EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Targeting of YAP1 by microRNA-15a and microRNA-16-1 exerts tumor suppressor function in gastric adenocarcinoma.

BACKGROUND: MicroRNAs (miRNAs) have been reported to play an important role in tumorigenesis. In this study, the role of miR-15a and miR-16-1 in gastric adenocarcinoma (GAC) was investigated. METHODS: The expression of miR-15a and miR-16-1 in cell lines and primary tumors was examined by miRNA qRT-PCR. Proliferative assays, colony formation, cell invasion and migration, flow cytometry analysis and in vivo study were performed by ectopic expression of miR-15a and miR-16-1. The putative target genes of miR-15a and miR-16-1 were explored by TargetScan and further validated. RESULTS: We found that miR-15a and miR-16-1 were down-regulated in GAC cell lines and primary tumor samples compared with normal gastric epithelium. Functional study demonstrated that ectopic expression of miR-15a and miR-16-1 suppressed cell proliferation, monolayer colony formation, invasion and migration, and xenograft formation in vivo. In addition, miR-15a and miR-16-1 induced G0/G1 cell cycle arrest which was further confirmed by Western blot and qRT-PCR of related cell cycle regulators. YAP1 was confirmed to be a functional target of miR-15a and miR-16-1 in GAC. YAP1 re-expression partly abrogated the inhibitory effect of miR-15a and miR-16-1 in GAC cells. In clinical samples, YAP1 protein expression shows negative correlation with miR-15a and miR-16-1 expression. CONCLUSION: In conclusion, targeting YAP1 by tumor suppressor miRNA miR-15a and miR-16-1 plays inhibitory effect and this might have a therapeutic potential in GAC.

Overexpression of HMGA1 deregulates tumor growth via cdc25A and alters migration/invasion through a cdc25A-independent pathway in medulloblastoma.

Overexpression of high mobility group AT-hook 1 (HMGA1) is common in human cancers. Little is known about the mechanisms underlying its deregulation and downstream targets, and information about its clinical and biological significance in medulloblastoma (MB) is lacking. Here, we demonstrated frequent genomic gain at 6p21.33-6p21.31 with copy number increase leading to overexpression of HMGA1 in MB. The overexpression correlated with a high proliferation index and poor prognosis. Moreover, we found that hsa-miR-124a targeted 3'UTR of HMGA1 and negatively modulated the expression in MB cells, indicating that loss/downregulation of hsa-miR-124a reported in our previous study could contribute to the overexpression. Regarding the biological significance of HMGA1, siRNA knockdown and ectopic expression studies revealed the crucial roles of HMGA1 in controlling MB cell growth and migration/invasion through modulation of apoptosis and formation of filopodia and stress fibers, respectively. Furthermore, we identified cdc25A as a target of HMGA1 and showed that physical interaction between HMGA1 and the cdc25A promoter is required for transcriptional upregulation. In clinical samples, HMGA1 and cdc25A were concordantly overexpressed. Functionally, cdc25A is involved in the HMGA1-mediated control of MB cell growth. Finally, netropsin, which competes with HMGA1 in DNA binding, reduced the expression of cdc25A by suppression of its promoter activity and inhibited in vitro and in vivo intracranial MB cell growth. In conclusion, our results delineate the mechanisms underlying the deregulation and reveal the functional significance of HMGA1 in controlling MB cell growth and migration/invasion. Importantly, the results highlight the therapeutic potential of targeting HMGA1 in MB patients.

Pseudoprogression of malignant glioma in Chinese patients receiving concomitant chemoradiotherapy.

