Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers

A large number of tiny noncoding RNAs have been cloned and named microRNAs (miRs). Recently, we have reported that miR-15a and miR-16a, located at 13q14, are frequently deleted and/or down-regulated in patients with B cell chronic lymphocytic leukemia, a disorder characterized by increased survival. To further investigate the possible involvement of miRs in human cancers on a genome-wide basis, we have mapped 186 miRs and compared their location to the location of previous reported nonrandom genetic alterations. Here, we show that miR genes are frequently located at fragile sites, as well as in minimal regions of loss of heterozygosity, minimal regions of amplification (minimal amplicons), or common breakpoint regions. Overall, 98 of 186 (52.5%) of miR genes are in cancer-associated genomic regions or in fragile sites. Moreover, by Northern blotting, we have shown that several miRs located in deleted regions have low levels of expression in cancer samples. These data provide a catalog of miR genes that may have roles in cancer and argue that the full complement of miRs in a genome may be extensively involved in cancers.     

Functional dissection of the chromosome 13q14 tumor-suppressor locus using transgenic mouse lines

Deletion of chromosomal region 13q14 represents the most common genetic aberration in B-cell chronic lymphocytic leukemia (CLL). 13q14 deletions are commonly large and heterogeneous in size and affect multiple genes. We recently found that targeted deletion in mice of the 0.11 megabase (mb)-long minimal deleted region (MDR) encompassing the DLEU2/miR-15a/16-1 cluster recapitulates the spectrum of CLL-associated lymphoproliferations in humans, including CLL, CD5(+) monoclonal B-cell lymphocytosis, and CD5(-) non-Hodgkin lymphomas. In the present study, we demonstrate that additional deletion of the 0.69-mb large genomic region telomeric to the MDR called the common deleted region (CDR) changed the spectrum of lymphoproliferations developing in CDR- versus MDR-deleted mice in that the number of CLL among B-cell lymphoproliferations was significantly elevated in the former. In addition, CDR-deleted mice seemed to succumb to their disease faster than MDR-deleted mice. Comparing HCDR3 regions of CD5(+) lymphoproliferations derived from this and published CLL mouse models, 44% (29 of 66) of junctions could be assigned to 8 sets of highly similar HCDR3 regions, demonstrating that CLL developing in mice frequently expresses almost identical, stereotypic Ag receptors. These results suggest that the size of 13q14 deletions influences the phenotype of the developing lymphoproliferations and potentially the severity of disease, suggesting a tumor-suppressor function for genetic elements in addition to DLEU2/miR-15a/16-1.     

p16INK4A and p15INK4B gene deletions in primary leukemias

The 9p21 locus has been deleted at a high frequency in a wide variety of tumors. Recently, two genes, p16INK4A and p15INK4B (also called MTS1 and MTS2), have been localized in close proximity at the 9p21 locus, encoding cyclin-dependent kinases 4/6 inhibitors of relative molecular mass 16 kD and 15 kD, respectively and also found to be deleted at a high frequency in tumor cell lines. We analyzed p16INK4A and p15INK4B genes in 178 cases of primary leukemias including 81 cases of chronic lymphocytic leukemia (CLL), seven of hairy cell leukemia (HCL), seven of chronic myelogenous leukemia (CML), 43 of acute myelogenous leukemia (AML), 27 of acute lymphoblastic leukemia (ALL), and 13 of myelodysplastic syndrome (MDS) by Southern blot analyses. The ALL cases showed a relatively high frequency of homozygous deletions (22%, 6 of 27) at the p16INK4A gene locus. Interestingly, of the six cases with p16INK4A homozygous deletions, only three showed homozygous deletions at the p15INK4B gene. In 81 CLL patients, we detected one homozygous and five heterozygous deletions at both the p16INK4A and p15INK4B genes and two heterozygous deletions at the p16INK4A gene alone. Deletion of these two genes in AML cases is relatively low (9%). We did not detect deletions in any of the MDS, HCL, and CML cases examined. Sequence analyses of p16INK4A gene of six CLL cases with heterozygous deletion at this locus showed a 27-bp deletion at the splice acceptor site of intron 1 in one case and changes in the coding sequence in three other cases. The data presented in this report showed that (1) p16INK4A and p15INK4B genes are preferentially deleted homozygously in ALL and heterozygously in CLL cases with frequent mutation in the second allele, and (2) p16INK4A gene appears to be more frequently deleted than p15INK4B gene.     

