Reduced expression of microRNA­206 regulates cell proliferation via cyclinD2 in gliomas.

MicroRNAs are short single­stranded non­coding RNA molecules that function as regulators of tumor progression, including regulation of glioblastoma multiforme, which is a World Health Organization grade Ⅳ glioma. Based on the results of a microRNA microarray, which included 198 patients with glioma from the Chinese Glioma Genome Atlas data set, it was observed that microRNA­206 (miR­206) was downregulated in high-grade (grades Ⅲ and Ⅳ) gliomas compared with grade II gliomas. In addition, high expression of miR­206 was associated with longer overall survival time in glioma patients. The present study aimed to investigate the biological functions of miR­206 in glioma progression in vitro using the LN229 glioma cell line. Cell proliferation was observed to be inhibited subsequent to transfection with miR­206. It was suggested that miR­206 induced cell cycle G1/S phase arrest by suppressing the expression of cyclinD2. The results of the present study concluded that miR­206 inhibits glioma progression via the regulation of cyclinD2 and that miR­206 may be a novel biomarker with potential for use as a therapeutic target in gliomas.

DNMT3A mutations and prognostic significance in childhood acute lymphoblastic leukemia.

Little is known about DNMT3A mutations in childhood acute lymphoblastic leukemia (ALL). We screened for DNMT3A mutations in exon 23 and its adjacent intron regions in diagnostic samples of 201 children with ALL. The cDNA samples from 82 patients were also sequenced to identify other mutations in the entire coding region. DNMT3A mutations were detected in exon 23 and its adjacent intron regions only in five patients (2.5%). There was only one mutation in exon 23 in two patients, respectively. In the other three patients, five intronic mutations were found. None of the mutations was found in the five corresponding complete remission samples. DNMT3A mutations were correlated with higher minimal residual disease at the end of remission induction (p = 0.078). Treatment outcome was obviously worse in patients with DNMT3A mutations than in other patients (p < 0.05). Thus, DNMT3A mutations can be found in a few children with ALL, and may have an adverse impact on prognosis.

Downregulation of TES by hypermethylation in glioblastoma reduces cell apoptosis and predicts poor clinical outcome.

BACKGROUND: Gliomas are the most common human brain tumors. Glioblastoma, also known as glioblastoma multiform (GBM), is the most aggressive, malignant, and lethal glioma. The investigation of prognostic and diagnostic molecular biomarkers in glioma patients to provide direction on clinical practice is urgent. Recent studies demonstrated that abnormal DNA methylation states play a key role in the pathogenesis of this kind of tumor. In this study, we want to identify a novel biomarker related to glioma initiation and find the role of the glioma-related gene. METHODS: We performed a methylation-specific microarray on the promoter region to identify methylation gene(s) that may affect outcome of GBM patients. Normal and GBM tissues were collected from Tiantan Hospital. Genomic DNA was extracted from these tissues and analyzed with a DNA promoter methylation microarray. Testis derived transcript (TES) protein expression was analyzed by immunohistochemistry in paraffin-embedded patient tissues. Western blotting was used to detect TES protein expression in the GBM cell line U251 with or without 5-aza-dC treatment. Cell apoptosis was evaluated by flow cytometry analysis using Annexin V/PI staining. RESULTS: We found that the TES promoter was hypermethylated in GBM compared to normal brain tissues under DNA promoter methylation microarray analysis. The GBM patients with TES hypermethylation had a short overall survival (P <0.05, log-rank test). Among GBM samples, reduced TES protein level was detected in 33 (89.2%) of 37 tumor tissues by immunohistochemical staining. Down regulation of TES was also correlated with worse patient outcome (P <0.05, log-rank test). Treatment on the GBM cell line U251 with 5-aza-dC can greatly increase TES expression, confirming the hypermethylation of TES promoter in GBM. Up-regulation of TES prompts U251 apoptosis significantly. This study demonstrated that both TES promoter hypermethylation and down-regulated protein expression significantly correlated with worse patient outcome. Treatment on the GBM cell line (U251) with 5-aza-dC can highly release TES expression resulting in significant apoptosis in these cells. CONCLUSIONS: Our findings suggest that the TES gene is a novel tumor suppressor gene and might represent a valuable prognostic marker for glioblastoma, indicating a potential target for future GBM therapy.

