RESUMO
Myostatin, a member of the TGF-beta superfamily, is a potent negative regulator of skeletal muscle and growth. Previously, we reported Mstn1 from zebrafish and studied its influence on muscle development. In this study, we identified another form of Myostatin protein which is referred to as Mstn2. The size of Mstn2 cDNA is 1342 bp with 109 and 132 bp of 5' and 3'-untranslated regions (UTRs), respectively. The coding region is 1101 bp encoding 367 amino acids. The identity between zebrafish Mstn1 and 2 is 66%. The phylogenetic tree revealed that the Mstn2 is an ancestral form of Mstn1. To study the functional aspects, we overexpressed mstn2 and noticed that embryos became less active and the juveniles with bent and curved phenotypes when compared to the control. The RT-PCR and in situ hybridization showed concurrent reduction of dystrophin associated protein complex (DAPC). In cryosection and in situ hybridization, we observed the disintegration of somites, lack of transverse myoseptum and loss of muscle integrity due to the failure of muscle attachment in mstn2 overexpressed embryos. Immunohistochemistry and western blot showed that there was a reduction of dystrophin, dystroglycan and sarcoglycan at translational level in overexpressed embryos. Taken together, these results indicate the suitability of zebrafish as an excellent animal model and our data provide the first in vivo evidence of muscle attachment failure by the overexpression of mstn2 and it leads to muscle loss which results in muscle dystrophy that may contribute to Duchenne syndrome and other muscle related diseases.
Assuntos
Regulação para Baixo/genética , Complexo de Proteínas Associadas Distrofina/genética , Distrofina/genética , Distrofia Muscular Animal/etiologia , Miostatina/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Distroglicanas/genética , Embrião não Mamífero , Músculo Esquelético/fisiopatologia , Miostatina/genética , Fenótipo , Sarcoglicanas/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Hepatocellular carcinoma (HCC) is one of the common cancers worldwide, caused by Hepatitis C virus (HCV) and hepatotoxins. Here we report the development of HCC in wild type as well as HCV core protein (HCP)-transgenic zebrafish upon treatment with a hepatotoxin, thioacetamide (TAA). Two-fold accelerated HCC development could be achieved in the TAA-treated transgenic fish, that is, the progression of the disease in TAA-treated wild type zebrafish developed HCC in 12 weeks whereas that of HCP-transgenic zebrafish shortened the HCC progression to 6 weeks. Histopathological observation showed the specific pathological features of HCC. The HCC progression was confirmed through RT-PCR that revealed an up and down regulation of different marker genes at various stages of HCC progression such as, steatohepatitis, fibrosis and HCC. Moreover, HCV core protein expressed in the HCP-transgenic zebrafish and TAA synergistically accelerate the HCC development. It must be mentioned that, this is the first report revealing HCV core protein along with TAA to induce HCC in zebrafish, particularly, in a short period of time comparing to mice model. As zebrafish has already been considered as a good human disease model and in this context, this HCC-zebrafish model may serve as a powerful preclinical platform to study the molecular events in hepatocarcinogenesis, therapeutic strategies and for evaluating chemoprevention strategies in HCC.
Assuntos
Hepacivirus/genética , Hepatopatias , Tioacetamida/toxicidade , Proteínas do Core Viral/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Doença Hepática Induzida por Substâncias e Drogas , Primers do DNA , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Fígado Gorduroso/virologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/virologia , Hepatopatias/genética , Hepatopatias/virologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Steatohepatitis has recently been increasing as a cofactor influencing the progression of fibrosis, cirrhosis, adenoma and carcinoma in liver; however, the mechanisms by which it contributes to liver injury remain uncertain. We induced steatohepatitis in zebrafish embryos using thioacetamide (TAA). TUNEL assay revealed significant increasing of apoptosis in liver after 5 days post fertilization and the increasing of apoptosis was observed to be associated with the up-regulation of apoptotic genes such as, bad, bax, P-38a, caspase-3 and 8, and JNK-1. Histological sections by oil red O stain showed the accumulation of fatty droplets which causes the pushing of the nucleus towards one side. Up-regulation of steatosis markers such as, ACC, adiponectin, PTL, CEBP- alpha and beta, SREBP-1 was also observed. Furthermore, the elevation of glutathione peroxidase in TAA treated embryos indicated that TAA induces lipid peroxidation which leads to causes liver damage. Zebrafish has already been considered as a good human disease model and in this context; TAA-treated zebrafish may serve as a good animal model to study the molecular pathogenesis of steatohepatitis. Moreover, non-availability of specific drugs to prevent steatohepatitis, this animal model may serve as a powerful preclinical platform to study the therapeutic strategies and for evaluating chemoprevention strategies for this disease.