RESUMO
In hepatocellular carcinoma (HCC), tumor specificity of gene therapy is of utmost importance to preserve liver function. MicroRNAs (miRNAs) are powerful negative regulators of gene expression and many are downregulated in human HCC. We identified seven miRNAs that are also downregulated in tumors in a rat hepatoma model (P<0.05) and attempted to improve tumor specificity by constructing a panel of luciferase-expressing vectors containing binding sites for these miRNAs. Attenuation of luciferase expression by the corresponding miRNAs was confirmed across various cell lines and in mouse liver. We then tested our vectors in tumor-bearing rats and identified two miRNAs, miR-26a and miR-122, that significantly decreased expression in liver compared with the control vector (6.40 and 0.26%, respectively; P<0.05). In tumor, miR-122 had a nonsignificant trend towards decreased (â¼50%) expression, whereas miR-26 had no significant effect on tumor expression. To our knowledge, this is the first work using differentially expressed miRNAs to de-target transgene expression in an orthotopic hepatoma model and to identify miR-26a, in addition to miR-122, for de-targeting liver. Considering the heterogeneity of miRNA expression in human HCC, this information will be important in guiding development of more personalized vectors for the treatment of this devastating disease.
Assuntos
Carcinoma Hepatocelular/genética , Vetores Genéticos , Neoplasias Hepáticas/genética , MicroRNAs/genética , Animais , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética/métodos , Células HEK293 , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas Experimentais , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Ratos , Ratos Endogâmicos BUF , TransgenesRESUMO
Ideal cancer gene therapies should have high tumor specificity and efficacy, and allow systemic administration to target metastases. We recently developed a bi-directional, two-step transcriptional amplification (TSTA) system driven by the tumor-specific Survivin promoter (pSurv) to amplify the correlated expression of both the reporter gene firefly luciferase (FL) and therapeutic gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we compare the specificity and potency of an adenovirus carrying this system (Ad-pSurv-TSTA-TRAIL-FL) to a nonspecific vector (Ad-pCMV-FL) in an orthotopic hepatocellular carcinoma (HCC) rat model after systemic administration. At 24 h after injection of Ad-pCMV-FL, bioluminescence imaging revealed a trend (P=0.30) towards greater FL expression in liver versus tumor. In striking contrast, Ad-pSurv-TSTA-TRAIL-FL showed increased FL activity within the tumor compared with the liver (P<0.01), a strong trend towards reduced liver expression compared with Ad-pCMV-FL (P=0.07), and importantly, similar FL levels within tumor compared with Ad-pCMV-FL (P=0.32). Hence, this vector shows potent, tumor-specific transgene expression even after extensive liver transduction and may be of significant value in avoiding hepatotoxicity in HCC patients. Future studies will explore the benefits of tumor-specific TRAIL expression in this model, the potential to target metastases and the extension of this vector for the treatment of other Survivin-positive tumors is warranted.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas Associadas aos Microtúbulos/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Adenoviridae/genética , Animais , Expressão Gênica , Marcação de Genes , Genes Reporter , Vetores Genéticos , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Regiões Promotoras Genéticas , Ratos , Sensibilidade e Especificidade , Survivina , TransgenesRESUMO
INTRODUCTION: In liver transplant recipients with Epstein-Barr virus (EBV) disease, we reported a low rate of acute rejection after stopping or markedly lowering immunosuppression. This observation led to the hypothesis that EBV, as a means of viral persistence, induces expression of antiapoptotic factors and these factors, in turn, confer protection to the transplanted organ. Bcl-2, an antiapoptotic factor induced by EBV in various host cells, is not normally expressed in the liver. We questioned whether bcl-2 is expressed in the transplanted liver and whether its expression is modified by EBV. MATERIALS AND METHODS: Retrospective liver biopsy specimen from liver transplant patients diagnosed with EBV (n=12) were examined for the presence of bcl-2 by immunohistochemistry and compared with EBV (-) transplant (n=15), and nontransplant (n=13) livers. RESULTS: The most significant finding was the presence of endothelial bcl-2 expression in the majority of EBV (+) transplant samples examined (67%) and its relative absence in the other two groups (P<0.005). There was also bcl-2 expression in the hepatocytes and lymphocytes of the majority of transplant liver samples, irrespective of EBV status. DISCUSSION: We have identified a strong association between EBV infection and endothelial bcl-2 expression in transplant livers. We also found that transplantation, in itself, was associated with bcl-2 expression in the hepatocytes and lymphocytes of liver allografts.