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  • 1
    In: Laboratory Investigation, Elsevier BV, Vol. 89, No. 6 ( 2009-06), p. 708-716
    Type of Medium: Online Resource
    ISSN: 0023-6837
    RVK:
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2009
    detail.hit.zdb_id: 2041329-4
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  • 2
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 3586-3586
    Abstract: Introduction Amplification of chromosome 13q31 is a frequent occurrence in lymphoma and solid tumors. The C13orf25 gene at 13q31.3 is the primary miRNA transcript for seven miRNAs. This specific cluster of miRNAs is sequentially related to the homologous miR-106a-92 cluster on chromosome X and the miR-106b-25 cluster on chromosome 7. The miR17-92 cluster has been shown to be over expressed in various non-Hodgkin Lymphoma (NHL). As a specific group of miRNAs which are derived from the same primary miRNA transcript, each miRNA in the cluster may be expected to show a similar expression level. However expression patterns vary markedly in different cancers. Aim of study To compare the expression pattern of the C13orf25 gene and the miRNAs in normal and malignant B-cells. Methods and Materials 51 cases of Ann Abour stage I and II primary diffuse large B-cell lymphoma (DLBCL) were collected together with 29 cases of B-CLL. All samples were examined for expression of miRNA miR-18a, -19, -20a, 17-3p, -17-5p and -92 and the C13orf 25 gene. Expression levels of the mature miRNAs were determined by qRT-PCR using Taqman miRNA assays. The level of C13orf25 was also determined by qRT-PCR using random primers and a Sybergreen probe. Results were compared by using 2−ΔCt and 2−ΔΔCt. Normal B-cell subpopulations were isolated from fresh tonsils obtained during routine pediatric tonsillectomies. B-cell subsets were stained accordingly and sorted by FACS. Results Comparison of the miRNA pattern (2−ΔCt) revealed that DLBCL have the highest expression level of miR-19b and that B-CLL shows a relative high expression level of miR-92. Normal naïve, germinal center and memory B-cells all show a similar expression pattern with miR-92 having the highest expression level. Remarkably a low expression level of miR-19b is seen in all three B-cell populations in contrast to DLBCL. Comparing the relative expression levels (2−ΔΔCt) of both NHL for each individual miRNA shows that B-CLL has a significantly higher level of expression for miR-19a. In DLBCL miR-17-5p, miR-18a and miR-20a have the highest expression levels. Currently 20 Mantle cell lymphoma are also being analyzed for the C13orf25 miRNAs cluster. Conclusion Our results show that both of the NHL B-cell malignancies and the B-cell subsets have different expression patterns of the individual levels of the 7 miRNAs contained in the same C13orf25 cluster. The relative expression levels of certain miRNAs from the same primary transcript are different in various NHL. Although there are no marked differences in the normal B-cell subsets for the C13orf25 cluster, further profiling revealed various different expression levels of other miRNAs. These findings may point towards a difference in processing efficiency or stability of miRNAs in the C13orf25 cluster leading to differences in pathogenesis specific for each malignancy even in cells of similar origin and differentiation stage.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 3
    In: Blood, American Society of Hematology, Vol. 112, No. 11 ( 2008-11-16), p. 1459-1459
    Abstract: MiRNAs are a new class of small RNAs, of 19–23 nucleotides that were discovered less than two decades ago. These tiny RNAs can negatively regulate genes at the post-transcriptional level by either triggering translational repression or direct cleavage of mRNAs. It has become evident that miRNAs are involved in hematopoiesis and that the aberrant expression of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to characterize the miRNA profile of naïve, germinal center and memory B cells sorted from tonsils and review expression of selected miRNAs in tonsils and in B cell malignancies by miRNA in situ hybridization (ISH). Quantitative (q)RT-PCR profiling revealed that several miRNAs were elevated in germinal center B cells, including miR-17–5p, miR-106a and miR-181b. miR-150 was one of the most abundant miRNAs in all subsets, but the expression level was more than 10 fold lower in germinal center B cell as compared to the other two subsets. MiRNA ISH on tonsillar tissue sections confirmed findings from the profiling work, and at the same time depicted differences in staining intensities within germinal centers. According to miRNA ISH, expression levels of miR-17-5p, miR-106a, and miR-181b were indeed higher in germinal center B cells as compared to