Publicação
The impact of Lama2-deficiency on cell cycle regulation and survival
| Resumo: | Laminin α2 chain-deficient congenital muscular dystrophy (LAMA2-CMD) is caused by recessive mutations in the LAMA2 gene. This neuromuscular disease is diagnosed at birth or within the first few months of life and is characterized by hypotonia and severe muscle weakness. Using the dyW mouse model of LAMA2-CMD, previous studies by the host laboratory showed that the onset of this muscular dystrophy occurs in utero, more specifically between embryonic days 17.5 and 18.5 (E17.5- E18.5), where it is observed a reduction in the size of muscle fibers and lower expression of the myogenic factors Pax7 and Myogenin. These alterations may be related to changes in cell cycle and cell survival regulation, as revealed by the overactivation of the transcription factor STAT3 and increased expression of the respective target gene Pim1. To investigate how Lama2-deficiency impacts on cell cycle regulation and survival, we used RT-qPCR and Western Blot to analyze the expression of genes and proteins involved in different cell fate pathways, comparing epaxial muscles of wildtype (wt) and dyW mice at E17.5 and E18.5. We also generated C2C12 myoblast cell lines deficient for Lama2 and for both Lama2 and p53, a master transcription regulator of cell cycle and survival. Using these in vitro models, pathways linked to proliferation/cell cycle were analyzed by RT-qPCR, immunofluorescence and Resazurin assay. Our results suggest a difference in genes linked to cell cycle regulation, proliferation, autophagy and senescence. C2C12 Lama2 knockouts revealed a decrease in proliferation, possibly explained by cell cycle arrest at G1/G0 phase, which was partially rescued by the co-deletion of Lama2 and p53. This work highlighted alterations in terms of cell fate regulation, involved in LAMA CMD onset, and resulted in the generation of an in vitro model, both important to gain further mechanistic insights about this disorder and to develop efficient therapies. |
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| Autores principais: | Melo, Catarina Elisa Mineiro de |
| Assunto: | Distrofia muscular congénita deficiente na cadeia α2 da laminina Músculo esquelético Ciclo celular e sobrevivência CRISPR Proliferação Teses de mestrado - 2022 |
| Ano: | 2022 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Laminin α2 chain-deficient congenital muscular dystrophy (LAMA2-CMD) is caused by recessive mutations in the LAMA2 gene. This neuromuscular disease is diagnosed at birth or within the first few months of life and is characterized by hypotonia and severe muscle weakness. Using the dyW mouse model of LAMA2-CMD, previous studies by the host laboratory showed that the onset of this muscular dystrophy occurs in utero, more specifically between embryonic days 17.5 and 18.5 (E17.5- E18.5), where it is observed a reduction in the size of muscle fibers and lower expression of the myogenic factors Pax7 and Myogenin. These alterations may be related to changes in cell cycle and cell survival regulation, as revealed by the overactivation of the transcription factor STAT3 and increased expression of the respective target gene Pim1. To investigate how Lama2-deficiency impacts on cell cycle regulation and survival, we used RT-qPCR and Western Blot to analyze the expression of genes and proteins involved in different cell fate pathways, comparing epaxial muscles of wildtype (wt) and dyW mice at E17.5 and E18.5. We also generated C2C12 myoblast cell lines deficient for Lama2 and for both Lama2 and p53, a master transcription regulator of cell cycle and survival. Using these in vitro models, pathways linked to proliferation/cell cycle were analyzed by RT-qPCR, immunofluorescence and Resazurin assay. Our results suggest a difference in genes linked to cell cycle regulation, proliferation, autophagy and senescence. C2C12 Lama2 knockouts revealed a decrease in proliferation, possibly explained by cell cycle arrest at G1/G0 phase, which was partially rescued by the co-deletion of Lama2 and p53. This work highlighted alterations in terms of cell fate regulation, involved in LAMA CMD onset, and resulted in the generation of an in vitro model, both important to gain further mechanistic insights about this disorder and to develop efficient therapies. |
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