MicroRNA-320a suppresses in GBM patients and modulates glioma cell functions by targeting IGF-1R.

Glioblastoma (GBM) is the most aggressive and malignant glioma. Currently, a few modern surgical and medical therapeutic strategies are applied for GBM, but the prognosis of GBM patients remains poor, and the average median survival time is only 14.6 months. In this study, we for the first time found that the levels of miR-320a were decreased in both GBM patients and glioma cells. In GBM patients, elevated miR-320a expression was associated with better prognosis. In addition, insulin-like growth factor-1 receptor (IGF-1R) was identified as a key direct target of miR-320a. Overexpression of miR-320a led to the inhibition of cell proliferation, migration, invasion, as well as tumorigenesis by targeting IGF-1R, and thus regulated the signaling pathways downstream, including PI3K/AKT and MAPK/ERK. In tumor orthotopic xenograft experiment, the tumor growth was depressed and survival time of mice model was prolonged when miR-320a was overexpressed. Therefore, our results suggested that miR-320a could suppress tumor development and growth by targeting IGF-1R, and miR-320a might serve as a new effective target for anti-cancer therapy strategies.

Integrated analysis using methylation and gene expression microarrays reveals PDE4C as a prognostic biomarker in human glioma.

Hypermethylation of tumor suppressor promoters is generally accepted to indicate poor prognosis in glioma; however, the DNA methylation patterns associated with different glioma prognoses remain to be elucidated. In the present study, promoter methylation and gene expression microarrays were used to screen candidate genes between different grades of glioma. Survival analysis was performed using the Kaplan­Meier (KM) method. Promoter methylation and protein expression of phosphodiesterase 4C (PDE4C) was examined in different grade gliomas and the correlation between PDE4C and wild-type (WT) p53 was evaluated in glioma cell lines. In addition, gene ontology and gene set variation analysis were used to examine PDE4C function. We found PDE4C exhibited promoter hypermethylation in high-grade glioma samples and hypomethylation in low-grade glioma, with PDE4C expression levels showing the reverse. This indicated PDE4C may be a candidate glioma biomarker. Through studies of PDE4C methylation and expression status in an independent cohort of 124 patient samples (56 low-grade and 63 high-grade glioma and 5 normal brain), we identified PDE4C as having significant promoter methylation and lower expression in high-grade glioma. Hypermethylation and reduced PDE4C protein expression were associated with grade progression and overall survival. In glioma cell lines, PDE4C was upregulated by demethylation treatment with 5-Aza-2'-deoxycytidine and WT p53 expression was downregulated after PDE4C siRNA suppression. Finally, we found PDE4C promoted apoptosis and inhibited migration in a U87 cell line. On the basis of these observations and the results from subset analysis, it is reasonable to conclude that PDE4C may function as a tumor suppressor by promoting apoptosis through the WT p53 pathway and inhibiting cell migration. The data show that PDE4C is downregulated through promoter hypermethylation in glioma.

SOCS3 promoter hypermethylation is a favorable prognosticator and a novel indicator for G-CIMP-positive GBM patients.

BACKGROUND: Hypermethylation of the suppressor of cytokine signaling 3(SOCS3) promoter has been reported to predict a poor prognosis in several cancers including glioblastoma multiforme (GBM). We explored the function of SOCS3 promoter hypermethylation in GBM cohorts, including analysis of the CpG island methylator phenotype (CIMP), when a large number of gene loci are simultaneously hypermethylated. METHODS: A whole genome promoter methylation profile was performed in a cohort of 33 GBM samples, with 13 long-term survivors (LTS; overall survival ≥ 18 months) and 20 short-term survivors (STS; overall survival ≤ 9 months). The SOCS3 promoter methylation status was compared between the two groups. In addition, we investigated the relationship of SOCS3 promoter methylation and G-CIMP status. RESULTS: Interestingly, in our present study, we found that SOCS3 promoter methylation was statistically significantly higher in the 13 LTS than that in the 20 STS. Furthermore, high SOCS3 promoter methylation detected via pyro-sequencing predicted a better prognosis in an independent cohort containing 62 GBM patients. This correlation was validated by the dataset from the Cancer Genome Atlas(TCGA) and the Chinese Cancer Genome Atlas(CGGA). In addition, we found that hypermethylation of the SOCS3 promoter was tightly associated with the G-CIMP-positive GBM patients. CONCLUSIONS: Using a total of 359 clinical samples, we demonstrate that SOCS3 promoter hypermethylation status has a favorable prognostic value in GBM patients because of whole genome methylation status. Particularly, the hypermethylation of the SOCS3 promoter indicates positive G-CIMP status.

