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The micro-RNA 199b-5p regulatory circuit involves Hes1, CD15, and epigenetic modifications in medulloblastoma.

ABSTRACT: Micro-RNA (miR) 199b-5p targets Hes1 in medulloblastoma, one of the downstream effectors of both the canonical Notch and noncanonical Sonic Hedgehog pathways. In medulloblastoma patients, expression of miR-199b-5p is significantly decreased in metastatic cases, thus suggesting a downregulation mechanism. We studied this mechanism, which is mediated mostly by Hes1 and epigenetic promoter modifications. The miR-199b-5p promoter region was characterized, which identified a Hes1 binding site, thus demonstrating a negative feedback loop of regulation. MiR-199b-5p was shown to be downregulated in several medulloblastoma cell lines and in tumors by epigenetic methylation of a cytosine-phosphate-guanine island upstream of the miR-199b-5p promoter. Furthermore, the cluster of differention (CD) carbohydrate antigen CD15, a marker of medulloblastoma tumor-propagating cells, is an additional direct target of miR-199b-5p. Most importantly, regulation of miR-199b-5p expression in these CD15+/CD133+ tumor-propagating cells was influenced by only Hes1 expression and not by any epigenetic mechanism of regulation. Moreover, reverse-phase protein array analysis showed both the Akt and extracellular-signal-regulated kinase pathways as being mainly negatively regulated by miR-199b-5p expression in several medulloblastoma cell lines and in primary cell cultures. We present here the finely tuned regulation of miR-199b-5p in medulloblastoma, underlining its crucial role by its additional targeting of CD15.

SUBMITTER: Andolfo I

PROVIDER: S-EPMC3337299 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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The micro-RNA 199b-5p regulatory circuit involves Hes1, CD15, and epigenetic modifications in medulloblastoma.

Andolfo Immacolata I Liguori Lucia L De Antonellis Pasqualino P Cusanelli Emilio E Marinaro Federica F Pistollato Francesca F Garzia Livia L De Vita Gennaro G Petrosino Giuseppe G Accordi Benedetta B Migliorati Roberta R Basso Giuseppe G Iolascon Achille A Cinalli Giuseppe G Zollo Massimo M

Neuro-oncology 20120312 5

Micro-RNA (miR) 199b-5p targets Hes1 in medulloblastoma, one of the downstream effectors of both the canonical Notch and noncanonical Sonic Hedgehog pathways. In medulloblastoma patients, expression of miR-199b-5p is significantly decreased in metastatic cases, thus suggesting a downregulation mechanism. We studied this mechanism, which is mediated mostly by Hes1 and epigenetic promoter modifications. The miR-199b-5p promoter region was characterized, which identified a Hes1 binding site, thus d ...[more]

PMID: 22411914

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Project description:BackgroundThrough negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3-4 years and 8-9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies.Methodology/principal findingsIn a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept.Conclusions/significanceDespite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

Project description:Epithelial ovarian cancer is a highly lethal and aggressive gynecological malignancy. The high mortality rate is due in part to the fact that many advanced cancer patients become refractory to current chemotherapeutic agents, leading to tumor recurrence and death. However, the underlying mechanisms leading to chemoresistance remain obscure. Here, we report that the loss of miR-199b-5p due to progressive epigenetic silencing leads to the activation of the JAG1-mediated Notch1 signaling cascade, thereby leading to the development of acquired chemoresistance in ovarian cancer. Using miRCURY LNA™ microRNA array and Q-PCR analyses of two pairs of cisplatin-sensitive and -resistant ovarian cancer cell lines, we identified miR-199b-5p as significantly down-regulated in cisplatin-resistant ovarian cancer cells and confirmed that miR-199b-5p is clinically associated with advanced and poor survival ovarian cancers. Interestingly, the loss of miR-199b-5p could be restored by 5-Aza-dC-mediated demethylation, and methylated specific PCR (MS-PCR), bisulfite-sequencing and pyrosequencing revealed that the promoter region of miR-199b-5p was hypermethylated. Computational and mechanistic analyses identified JAG1 as a primary target of miR-199b-5p. Notably, the reduced expression of miR-199b-5p was found to be inversely correlated with the increased expression of JAG1 using an ovarian cancer tissue array. Enforced expression of miR-199b-5p sensitized ovarian cancer cells to cisplatin-induced cytotoxicity both in vitro and in vivo. Conversely, re-expression of miR-199b-5p and siRNA-mediated JAG1 knockdown or treatment with Notch specific inhibitor ?-secretase (GSI) attenuated JAG1-Notch1 signaling activity, thereby enhancing cisplatin-mediated cell cytotoxicity. Taken together, our study suggests that the epigenetic silencing of miR-199b-5p during tumor progression is significantly associated with acquired chemoresistance in ovarian cancer through the activation of JAG1-Notch1 signaling.

Project description:Objective Animal models of post-traumatic osteoarthritis (PTOA) recapitulate the pathological changes observed in human PTOA. Here we aimed to compare the cartilage transcriptome responses of a non-surgical, mechanically induced rupture of the anterior cruciate ligament (ACL) model and the surgical destabilisation of the medial meniscus (DMM) model. Methods Skeletally mature male C57Bl6 mice were subjected to either the non-surgical, mechanical ACL rupture or surgical DMM models and transcriptome profiling performed on micro-dissected cartilage at day 7 and 42 post-procedure, respectively; in general, naïve animals served as controls. MicroRNA profiling was also performed on the ACL rupture model. Expression levels of a miRNA of interest, miR-199-5p, were inhibited in primary human articular chondrocytes (HAC) with RNA-seq and 3’UTR assays used to identify and valid potential target genes. Results The number of differentially expressed genes between the two models were comparable and highly correlative (Spearman R =0.8, P<2.2E-16). Gene ontology enrichment analysis identified similarly enriched pathways, containing anabolic terms including ‘extracellular matrix organisation’ enriched for the upregulated genes. Within the ACL rupture miRNA transcriptome, miR-199-5p family members were amongst the most abundantly, and differentially expressed, which was replicated in the DMM cartilage by qRT-PCR. Inhibition of miR-199-5p in HAC led to a comparable transcriptome response to that observed in both human OA damaged vs intact cartilage and murine DMM cartilage datasets. Several genes, including GIT1, NCEH1, SOS2 and ECE1 were all experimentally verified as targets. Conclusion For the first time, we have characterised both the mRNA and miRNA articular cartilage signature in the ACL rupture model and demonstrated highly correlative responses with the DMM PTOA model. These data support the use of the ACL rupture model as a non-invasive alternative to DMM.

Dataset Information

The micro-RNA 199b-5p regulatory circuit involves Hes1, CD15, and epigenetic modifications in medulloblastoma.

Publications

The micro-RNA 199b-5p regulatory circuit involves Hes1, CD15, and epigenetic modifications in medulloblastoma.

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