OBJECTIVES: To investigate the frequency of pseudoprogression of glioblastoma in Chinese patients receiving concomitant chemoradiotherapy and investigate its association with pseudoprogression and tumour molecular marker O(6)-methylguanine-DNA methyltransferase promoter methylation status. DESIGN: Case series with internal comparisons. SETTING: University teaching hospital, Hong Kong. PATIENTS: Patients with glioblastoma treated with concomitant chemoradiotherapy during April 2005 to June 2010 were reviewed. Magnetic resonance imaging brain scans, pre- and post-concomitant chemoradiotherapy and 3-monthly thereafter were reviewed by an independent neuroradiologist according to Macdonald's criteria. Relevant patient information (clinical condition, performance score, development of new neurological deficits, use of steroids, and survival) was retrieved. For each patient, O(6)-methylguanine-DNA methyltransferase methylation status was investigated with genomic DNA from formalin-fixed or paraffin-embedded sections of tumour tissues by methylation-specific polymerase chain reaction. RESULTS: During the study period, 28 primary glioblastoma patients underwent concomitant chemoradiotherapy. The mean age of the patients was 48 (range, 16-71) years. Thirteen patients (13/28, 46%) developed early radiological progression of the tumour after completion of concomitant chemoradiotherapy, of whom five (39%) were subsequently found to have had pseudoprogression. Patients with pseudoprogression showed a trend towards longer survival (22 months in pseudoprogression vs 17 months in all others vs 11 months in those with genuine progression). Among the 27 patients tested for O(6)-methylguanine-DNA methyltransferase promoter status, 12 (44%) were methylated. Two (2/12, 17%) in the methylated group had pseudoprogression, while three (3/15, 20%) in the unmethylated group had pseudoprogression. CONCLUSIONS: Nearly half of all patients (46%) developed early radiological progression (within 3 months of completing concomitant chemoradiotherapy). Moreover, about one in three of such patients had pseudoprogression. Pseudoprogression is an important clinical condition to be aware of to prevent premature termination of an effective treatment.

Association of molecular marker O(6)Methylguanine DNA methyltransferase and concomitant chemoradiotherapy with survival in Southern Chinese glioblastoma patients.

OBJECTIVES: (1) To compare the survival of concomitant chemotherapy and radiotherapy with radiotherapy alone in Chinese patients with primary glioblastoma. (2) To determine the methylation status of O(6)Methylguanine DNA methyltransferase in Chinese primary glioblastoma, and to assess the prognostic value of O(6)Methylguanine DNA methyltransferase methylation status in such patients. DESIGN: Retrospective correlative analysis. SETTING: University teaching hospital, Hong Kong. PATIENTS: Patients diagnosed with histologically proven primary glioblastoma in the period of March 2005 to June 2007 were recruited. Genomic DNA was isolated from formalin-fixed and paraffin-embedded sections of glioblastoma tissues. Methylation-specific polymerase chain reaction for O(6)Methylguanine DNA methyltransferase was performed. Patients' information at presentation was collected (age, performance status, steroid use, extent of resection, complications, radiotherapy data, use of chemotherapy). Primary outcome was measured by overall survival while secondary outcome was measured by progression-free survival. Overall and progression-free survivals were estimated by the Kaplan-Meier technique. Outcomes were assessed for groups with and without concomitant chemoradiotherapy and for groups with and without O(6)Methylguanine DNA methyltransferase methylation. RESULTS: A total of 35 glioblastoma patients were recruited; 27 were male and 8 female. Their mean age was 50 years. In all, 17 received concomitant chemoradiotherapy, and 18 received radiotherapy only. Their median overall survival was 12 (range, 7-17) months and the median progression-free survival was 5 (range, 3-6) months. In the radiotherapy alone group, the median progression-free survival and overall survival was 4 (range, 3-5) months and 6 (range, 2-10) months, respectively. In the concomitant radiochemotherapy group, the median progression-free survival and overall survival was 6 (range, 2-10) months and 13 (range, 8-18) months, respectively. Fifteen (43%) of the tumour samples showed methylation of O(6)Methylguanine DNA methyltransferase. There was a trend towards overall longer survival in the group with methylated tumours compared to those with unmethylated tumours; respective values for median survival (ranges) were 17 (13-21) versus 10 (6-14) months (P=0.105). CONCLUSIONS: Our single-centre results indicated that Chinese glioblastoma patients who had received concomitant chemoradiotherapy showed a trend towards longer overall survival compared to those receiving radiotherapy alone. Approximately 43% of our Chinese glioblastoma samples showed methylation of O(6)Methylguanine DNA methyltransferase. O(6)Methylguanine DNA methyltransferase methylation may be a significant prognostic factor in Chinese glioblastoma patients.

miR-375 is involved in Hippo pathway by targeting YAP1/TEAD4-CTGF axis in gastric carcinogenesis.