Expression of SPRR3 is associated with tumor cell proliferation and invasion in glioblastoma multiforme.

Esophagin, also known as small proline-rich protein 3 (SPRR3), has been demonstrated to be important in the initiation and progression of numerous types of tumor, including colorectal and breast cancer. However, studies concerning the biological functions of SPRR3 in glioblastoma multiforme (GBM) are limited. Therefore, we aimed to identify the functions and molecular mechanisms underlying the role of SPRR3 in GBM. Hypomethylation of SPRR3 was observed and associated with a poor clinical outcome in GBM patients compared with healthy individuals by using gene methylation profiling. The present study was performed to investigate the expression status and effects of SPRR3 in GBM. The U251 cell line was used in the functional analyses. Cell growth was examined by MTT and colony formation assay. Cell invasion was measured using the Transwell invasion assay. The expression of SPRR3 in tissue samples was examined by immunohistochemistry. The results revealed that the overexpression of SPRR3 accelerates U251 cell proliferation and invasion. It was also observed that SPRR3 was markedly upregulated in 72.7% of GBM samples (24/33) compared with the normal tissue. These results suggest that an increased expression of SPRR3 is involved in tumorigenesis.

Epigenetic silencing of KAZALD1 confers a better prognosis and is associated with malignant transformation/progression in glioma.

In order to more thoroughly analyze aberrant DNA methylation in glioma, we applied a large cohort methylation microarray including 119 glioma samples. Six genes, ADCY1, KAZALD1, KLF4, SLMAP, TETRAN and TP53INP1, were screened out through significance analysis of microarray (SAM), survival Cox-regression and certain other pre-set conditions. We focused on the KAZALD1 oncogene. KAZALD1, also known as IGFBP-rP10, belongs to the IGFBP family. We found that KAZALD1 was hypomethylated in high-grade glioma (anaplastic gliomas and glioblastomas) compared to low-grade glioma (astrocytoma, oligodendrocytoma and oligoastrocytoma) using methylation microarrays (p<0.001). Immunohistochemistry (IHC) of 91 glioma samples showed that the KAZALD1 expression scores of high-grade glioma samples were higher compared to the scores of low-grade gliomas (p<0.001). In high-grade gliomas, overall survival (OS) was shorter for patients with KAZALD1 hypomethylation or overexpression compared to those without. Decreased KAZALD1 expression in glioma inhibited cell proliferation and invasion both in vitro and in vivo. On the basis of these observations and the results from subset analysis, it is reasonable to conclude that KAZALD1 promoter hypomethylation is an important prognostic biomarker in glioma. KAZALD1 promotes glioma malignant progression through invasion and proliferation.

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients.

BACKGROUND: Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets. METHODS: We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors. RESULTS: Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013). CONCLUSIONS: Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.

Inhibition of caspase-8 activity caused by overexpression of BCL10 contributes to the pathogenesis of high-grade MALT lymphoma.

BACKGROUND: Mucosa-associated lymphoid tissue (MALT) lymphoma comprises approximately 8% of all non-Hodgkin lymphomas and is the most common lymphoma in the gastro-intestinal tract. It is caused by genetic abnormalities or bacterial infections/chronic inflammation. B-cell lymphoma/leukemia 10 (BCL10) overexpression and nuclear expression have been associated with high-grade MALT lymphomas with genetic abnormalities that are unresponsive to Helicobacter pylori eradication treatment. To explore the molecular mechanism of BCL10 overexpression on the pathogenesis and malignant phenotype of MALT lymphoma, we generated EµSR-BCL10 transgenic mice. PROCEDURE: By generation of heterozygous and homozygous EuSR-BCL10 mice and showing BCL10 expression levels in these mice, we quantitatively examined relation of MZ B cell expansion and inhibition of caspase-8 activity with BCL10 protein level. We also investigated API2 and caspase-8 expression by Western blot and their interaction with BCL10 by co-immunoprecipitation. RESULTS: MZ B-cell expansion is directly related to BCL10 protein level in a dose-dependent manner. The activity of caspases-8 and -3, but not caspase-9, was inhibited with increasing of BCL10 protein level. Expanded MZ B cells showed selective survival under stimulation of anti-immunoglobulin M, but not dexamethasone, γ-irradiation, or anti-CD95, implying that overexpressed BCL10 exerts anti-apoptotic effects through B-cell antigen receptor (BCR) pathway. Overexpressed BCL10 protein co-immunoprecipitated with caspase-8 and API2 protein, suggesting an in vivo interaction of them. CONCLUSION: Our data demonstrate a novel effect of overexpressed BCL10 in the pathogenesis of high-grade MALT lymphoma by increasing expression of API2 and it then forming a protein complex with BCL10/caspase-8 leading to caspase-8 activity suppression.