Homozygous deletions of the p15 (MTS2) and p16 (CDKN2/MTS1) genes in adult T-cell leukemia

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type I (HTLV-I). Twenty to 40 years often elapse from viral infection to overt ATL, suggesting that other genetic events must occur to produce frank leukemia. The p15 (MTS2) and p16 (CDKN2/MTS1) genes located on chromosome 9p have been implicated as candidate tumor-suppressor genes in several types of tumors. We examined for alterations of these genes in ATL using Southern blot and polymerase chain reaction-single-strand conformation polymorphism analyses. Both p15 and p16 genes were homozygously deleted in 4 of 23 acute/lymphomatous ATL (17%). An additional 3 (13%) and 4 (17%) acute/lymphomatous samples had hemizygous deletions in at least one exon of p15 and p16, respectively. One of 14 chronic ATL samples had a homozygously deleted p16 gene and another had a hemizygous deletion of p16. Neither homozygous nor hemizygous deletions of the p15 gene were found in chronic ATL. In total, 10 of 37 (27%) ATL samples had loss of the p15 and/or p16 genes. No point mutations of the p15 and p16 genes were found. The ATL patient with a homozygously deleted p16 in the chronic phase rapidly progressed to acute ATL and died within 6 months of the initial diagnosis. One instructive patient had no detectable deletion of the p15 and p16 genes during the chronic phase of ATL but had a homozygous deletions of both genes when she progressed to acute ATL. Our results suggest an association of p15/p16 deletions with development of acute ATL.     

Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays

Recurrent genomic aberrations are important prognostic parameters in chronic lymphocytic leukemia (CLL). High-resolution 10k and 50k Affymetrix SNP arrays were evaluated as a diagnostic tool for CLL and revealed chromosomal imbalances in 65.6% and 81.5% of 70 consecutive cases, respectively. Among the prognostically important aberrations, the del13q14 was present in 36 (51.4%), trisomy 12 in 9 (12.8%), del11q22 in 9 (12.8%), and del17p13 in 4 cases (5.7%). A prominent clustering of breakpoints on both sides of the MIRN15A/MIRN16-1 genes indicated the presence of recombination hot spots in the 13q14 region. Patients with a monoallelic del13q14 had slower lymphocyte growth kinetics (P=.002) than patients with biallelic deletions. In 4 CLL cases with unmutated VH genes, a common minimal 3.5-Mb gain of 2p16 spanning the REL and BCL11A oncogenes was identified, implicating these genes in the pathogenesis of CLL. Twenty-four large (>10 Mb) copy-neutral regions with loss of heterozygosity were identified in 14 cases. These regions with loss of heterozygosity are not detectable by alternative methods and may harbor novel imprinted genes or loss-of-function alleles that may be important for the pathogenesis of CLL. Genomic profiling with SNP arrays is a convenient and efficient screening method for simultaneous genome-wide detection of chromosomal aberrations.     

B-CLL and bcl-2 gene

Most of human follicular lymphomas (approximately 90% in U.S.A. or approximately 30% in Japan) possess the t(14; 18) chromosome translocation that directly involves the IgH locus on chromosome 14 and the bcl-2 gene on chromosome 18. The t(14; 18) chromosome translocation occurs nearly exclusively at two hot spots, a major breakpoint clustering region (mbr) within the 2nd exon noncoding region and the minor breakpoint clustering region (mcr) within the 3' flanking region of the bcl-2 gene. Here we show that the rearrangement of the bcl-2 gene occurs in a significant fraction (approximately 10%) of B-CLL. All of the rearranged bcl-2 genes were juxtaposed with Ig lambda or Ig kappa genes, implying that the bcl-2 gene is preferentially linked to the IgL genes in CLL.     

