Down-regulation of PAX3 gene expression in rhabdomyosarcoma and melanoma

Mashud, Ahmed Abdallah (2014) Down-regulation of PAX3 gene expression in rhabdomyosarcoma and melanoma. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

The PAX3 gene as a member of the paired homeodomain family of transcription factors plays a crucial role during embryonal development by regulating the early development of neural structures, derivatives of the neural crest and skeletal muscles. Following embryonal development, the PAX3 expression is switched off. Mutations in the PAX3 gene are commonly associated with Waardenburg’s syndrome and in Craniofacial-hand syndrome. Aberrant re-expression of PAX3 after embryogenesis plays a key role in the onset, growth, survival and progression of rhabdomyosarcoma, melanoma and neuroblastoma. Alternative splicing of PAX3 results in seven transcript variants (PAX3a, PAX3b, PAX3c, PAX3d, PAX3e, PAX3g and PAX3h), the interactions of which with other downstream targets, make it difficult for manipulation and the development of potent chemotherapeutic regimens to effectively treat malignant tumours including rhabdomyosarcoma, melanoma and neuroblastoma which have unfavourable prognostic outcomes. This research was aimed at down-regulating PAX3 gene expression in human rhabdomyosarcoma and melanoma cell lines, subsequently identifying the downstream target genes of PAX3 and determining the effects on cell growth and survival. The expression of PAX3 in human rhabdomyosarcoma and human melanoma cell lines was significantly down-regulated using novel pre-designed PAX3 small interference RNA molecules, at a final concentration of 0.5μM in an in vitro transient transfection. The three prime Affymetrix microarray analyses showed more than a four-fold and a two-fold down-regulation of PAX3 gene expression in the human JR1 embryonal rhabdomyosarcoma and RH30 alveolar rhabdomyosarcoma cell lines respectively, whilst in the human A375 melanoma cell line, over an eight-fold down-regulation of PAX3 expression was demonstrated relative to negative control cells. A quantitative RT-PCR analysis, which was used in validating results of the Affymetrix array, confirmed the knockdown of PAX3 in both human rhabdomyosarcoma and melanoma cell lines. A semi-quantitative RT-PCR analysis of gene expression revealed at least 90% down-regulation of all PAX3 variant expression in JR1, RH30 and A375 cell lines relative to negative controls cells. Higher levels of gene silencing were observed in the JR1 cell line than in either RH30 or A375 cell lines. Western blotting analysis, which quantified the level of PAX3 gene knockdown, indicated a 98%, 92% and 90% reduction of PAX3 protein in JR1, RH30 and A375 cell lines respectively. This down-regulation of PAX3 expression significantly inhibited tumour cell growth, proliferation, migration, adhesion, invasion, and induced apoptosis of JR1, RH30 and A375 cell lines in vitro. These results were explainable by the particular genes that were up- or down-regulated by PAX3, which were correlated with the microarray results and the quantitative RT-PCR experiments. The expression of PAX3 gene has been previously demonstrated to promote tumourigenesis of rhabdomyosarcoma and melanoma. Results of this present study suggest that down-regulation of PAX3 might inhibit the progression of rhabdomyosarcoma and melanoma and PAX3 thus could be a suitable target for the development of potent chemotherapy. Silencing of PAX3 in these cell lines resulted in the alteration of expression of a host of downstream target genes, which PAX3 uses in the modulation of cellular activities, including cell growth, proliferation, migration, adhesion, metastatic invasion and apoptosis of the rhabdomyosarcoma and melanoma cell lines.