naïve and memory B cells in the mantle zone. Surprisingly, we also observed gradual decrease of miR-17-5p, miR-106a, and miR-181b staining from dark to light zone in the germinal centers. Moreover, miRNA ISH with a probe for miR-150 demonstrated an interesting staining pattern in lymph node tissue sections. Naïve and memory B cells located in the mantle zone showed a higher miR-150 expression as compared to most of the cells in the germinal centers. However, within the germinal centers a minority of cells showed a much stronger cytoplasmic staining in part of the blasts located specifically in the dark zone. This indicated that part of the centroblasts have a high expression level of miR-150. The level of miR-150 was surprisingly low in 22 B cell lymphoma cell lines, irrespective of germinal center or non germinal center B cell origin. This seemingly negative association of miR-150 with proliferation suggests a role in B cell growth/death. We observed an inverse expression pattern of miR-150 and Survivin in the germinal centers by miRNA ISH and immunohistochemistry. Moreover, induction of miR-150 using synthetic mature miR-150 duplex resulted in reduced Survivin expression levels. Our results suggested that aside the experimentally proven target c-Myb, Survivin may also be regulated by miR-150. In conclusion, we have revealed a unique miRNA profile of naïve, germinal center and memory B cells sorted from normal tonsils and the results were confirmed by miRNA ISH. Within the germinal centers a marked difference was observed between the light zone and the dark zone.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2008
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 4
    In: Blood, American Society of Hematology, Vol. 110, No. 11 ( 2007-11-16), p. 381-381
    Abstract: Introduction Classical Hodgkin lymphoma (cHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) differs not only in the form of histology and reactive background but also in the phenotypes of the tumor cells. Although tumor cells from both HL subtypes are originated from the germinal center (GC) B cell, gene expression studies show that lymphocytic and histiocytic (L & H) cells from NLPHL resembles normal B cells while Hodgkin/Reed-Sternberg cells (H/RS) from cHL demonstrate a loss of B cell phenotype and have significant overlap with primary mediastinal B cell lymphoma (PMBL). Recently, a new class of small RNAs, namely the micro-RNAs (miRNAs), has been identified. It is now known that at the post-transcriptional level, miRNAs negatively regulate gene expression in a sequence specific manner. Unique miRNAs expression patterns have been reported in various tissue types and also during a wide range of physiological states, such as cell proliferation, development, differentiation, apoptosis and hypoxia. As miRNAs play important roles in many cellular processes, it is proposed that there is a link between aberrant miRNA expression and loss of B cell phenotype in cHL. Methods In this study, miRNA profiles from cell lines of various B cell lymphoma subtypes were examined by qRT-PCR. Also, several B cell subsets were sorted from tonsil by FACS and the miRNA profiles studied by qRT-PCR. Some of the miRNAs are analyzed by in situ hybridization (ISH) in both HL tissue and tonsil samples. Results The miRNA profiling data indicated that cHL cell lines cluster together with PMBL while DEV, an NLPHL cell line, clusters together with CB. Upon validation of differentially expressed miRNAs on a cell line panel of 33 cell lines by monoplex qRT-PCR, 5/8 miRNAs identified as differentially expressed between cHL and GC B cells, were confirmed. Four out of six miRNAs differentially expressed between cHL and PMBL, were also confirmed as being differentially expressed in a larger cell panel. A high degree of overlap was observed between the most abundantly expressed miRNAs in the four HL cell lines. Expression of these miRNAs in HRS cells was verified by ISH in HL tissue samples. miRNA profiles of naive, GC and memory B cells display unique patterns. The overall miRNA expression levels were much lower than observed in the cell lines. Results of miRNA ISH in tonsil tissue demonstrated a specific staining pattern for each miRNA. These data indicate that miRNAs are particularly important for subsets of lymphocytes. Conclusion Several miRNAs that are expressed specifically in Hodgkin lymphoma have been identified. However, the effect of the aberrant expressions of these miRNAs in HL is yet to be elucidated, as the targets of these miRNAs remain unknown.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    RVK:
    RVK:
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2007
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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