RESUMO
BACKGROUND: Because of increased resistance to apoptosis in tumor cells, inhibition of specific anti-apoptotic factors may provide a rational approach for the development of novel therapeutic strategies. Livin, a novel inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poorly prognostic significance. However, no data are available concerning the significance of livin in gastric cancer. In this study, we detected the expression of livin in human gastric carcinoma and investigated the apoptotic susceptibility of SGC - 7901 cell by shRNA-mediated silencing of the livin gene. METHODS: The mRNA and protein expression of livin were analyzed by RT-PCR and western blot assay. The relationship between livin expression and clinical pathologic parameters was investigated. The small interfering RNA eukaryotic expression vector specific to livin was constructed by gene recombination, and the nucleic acid was sequenced. Then it was transfected into SGC-7901 cells by Lipofectamin 2000. RT-PCR and Western blot assay were used to validate gene-silencing efficiency of livin in SGC-7901 cells. Stable clones were obtained by G418 screening. The cell apoptosis was assessed by flow cytometry (FCM). Cell growth state and 50 % inhibition concentration (IC50) of 5-FU and cisplatin was determined by MTT method. RESULTS: The expression of livin mRNA and protein were detected in 19 of 40 gastric carcinoma cases (47.5%) and SGC-7901 cells. No expression of livin was detected in tumor adjacent tissues and benign gastric lesion. The positive correlation was found between livin expression and poor differentiation of tumors as well as lymph node metastases (P<0.05). Four small interfering RNA eukaryotic expression vector specific to livin were constructed by gene recombination. And one of them can efficiently decrease the expression of livin, the inhibition of the gene was not less than 70% (P<0.01). The recombinated plasmids were extracted and transfected gastric cancer cells. The stable clones were obtained by G418 screening, and were amplified and cultured. When livin gene was silenced, the reproductive activity of the gastric cancer cells was significantly lower than the control groups(P<0.05). The study also showed that IC50 of 5-Fu and cisplatin on gastric cancer cells treated by shRNA was decreased and the cells were more susceptible to proapoptotic stimuli (5-Fu and cisplatin) (P<0.01). CONCLUSIONS: Livin is overexpressed in gastric carcinoma with a relationship to tumor differentiation and lymph node metastases, which is suggested to be one of the molecular prognostic factors for some cases of gastric cancer. ShRNA can inhibit livin expression in SGC-7901 cells and induce cell apoptosis. Livin may serve as a new target for apoptosis-inducing therapy of gastric cancer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Proteínas Inibidoras de Apoptose/genética , Proteínas de Neoplasias/genética , Neoplasias Gástricas/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/genética , Adulto , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/genética , Western Blotting , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/farmacologia , Feminino , Citometria de Fluxo , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Concentração Inibidora 50 , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Proteolytic cleavages play an important role in reovirus infection during entry into cells. The effects of protease digestion on the morphology, infectivity and polypeptide composition of grass carp reovirus (GCRV) were investigated. Following treatment with chymotrypsin, the different subviral particles of GCRV were isolated using density gradient centrifugation and examined by electron microscope (EM). Analysis of protein components revealed that the viral outer capsid was composed of VP5 and VP7. Of particular note, VP5 was found to primarily exist within virions as cleaved fragments, which was consistent with observations for its analogue mu1/mu1C, generated by autolysis of mu1 at the mu1N/mu1C junction for mammalian orthoreoviruses (MRVs). Meanwhile, both trypsin- and chymotrypsin-treated GCRV particles appeared to have an enhanced infectivity. Moreover, the corresponding assays between infectivity and protein component indicated that the enhancement of infectivity was correlated to the complete digestion of the outer capsid protein VP7 and partial cleavage of VP5. Overall, the results presented in this paper provided strong evidence that the proteins VP5 and VP7 of GCRV play an indispensable role in viral infection.