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MicroRNAs and Metabolites in Serum Change after Chemotherapy and Impact on Haematopoietic Progenitor Cells

ABSTRACT: Hematopoietic reconstitution after chemotherapy and hematopoietic stem cell transplantation (HSCT) requires massive activation of the stem cell pool. In a previous study, we have shown that there are systemically released factors in patient serum after HSCT that may play an important role in hematopoietic recovery, and that these serum samples significantly stimulate hematopoietic stem and progenitor cell (HPC) in vitro expansion. Aberrant microRNA (miRNA) expression is a feature of leukemia and miRNAs also play a significant role in normal hematopoiesis and differentiation. In this study, we have addressed the question if miRNAs and metabolic pathways are involved in HPC activation after HSCT. Serum from patients before chemotherapy and after chemotherapy during aplasia was used as supplement for in vitro cultivation of CD34+ HPCs. Serum taken after chemotherapy significantly enhanced HSC proliferation and maintained a primitive CD34+ immunophenotype. Microarray analysis revealed that 23 miRNAs are differentially expressed in serum before and after chemotherapy - particularly miRNA-320 and miRNA-1275 were down-regulated and miRNA-3663-3p was up-regulated. This suggest that hematopoietic regeneration after chemotherapy might be tightly regulated by miRNAs and this opens new perspectives for in vivo stimulation of the stem cell pool.

ORGANISM(S): Homo sapiens

PROVIDER: GSE57570 | GEO | 2015/06/01

SECONDARY ACCESSION(S): PRJNA246720

REPOSITORIES: GEO

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Project description:MicroRNAs (miRNAs) have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous methods have been established to identify and quantify miRNAs. The shortness of miRNA sequence results in a high dynamic range of melting temperatures and, moreover, impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray based approach for global and absolute quantification of miRNAs. The method relies on an equimolar pool of about 1000 synthetic miRNAs of known concentration which is used as an universal reference and labeled and hybridized in a dual colour approach on the same array as the sample of interest. Each single miRNA is quantified with respect to the universal reference outbalancing bias related to sequence, labeling, hybridization or signal detection method. We demonstrate the accuracy of the method by various spike in experiments. Further, we quantified miRNA copy numbers in liver samples and CD34(+)CD133(-) hematopoietic stem cells. The RNA extracted from 7 x 10^ 5 to 1 x 10^ 6 CD34(+)/CD133(-) cells of three different donors was analyzed. 1 µg of respective total RNA was mixed with 2.5 fmol of each of 18 RNA oligonucleotides reverse complement to miRControl 3 probes and subsequently fluorescently labelled by 3’ ligation. Total RNA mix was hybridized in a dual colour approach to microarrays versus a second labelled synthetic miRNA pool. The synthetic miRNA pool consisted of 2.5 fmol of each of 954 non redundant miRNAs sequences and miRControl 3 sequences. The array data was normalized by calculating the median of the miRControl 3 present in the CD34(+)/CD133(-) and UR sample. The miRNA amount was calculated with respect to the corresponding miRNA in the UR.

Project description:CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). From each patient only one sample was analyzed. Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays according to manufacturer's instructions. Following quality control and normalization 9 samples of G-CSF-mobilized CD34+ cells were compared with 7 samples of PEG-G-CSF-mobilized cells were compared by significance analysis of microarrays (SAM). The aim was to figure out transcriptional differences resulting from various pharmacokinetics of the same drug (G-CSF: fluctuating serum levels; PEG-G-CSF: continously high serum levels). Peripheral blood stem and progenitor cells (PBSC) are widely used for autologous and allogeneic hematopoietic stem cell transplantation. PBSC can be mobilized into the peripheral blood using cytokines, cytotoxic chemotherapy or a combination of both. Granulocyte colony stimulating factor (G-CSF) has become the most commonly administered cytokine for PBSC mobilization because of its high potency and lack of serious toxicity. Recently, a modified form of recombinant human G-CSF has been introduced. This new compound is pegylated G-CSF (Peg-G-CSF) which possesses a substantially longer half-life than the unconjugated drug because of its reduced renal excretion and therefore provides the basis for continuous G-CSF serum-levels after a single injection. The use of Peg-G-CSF in patients who had received a cytotoxic chemotherapy was accompanied by mobilization kinetics different from those observed in patients who had received unconjugated G-CSF. In particular, more rapid leukocyte recovery and occurrence of CD34-positive cells in the peripheral blood was seen. These findings are presumably related to the more even and continuously high serum level of G-CSF maintained by Peg-G-CSF. Using microarray technology and functional assays, we examined whether pegylation of G-CSF and its different pharmacokinetics results in addition to an earlier leucocyte recovery in transcriptional and functional changes in CD34+ cells obtained from patients who had received either G-CSF or Peg-G-CSF following a standard cytotoxic chemotherapy for PBSC mobilisation. CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). 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 339 genes were significantly differentially expressed (q value <5%; fold change > 1.2). Peg-G-CSF-mobilized CD34+ cells showed an expression pattern of more early progenitor cells as early stem cell markers such as HOX genes were higher expressed and differentiation-associated genes were lower expressed in comparison with G-CSF-mobilized cells. Moreover, Peg-G-CSF-mobilized CD34+ cells had a greater expression level of cell-cycle-promoting genes suggesting a greater cycle activity of these cells. In conclusion, despite the similar active drug component different pharmacokinetics of Peg-G-CSF and G-CSF result in distinct molecular phenotypes reflecting different functional characteristics.

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MicroRNAs and Metabolites in Serum Change after Chemotherapy and Impact on Haematopoietic Progenitor Cells

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