miR-375 is a tumor-suppressive microRNA (miRNA) in gastric cancer (GC). However, its molecular mechanism remains unclear. The aim of this study is to comprehensively investigate how miR-375 is involved in Hippo pathway by targeting multiple oncogenes. miR-375 expression in gastric cancer cell lines and primary GC was investigated by qRT-PCR. The regulation of YAP1, TEAD4, and CTGF expression by miR-375 was evaluated by qRT-PCR, western blot, and luciferase reporter assays, respectively. The functional roles of the related genes were examined by siRNA-mediated knockdown or ectopic expression assays. The clinical significance and expression correlation analysis of miR-375, YAP1, and CTGF were performed in primary GCs. TCGA cohort was also used to analyze the expression correlation of YAP1, TEAD4, CTGF, and miR-375 in primary GCs. miR-375 was down-regulated in GC due to promoter methylation and histone deacetylation. miR-375 downregulation was associated with unfavorable outcome and lymph node metastasis. Ectopic expression of miR-375 inhibited tumor growth in vitro and in vivo. Three components of Hippo pathway, YAP1, TEAD4 and CTGF, were revealed to be direct targets of miR-375. The expression of three genes showed a negative correlation with miR-375 expression and YAP1 re-expression partly abolished the tumor-suppressive effect of miR-375. Furthermore, CTGF was confirmed to be the key downstream of Hippo-YAP1 cascade and its knockdown phenocopied siYAP1 or miR-375 overexpression. YAP1 nuclear accumulation was positively correlated with CTGF cytoplasmic expression in primary GC tissues. Verteporfin exerted an anti-oncogenic effect in GC cell lines by quenching CTGF expression through YAP1 degradation. In short, miR-375 was involved in the Hippo pathway by targeting YAP1-TEAD4-CTGF axis and enriched our knowledge on the miRNA dysregulation in gastric tumorigenesis.

Detection of oncogene amplifications in medulloblastomas by comparative genomic hybridization and array-based comparative genomic hybridization.

OBJECT: Few studies have been conducted to investigate the genomic survey of oncogene amplification in medulloblastoma. Low frequency of N-myc, C-myc, and epidermal grow factor receptor (EGFR) gene amplification (< 10%) has been reported in medulloblastoma. Previous comparative genomic hybridization (CGH) study of primary medulloblastomas has revealed chromosomal amplification on 2p21, 3p, 5p15.3, 7q, 8q24, 11q22.3, and 17q. The aim of this study was to detect common oncogenes involved in medulloblastoma tumorigenesis. METHODS: The authors studied a series of 14 samples by performing CGH and array-based CGH. The CGH analysis detected nonrandom losses on 8p, 17p, 16q, 8q, and 1p, whereas gains were found on 17q, 12q, 7q, and 1p. Array-based CGH was conducted to investigate amplification of 58 oncogenes throughout the genome of these samples. Gene amplifications identified for the first time included PGY1 at 7q21.1, MDM2 at 12q14.3-q15, and ERBB2 at 17q21.2. The highest frequencies of oncogene gain were detected in D17S1670 (61.5%), PIK3CA (46.2%), PGY1 (38.5%), MET (38.5%), ERBB2 (38.5%), and CSE1L (38.5%). The gain in gene copy numbers was confirmed in 34 additional archival medulloblastoma cases by using fluorescence in situ hybridization analysis. CONCLUSIONS: This is the first genome-wide survey of multiple oncogene amplifications involved in the development of medulloblastoma. Gains of several candidate oncogenes such as D17S1670, ERBB2, PIK3CA, PGY1, MET, and CSE1L were frequently detected. These genes may be used as molecular markers and therapeutic targets of medulloblastomas.

Chromosome 14q may harbor multiple tumor suppressor genes in primary glioblastoma multiforme.

OBJECTIVE: To evaluate whether deletion of chromosome 14q is involved in the carcinogenesis of primary glioblastoma multiforme and to identify possibly common deletion regions. METHJODS: Fourteen fluorescent dye-labeled polymorphic markers were used and polymerase chain reaction-based microsatellite analysis was employed to investigate loss of heterozygosity (LOH) on chromosome 14q in 20 primary glioblastoma multiforme (GBM). RESULTS: Ten of twenty (50%) GBM displayed LOH at one or more of the markers on chromosome 14q. Five tumors showed either LOH or non-informative on all markers tested. The most frequent LOH was observed at locus D14S65 (57.1%) on 14q32.1, and in the chromosomal region spanning from D14S63 (47.1%) to D14S74 (46.7%) on 14q23-31. None of the informative loci exhibited microsatellite instability. CONCLUSIONS: Allelic deletion on chromosome 14q plays an important role in the pathogenesis of GBM. Chromosomal regions at locus D14S65 on 14q32.1 and spanning from D14S63 to D14S74 on 14q23-31 may harbor multiple tumor suppressor genes associated with GBM.