Chronic lymphocytic leukemia B cells of more than 1% of patients express virtually identical immunoglobulins

We examined the immunoglobulin (Ig) heavy chain variable region genes (V(H) genes) used by leukemia cells of 1220 unrelated patients with chronic lymphocytic leukemia (CLL). We found 1188 (97%) expressed Ig encoded by a single Ig V(H) subgroup, the most common of which was V(H)3 (571 or 48.1%), followed by V(H)1 (319 or 26.8%) and V(H)4 (241 or 20.2%). Using allele-specific primers, we found 13.8% of all samples (n = 164) used one major V(H)1-69 allele, designated 51p1, 163 of which were not somatically mutated. For these cases, there was marked restriction in the structure of the Ig third complementarity determining regions (CDR3s), which were encoded by a small number of unmutated D and J(H) gene segments. Strikingly, 15 of the 163 cases had virtually identical CDR3s encoded by the second reading frame of D3-16 and J(H)3. Further analysis revealed that each of these 15 samples used the same unmutated Ig kappa light-chain gene, namely A27. These data reveal that approximately 1.3% (15/1220) of all patients had leukemia cells that expressed virtually identical Ig. This finding provides compelling evidence that the Ig expressed by CLL B cells are highly selected and not representative of the Ig expressed by naive B cells.     

A narrow deletion of 7q is common to HCL, and SMZL, but not CLL

To further characterise the genetic background of the two closely related B-lymphocytic malignancies hairy cell leukaemia (HCL), and splenic marginal zone lymphoma (SMZL) we have identified characteristic copy number imbalances by comparative genomic hybridisation (CGH). Based on these findings, areas of special interest were fine mapped, and relevant probes constructed for use in interphase-fluorescence in situ hybridisation (FISH) investigations. Thus, using the CGH data from 52 HCL and 61 SMZL patients, we identified the characteristic profiles of copy number imbalances for both diseases. These were a gain of 5q13-31 (19%) and loss of 7q22-q35 (6%) for HCL, and gain of 3q25 (28%), loss of 7q31 (16%), and gain of 12q15 (16%) for SMZL. A partial loss of 7q unusual for low-malignant B-cell diseases was found to be common to the two diseases. This loss was therefore fine mapped with BAC/PAC clones. Fine mapping revealed that in SMZL the minimal lost region covers 11.4 Mb spanning from 7q31.33 to 7q33 located between sequence tagged site (STS)-markers SHGC-3275 and D7S725. This area was distinct from the commonly deleted 7q region of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML). A FISH probe specific for the 7q region was constructed. Using this probe in an interphase-FISH investigation we showed the minimal lost 7q-region of HCL and SMZL to be one and the same. In one HCL case, this investigation furthermore showed the extent of the deleted region to be below the detection limit of CGH, whereas interphase-FISH screening of 36 chronic lymphocytic leukaemia (CLL) cases showed no deletion of the 7q area. In conclusion, we have identified characteristic profiles of copy number imbalances in HCL and SMZL and fine mapped the minimal extent of a commonly lost 7q area of special interest. We hypothesise that this region may contain (a) gene(s) important for the pathology of HCL and SMZL.     

Cocktail of eternity: HDAC meets miR

In this issue of Blood, Sampath and colleagues provide an important missing link in how microRNAs (miRs) can be silenced in chronic lymphocytic leukemia (CLL):histone deacetylases (HDACs) that are overexpressed in CLL block critical miRs in the malignant B cell resulting in pro-survival signals. Thus,HDAC inhibition is an attractive new therapeutic strategy in CLL.     

13q deletions in lymphoid malignancies

Previous studies have indicated that a candidate tumor suppressor gene resides telomeric of the RB1 gene at 13q14, a region that is commonly deleted in B-cell chronic lymphocytic leukemia (B-CLL). In this study, we have evaluated the frequency and minimal region of overlap for 13q deletions in malignant cells from various lymphoid neoplasms. We observed losses at 13q14 in 33/75 (44%) B-CLL cases, four of 16 (25%) non-Hodgkin's lymphoma (NHL) cases, eight of 29 (28%) patients with acute lymphocytic leukemia (ALL), and one of 15 T-cell lines. In some ALL cases, inactivation of the RB1 gene is suggested as the important event in the 13q deletions. The most commonly deleted marker in CLL and NHL was D13S319, between RBkpt and the D13S25 loci. Homozygous deletions of this marker were observed in 10 of 75 B-CLL cases, in six of which the homozygous deletions included only the D13S319 locus. Our data suggest that 13q deletions are common in lymphoid neoplasms, and that deletion of a candidate tumor suppressor gene(s) in the vicinity of the D13S319 marker is a tumorigenic event in these diseases.     