Role of interleukin-10 and transforming growth factor beta 1 in otitis media with effusion.

BACKGROUND: Otitis media with effusion (OME) is a disease with complicated pathogeneses which are not clearly known. Increasing interest has been focused on immunological cells, cytokines and their roles in chronic inflammatory states. This study was designed to disclose the existence and roles of interleukin-10 (IL-10) and transforming growth factor beta1 (TGF-beta1) in the cause of OME in adults, and to investigate the probable role of Foxp3(+)CD4(+)CD25(+) T cells in OME. METHODS: The concentrations of IL-10 and TGF-beta1 in the middle ear effusions (MEEs) and plasmas of 36 adults (45 ears) with OME were measured by means of enzyme linked immunosorbent assay (ELISA). As contrast, the concentrations of IL-10 and TGF-beta1 in the plasma of 30 normal volunteers were measured using the same method. Furthermore, the proportion of Foxp3(+)CD4(+)CD25(+) T cells in CD4(+) T cells of blood was tested by flow cytometry. RESULTS: (1) The concentrations of IL-10 in all MEEs and plasmas of the chronic OME patients were higher than those in patients with acute OME (both P < 0.05), so was TGF-beta1 (both P < 0.01). The concentration of IL-10 in MEEs was significantly higher than that in plasmas, not only in acute OME (P < 0.01), but also in chronic OME (P < 0.01). In chronic OME, the concentration of TGF-beta1 in MEEs had no statistical difference with those in plasmas of the same patients. However, the concentration of TGF-beta1 in plasmas of patients with chronic OME was significantly higher than that in plasmas of normal volunteers (P < 0.01). (2) The concentrations of IL-10 and TGF-beta1 in MEEs of the patients who had been treated more than once were higher than those MEEs of the patients who were treated for the first time, respectively (P < 0.05, P < 0.01). The level of TGF-beta1 in plasmas of the patients who had been treated more than once was higher than in those of the patients who were treated firstly (P < 0.05), while the level of IL-10 in plasmas had no difference. The concentration of IL-10 in mucoid MEEs was higher than those in serous ones (P < 0.05), while TGF-beta1 had no statistical difference between mucoid and serous MEEs (P > 0.05). The concentration of IL-10 in MEEs had a strong correlation with the duration of the illness (r = 0.547, P < 0.01). The same correlation was also found between the concentration of TGF-beta1 in MEEs and the times patients being treated (r = 0.579, P < 0.01). (3) The proportion of Foxp3(+)CD4(+)CD25(+)T/CD4(+) T cells in the blood of chronic OME was not only significantly higher than that in the acute OME (P < 0.01), but also higher than that in normal volunteers (P < 0.01). In chronic OME, there was a correlation between the proportion of Foxp3(+)CD4(+)CD25(+) T/CD4(+) T cells in the blood and the concentration of IL-10 in the plasmas (r = 0.602, P < 0.05). CONCLUSIONS: IL-10 and TGF-beta1, as two important immunoregulatory mediators, participate in middle ear inflammatory response, especially in chronic course of OME in adults. Foxp3(+)CD4(+)CD25(+) T cells may play some immunoregulatory roles in the course of this disease.

Emu-BCL10 mice exhibit constitutive activation of both canonical and noncanonical NF-kappaB pathways generating marginal zone (MZ) B-cell expansion as a precursor to splenic MZ lymphoma.