Genome-wide allelotype study of primary glioblastoma multiforme.

OBJECTIVE: To investigate the molecular genetic pathogenesis of primary glioblastoma multiforme (GBM) and identify which chromosomes or chromosomal regions of the entire genome may harbor tumor suppressor genes (TSGs) associated with GBM. METHODS: A high-resolution allelotype study of 21 cases of primary GBM was performed by PCR-based loss of heterozygosity (LOH) analysis. Three hundred and eighty-two fluorescent dye-labeled microsatellite markers covering all 22 autosomes were applied. The mean genetic distance between two flanking markers was about 10 cM. RESULTS: LOH was observed on all 39 nonacrocentric autosomal arms examined in this study. The LOH frequencies of 10q, 10p, 9p, 17p and 13q were the highest (> 50%). Furthermore, high LOH frequencies were detected in the regions containing known TSGs including PTEN, DMBT1, p16, p15, p53 and RB; the LOH frequencies on 14q, 3q, 22q, 11p, 9q, 19q were also high (> 40.5%). Our study observed the following commonly deleted regions: 9p22-23, 10p12.2-14, 10q21.3, 13q12.1-14.1, 13q14.3-31, 17p11.2-12, 17p13, 3q25.2-26.2, 11p12-13, 14q13-31, 14q32.1, 14q11.1-13, 22q13.3, 4q35, 4q31.1-31.2, 6q27 and 6q21-23.3. CONCLUSIONS: The molecular pathogenesis of GBM is very complicated and associated with a variety of genetic abnormalities on many chromosomal arms. The most closely related chromosomal arms to the pathogenesis of GBM are 10q, 10p, 9p, 17p and 13q. Besides the well-known TSGs including PTEN, DMBT1, p16, p15, p53 and RB, multiple unknown TSGs associated with GBM may be present on the commonly deleted regions detected in the present study.

[An allelotype study of primary and corresponding recurrent glioblastoma multiforme].

OBJECTIVE: To investigate molecular genetic alterations associated with primary and corresponding recurrent glioblastoma multiforme(GBM) and to identify which chromosomal regions of the whole genome may be involved in the recurrence of primary GBM. METHODS: A high-resolution allelotyping study of one patient's primary GBM and corresponding recurrent GBM was performed by PCR-based loss of heterozygosity(LOH) analysis with the use of 382 fluorescent dye-labeled polymorphic microsatellite markers covering all 22 autosomes. The mean genetic distance between two flanking markers is 10 cM. RESULTS: LOH at locus D9S157 on 9p21 and at loci D10S537, D10S185, D10S192, D10S597, D10S587, D10S217 on 10q21.3-26.3 was observed in the primary GBM. As for corresponding recurrent tumor, LOH was observed not only in expanded regions on 9p21 and 10q21.3-26.3 but also on multiple other chromosomal arms, including 1q, 7p,7q, 21q, 20p, 20q, 10p, 19p, 19q. CONCLUSION: Chromosome 9p and 10q may be involved in the development of this GBM. Although histopathological diagnoses of the primary and corresponding recurrent tumor are identical, the recurrence of GBM is characterized by an increased involvement of molecular genetic abnormalities and may be accompanied by inactivation of more tumor suppressor genes.

[An allelotype study of human glioblastoma].

OBJECTIVE: To reveal the molecular genetic mechanisms for the pathogenesis of glioblastoma (GBM) and determine which chromosomes or chromosomal regions may play a role in the pathogenesis of GBM or may harbor tumor suppressor genes (TSGs) associated GBM. METHODS: An allelotype study of 21 cases of GBM was performed by polymerase chain reaction and loss of heterozygosity (LOH) analysis. Three hundred and eighty-two microsatellite markers covering all 22 autosomes were used. The mean genetic distance between two flanking markers is about 10 cM. Fluorescent dye-labeled primers and Perkin Elmer 377 DNA Sequencer were applied. RESULTS: LOH was observed on all chromosomal arms examined in this study. The LOH frequencies of 10q, 10p, 13q, 17p and 9p were the highest (>50%), on which high LOH frequencies were detected at the regions resided by the known TSGs including PTEN, DMBT1, p16, p15, p53 and Rb. The following commonly deleted regions were detected: 9p22-23, 10p12.2-14, 10q21.3, 13q12.1-14.1, 13q14.3-31, 17p11.2-12, 17p13, 3q24-27, 11p12-13, 14q31-32.3, 14q21-24.1, 22q13.2-13.3, 4q35, 4q31.1-31.2, 6qtel, 6q16.3. CONCLUSION: This study demonstrated that the pathogenesis of GBM is very complicated and associated with various molecular genetic abnormalities on lots of chromosomes. The chromosomal arms most closely relevant to the pathogenesis of GBM are 10q, 10p, 9p, 17p and 13q. Besides the well-known TSGs, such as PTEN, DMBT1, p16, p15, p53 and Rb, multiple unknown TSGs associated with GBM may be present on the commonly deleted regions observed for the first time in this study.

