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Inibizione selettiva del gene MYCN mediante PNA (acidi peptido nucleici) anti-gene nel rabdomiosarcoma umano

By Stefania Purgato

Abstract

MYCN oncogene amplification/expression is a feature of many childhood tumors, and some adult\ud tumors, and it is associated with poor prognosis. While MYC expression is ubiquitary, MYCN has a\ud restricted expression after birth and it is an ideal target for an effective therapy. PNAs belong to the\ud latest class of nucleic acid-based therapeutics, and they can bind chromosomal DNA and block gene\ud transcription (anti-gene activity). We have developed an anti-gene PNA that targets specifically the\ud MYCN gene to block its transcription. We report for the first time MYCN targeted inhibition in\ud Rhabdomyosarcoma (RMS) by the anti-MYCN-PNA in RMS cell lines (four ARMS and four\ud ERMS) and in a xenograft RMS mouse model. Rhabdomyosarcoma is the most common pediatric\ud soft-tissue sarcoma, comprising two main subgroups [Alveolar (ARMS) and Embryonal (ERMS)].\ud ARMS is associated with a poorer prognosis. MYCN amplification is a feature of both the ERMS\ud and ARMS, but the MYCN amplification and expression levels shows a significant correlation and\ud are greater in ARMS, in which they are associated with adverse outcome.\ud We found that MYCN mRNA and protein levels were higher in the four ARMS (RH30, RH4, RH28\ud and RMZ-RC2) than in the four ERMS (RH36, SMS-CTR, CCA and RD) cell lines. The potent\ud inhibition of MYCN transcription was highly specific, it did not affect the MYC expression, it was\ud followed by cell-growth inhibition in the RMS cell lines which correlated with the MYCN\ud expression rate, and it led to complete cell-growth inhibition in ARMS cells. We used a mutated-\ud PNA as control. MYCN silencing induced apoptosis. Global gene expression analysis (Affymetrix\ud microarrays) in ARMS cells treated with the anti-MYCN-PNA revealed genes specifically induced\ud or repressed, with both genes previously described as targets of N-myc or Myc, and new genes\ud undescribed as targets of N-myc or Myc (mainly involved in cell cycle, apoptosis, cell motility,\ud metastasis, angiogenesis and muscle development). The changes in the expression of the most\ud relevant genes were confirmed by Real-Time PCR and western blot, and their expression after the\ud MYCN silencing was evaluated in the other RMS cell lines. The in vivo study, using an ARMS\ud xenograft murine model evaluated by micro-PET, showed a complete elimination of the metabolic\ud tumor signal in most of the cases (70%) after anti-MYCN-PNA treatment (without toxicity),\ud whereas treatment with the mutated-PNA had no effect.\ud Our results strongly support the development of MYCN anti-gene therapy for the treatment of RMS,\ud particularly for poor prognosis ARMS, and of other MYCN-expressing tumors

Topics: BIO/14 Farmacologia
Year: 2008
DOI identifier: 10.6092/unibo
OAI identifier: oai:amsdottorato.cib.unibo.it:690
Provided by: AMS Tesi di Dottorato
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