BCL10, required for nuclear factor kappaB (NF-kappaB) activation during antigen-driven lymphocyte responses, is aberrantly expressed in mucosa-associated lymphoid tissue-type marginal zone (MZ) lymphomas because of chromosomal translocations. Emu-driven human BCL10 transgenic (Tg) mice, which we created and characterize here, had expanded populations of MZ B cells and reduced follicular and B1a cells. Splenic B cells from Tg mice exhibited constitutive activation of both canonical and noncanonical NF-kappaB signaling pathways is associated with increased expression of NF-kappaB target genes. These genes included Tnfsf13b, which encodes the B-cell activating factor (BAFF). In addition, levels of BAFF were significantly increased in sera from Tg mice. MZ B cells of Tg mice exhibited reduced turnover in vivo and enhanced survival in vitro, indicative of lymphoaccumulation rather than lymphoproliferation as the cause of MZ expansion. In vivo antibody responses to both T-independent, and especially T-dependent, antigens were significantly reduced in Tg mice. Mortality was accelerated in Tg animals, and some mice older than 8 months had histologic and molecular findings indicative of clonal splenic MZ lymphoma. These results suggest that, in addition to constitutive activation of BCL10 in MZ B cells, other genetic factors or environmental influences are required for short latency oncogenic transformation.

Growth retardation and premature aging phenotypes in mice with disruption of the SNF2-like gene, PASG.

Imperfect maintenance of genome integrity has been postulated to be an important cause of aging. Here we provide support for this hypothesis by demonstrating that the disruption of PASG (lsh), a SNF2-like factor that facilitates DNA methylation, causes global hypomethylation, developmental growth retardation and a premature aging phenotype. PASG mutant mice display signs of growth retardation and premature aging, including low birth weight, failure to thrive, graying and loss of hair, reduced skin fat deposition, osteoporosis, kyphosis, cachexia, and premature death. Fibroblasts derived from PASG mutant embryos show a replicative senescence phenotype. Both PASG mutant mice and fibroblasts demonstrate a markedly increased expression of senescence-associated tumor suppressor genes, such as p16(INK4a), that is independent of promoter methylation, but, instead, is associated with down-regulation of bmi-1, a negative regulator of p16(INK4a). These studies show that PASG is essential for properly maintaining DNA methylation and gene expression patterns that are required for normal growth and longevity. PASG mutant mice provide a useful model for the study of aging as well as the mechanisms regulating epigenetic patterning during development and postnatal life.

Activating mutations of RTK/ras signal transduction pathway in pediatric acute myeloid leukemia.

Activating mutations of receptor tyrosine kinases (RTKs) and their downstream affectors are common in acute myeloid leukemia (AML). We performed mutational analysis of FLT3, c-kit, c-fms, vascular endothelial growth factor (VEGF) receptors (Flt-1, KDR [kinase domain receptor]), and ras genes in a group of 91 pediatric patients with AML treated on Children's Cancer Group clinical trial CCG-2891. Forty-six percent of patients had activating mutations of FLT3 (24.5%), c-kit (3%), or ras (21%) genes. Mutation-positive patients had a higher median diagnostic white blood cell (WBC) count (71.5 vs 19.6 x 10(9)/L; P =.005) and lower complete remission rate (55% versus 76%; P =.046) than mutation-negative patients. The Kaplan-Meier estimate of overall survival (OS) for patients with and without an activating mutation was 34% versus 57%, respectively (P =.035). However, within this group, patients with FLT3/ALM (activation loop mutation) had good outcomes (OS, 86%). Exclusion of the FLT3/ALM from analysis decreased the OS for the remaining mutation-positive patients to 26% (P =.003). Ten of the 23 mutation-positive and 11 of the 34 mutation-negative patients received an allogeneic bone marrow transplant (BMT) in first complete remission (CR). In the mutation-positive group, the disease-free survival (DFS) for the allogeneic BMT recipients was 72% versus 23% for the 13 patients who received chemotherapy or autologous BMT (P =.01). DFS for the mutation-free patients with and without allogeneic BM transplantation was 55% and 40%, respectively (P =.38). Activating mutations in the RTK/ras signaling pathway are common in pediatric AML, and their presence may identify a population at higher risk of poor outcome who may benefit from allogeneic BM transplantation.