LncRNA miR503HG inhibits epithelial-mesenchymal transition and angiogenesis in hepatocellular carcinoma by enhancing PDCD4 via regulation of miR-15b

ObjectiveTo reveal the effect of lncRNA miR503HG on epithelial-mesenchymal transition (EMT) and angiogenesis in hepatocellular carcinoma (HCC).MethodsThe expressions of miR503HG, miR-15b and PDCD4 in HCC tissues and cell lines were measured. After cell transfection, Transwell assay tested the migration and invasion ability of HCC cells. qRT-PCR and Western blot detected the expressions of EMT markers (E-cad, N-cad, Vim and Snail-1). Matrigel-based tube formation assay assessed the angiogenesis capacity of human umbilical vein endothelial cells (HUVECs) cultured in conditioned medium of treated HCC cells. ELISA detected the level of VEGF in supernatant of HUVECs. RIP, RNA pulldown and dual-luciferase reporter assay were applied to verify the binding of miR-15b to miR503HG or PDCD4. pcDNA3.1-miR503HG-BEL-7404 cells or pcDNA3.1-BEL-7404 cells were implanted into nude mice for construction of HCC model in vivo.ResultsmiR503HG and PDCD4 were under-expressed and miR-15b was over-expressed in HCC cells and tissues. Up-regulation of miR503HG and PDCD4 or inhibition of miR-15b hindered migration, invasion and EMT of HCC cells and angiogenesis of HUVECs. Both miR503HG and PDCD4 could bind to miR-15b. Over-expression of miR503HG suppressed HCC growth and angiogenesis in nude mice.ConclusionLncRNA miR503HG suppresses EMT and angiogenesis in HCC via miR-15b/PDCD4 axis.     

Loss of the cyclin-dependent kinase 4-inhibitor (p16; MTS1) gene is frequent in and highly specific to lymphoid tumors in primary human hematopoietic malignancies

The cyclin-dependent kinase 4-inhibitor (CDK41; p16; or MTS1) gene has been proposed as a candidate for a tumor-suppressor gene located in chromosome 9p21, a frequently deleted region in a wide spectrum of human cancers, including leukemias. Recent studies disclosed that it was frequently deleted or mutated in a variety of primary human cancers, including acute lymphoblastic leukemia. The purpose of this study is to figure out the precise manners and frequencies of p16 gene inactivation in diverse hematopoietic tumor types and thus to clarify its significance in development of human hematopoietic malignancies. A total of 410 tumor specimens from patients with primary hematopoietic malignancies were examined for deletions of the p16 gene as well as the neighboring p15 gene and the nearby interferon alpha gene by Southern blot analysis. Tumor-specific mutations or small deletions of the p16 gene were also studied in 74 patients using single-strand conformation polymorphism analysis and direct sequencing. Loss of the p16 gene was most frequently observed among the three genes examined and was found in 59 of the 410 patients: 2 of 134 with acute myelocytic leukemia, 41 of 105 with acute lymphocytic leukemia, 2 of 15 with chronic lymphocytic leukemia, 5 of 14 with adult T-cell leukemia, 4 of 33 with non-Hodgkin's lymphoma, 3 of 8 with mixed-lineage leukemia, and 2 of 61 with chronic myelocytic leukemia. In 16 of the 59 patients, the p16 deletions occurred due to rearrangements within the small region between the p15 exon 2 and the p16 exon 2. Tumor-specific mutations or small deletions of the p16 gene were not detected in the 74 patients examined, including 12 of 14 patients with hemizygous deletions of the gene. Loss of the p16 gene is frequent in and highly specific to lymphoid malignancies (54 of 183 [30%] in lymphoid tumor v2 of 219 [1%] in myeloid tumors; P < .0001). The deletion analyses strongly suggest that the p16 gene is a tumor-suppressor gene located in chromosome 9p21 that is involved in development of human lymphoid tumors. Gene deletions but not minute mutations should be the predominant mechanism of p16 gene inactivation in these types of tumors.