TGM2 inhibition attenuates ID1 expression in CD44-high glioma-initiating cells.

BACKGROUND: CD44 is a molecular marker associated with cancer stem cell populations and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting glioma cells high in CD44. METHODS: Glioma-initiating cell lines were derived from fresh surgical glioblastoma samples. Expression of tissue transglutaminase 2 (TGM2) was attenuated through lentivirus-mediated short hairpin RNA knockdown. MTT assay [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to evaluate the growth inhibition induced by TGM2 inhibitor. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling was used to evaluate cell apoptosis following TGM2 inhibition. CD44(+) glioma stem cells were sorted by flow cytometry. A nude mice orthotopic xenograft model was used to evaluate the in vivo effect of TGM2 inhibitor. RESULTS: TGM2 was highly expressed in CD44-high glioblastoma tissues and tumor-derived glioma-initiating cell lines. TGM2 knockdown impaired cell proliferation and induced apoptosis in CD44-high glioma-initiating cell lines. Further studies indicated that expression of inhibitor of DNA binding 1 protein (ID1) is regulated by TGM2 and might be an important mediator for TGM2-regulated cell proliferation in CD44-high glioma-initiating cell lines. TGM2 inhibitor reduces ID1 expression, suppresses cell proliferation, and induces apoptosis in CD44-high glioma-initiating cell lines. Furthermore, TGM2 is highly expressed in CD44(+) glioma stem cells, while pharmacological inhibition of TGM2 activity preferentially eliminates CD44(+) glioma stem cells. Consistently, TGM2 inhibitor treatment reduced ID1 expression and induced apoptosis in our orthotopic mice xenograft model, which can be translated into prolonged median survival in tumor-bearing mice. CONCLUSIONS: TGM2 regulates ID1 expression in glioma-initiating cell lines high in CD44. Targeting TGM2 could be an effective strategy to treat gliomas with high CD44 expression.

Impact of experimental acute hyponatremia on severe traumatic brain injury in rats: influences on injuries, permeability of blood-brain barrier, ultrastructural features, and aquaporin-4 expression.

The effects of acute hyponatremia on severe traumatic brain injury (TBI) in 35 adult male Sprague-Dawley rats were studied in a replicated focal and diffuse injury rat model. Such effects were assessed by the cerebral contusion volume and axonal injury (AI) densities, determined by quantitative immunoreactivity of beta-amyloid precursor protein, by blood-brain barrier (BBB) permeability based on endogenous IgG immunostaining, and by ultrastructural features. Significant increase of contusion volume (P < 0.05) and of AI in the segment of corpus callosum beneath the contusion (P < 0.05) and ipsilateral thalamus (P < 0.05) were observed at 4 h postinjury during the hyponatremic phase. No change in BBB permeability was observed in the hyponatremia + TBI (HT) groups. Significant swelling of perivascular astrocytic foot processes in the HT groups was seen at 4 h (P < 0.01) and 1 day postinjury (P < 0.01) by quantitative image analysis of ultrastructures. However, attenuated swelling of perivascular astrocytic foot processes in severely edematous medulla oblongata with simultaneous swelling of perikaryal astrocytic processes was observed in the HT 1-day group. The ultrastructural features were also correlated with the down-regulation of aquaporin-4 (AQP4) mRNA expression (P < 0.05). Results suggest that acute hyponatremia acts as one of the secondary insults following severe TBI. Such exacerbation may not be attributable to further disruption of BBB permeability, but rather to the ischemia resulting from the swelling of perivascular astrocytic foot processes impeding microcirculation. Down-regulated AQP4 mRNA expression may be one of the molecular mechanisms maintaining water homeostasis in diffusely injured brain exposed to acute hyponatremia.