expression at the post-transcriptional level and thereby many fundamental biological processes. A number of methods, such as multiplex polymerase chain reaction, microarrays have been developed for profiling levels of known miRNAs. These methods lack ability to identify novel miRNAs and accurately determine expression at a range of concentration. Deep or massively parallel sequencing methods are providing suitable platforms for genome wide transcriptome analysis and have the ability to identify novel transcripts. The results of analysis of small RNA sequences obtained by Solexa technology of normal peripheral blood mononuclear cells, tumor cell lines K562 (chronic myelogenous leukemia) and HL60 (acute promyelogenous leukemia) are presented. Custom computation pipelines were used to generate expression profiles of known and for identification of novel miRNAs. Some of the highly expressed miRNAs in the leukocytes include several members of the let 7 family, mir-21, 103, 185, 191 and 320a. Comparison of the miRNA profiles of normal versus K562 cells or HL60 revealed a specific set of differentially expressed molecules. Correlation of the miRNA with that of mRNA expression profiles, obtained by microarray, revealed a set of target genes showing inverse correlation with miRNA levels. Our computational pipeline also predicted a number of novel miRNAs. Some of the predictions were validated by realtime RT-PCR and or RNAase protection assay.
Project description:We did microarray to compare the gene expression profile of peripheral blood mononuclear cells (PBMC from normal volunteer) and two leukemic cell lines that is K562 (Chronic myelogenous leukemia cell line), HL60 (Promyelocytic leukemia cell line) in order to find differentially expressed genes in these samples.