RESUMO
Transcriptional factor 3 (TCF3, also termed E2A), first reported to exert crucial functions during lymphocyte development, has been revealed to participate in the pathogenesis of human cancers. The aim of this work was to investigate the function of TCF3 in cervical cancer (CC) and the molecular interactions. The bioinformatics prediction suggested that TCF3 was highly expressed in CC and linked to poor prognosis. Increased TCF3 expression was identified in CC cell lines, and its downregulation reduced proliferation and migration of CC cells in vitro as well as growth of xenograft tumors in vivo. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that the TCF-3-related genes and genes showed differential expression between CC and normal tissues were mainly enriched in the Wnt/ß-catenin pathway. TCF3 bound to sirtuin 1 (SIRT1) promoter for transcriptional activation, and SIRT1 promoted deacetylation and nuclear translocation of ß-catenin in CC. SIRT1 overexpression blocked the role of TCF3 silencing and restored cell proliferation in vitro and tumor growth in vivo. Treatment with XAV-939, a ß-catenin inhibitor, significantly suppressed the cell proliferation and tumor growth induced by SIRT1 overexpression. In conclusion, this study demonstrates that TCF3 augments progression of CC by activating SIRT1-mediated ß-catenin signaling.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sirtuína 1/metabolismo , Neoplasias do Colo do Útero , beta Catenina , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Humanos , Sirtuína 1/genética , Fator 3 de Transcrição/metabolismo , Neoplasias do Colo do Útero/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
OBJECTIVE: To efficiently construct a replication-defective recombinant adenoviral vector using a two-step transformation procedure. METHODS: Plasmid pAdEasy-1 was linearized and transformed into E.coli BJ5183 to construct BJ5183pAdEasy-1 cells. Cytosine deaminase (CD) gene was obtained from plasmid pBS-CD, and subcloned into the shuttle plasmid to form transfer plamid of pAdtrackCMV-CD, which was then linearized and transformed into BJ5183pAdEasy-1 cells. The recombinant adenovirus plasmid DNA was extracted from the transformed bacteria and digested with Pac I after identification, followed by transfection into 293 packaging cells. PCR was used to detect target gene, and the titer and the infection rate of the recombinant Ad were measured with the aid of green fluorescent protein (GFP) reporter gene. The same recombinant AdCMV-CD was constructed in a one-step transformation method for comparison. RESULTS: The homologous recombination of the two-step transformation method resulted in a success rate of 76.5% (13/17), while the success rate of the one-step method was only 11.8% (2/17), showing significant difference between the two methods (P=0.000 17 by Fisher's exact test). CONCLUSION: The two-step transformation procedure is more efficient and convenient than one-step method.
Assuntos
Adenoviridae/genética , Citosina Desaminase/genética , Vírus Defeituosos/genética , Vetores Genéticos/genética , Transformação Genética , Terapia Genética , Recombinação GenéticaRESUMO
OBJECTIVE: To efficiently construct the recombinant adenovirus containing CD/TK fusion gene driven by vascular endothelial growth factor (VEGF) promoter using improved homologous recombination in bacteria. METHODS: Chemical transformation, instead of electroporation, of the plasmid pAdEasy-1 into E.coli BJ5183 strain was performed to prepare BJ5183pAdEasy-1 as the competent bacterium, in which pAdEasy-1 and pAdtrack-VEGFP-CDglyTK were recombined. Finally, the recombinant adenovirus of AdVEGFP-CDglyTK was constructed by transfecting 293 cells with linearized adenoviral genomes of pAdEasy-VEGFP-CDglyTK. RESULTS: This new transformation procedure generated recombinant plasmids in a yield of 60% (6/10), and the adenovirus AdVEGFP-CDglyTK was harvested 7-12 d after transfection. CONCLUSION: The improved homologous recombination in bacteria is efficient, convenient and easy to be carried out.
Assuntos
Citosina Desaminase/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Timidina Quinase/genética , Fator A de Crescimento do Endotélio Vascular/genética , Adenoviridae/genética , Plasmídeos , Recombinação Genética , TransfecçãoRESUMO
OBJECTIVE: To construct recombinant adenovirus containing double suicide gene driven by cytomegalovirus (CMV) promoter. METHODS: CD and TK gene were amplified by PCR and cloned into the shuttle vector pAdtrack CMV, and the resultant plasmid pAdtrack CMV-CDTK was linearized with PmeI and transformed into competent AdEasier-1 cells prepared by CaCl2 method. PacI digestion of identified recombinant plasmid DNA was performed before it was transected into 293 cells to package adenovirus, which was used to infect endothelial cells in vitro after adenovirus titer determination. RESULTS: Chemical transformation of linearized transfer plasmid into AdEasier-1 cells resulted in very high ratio (20/20) of positive clones of recombinant adenovirus containing. CD and TK fusion gene as confirmed by PCR test. CONCLUSION: The modified AdEasy system is more convenient and efficient for constructing recombinant adenovirus, which may facilitate further study of anti-tumor therapy targeting at the double suicide gene.
Assuntos
Adenoviridae/genética , Citomegalovirus/genética , Citosina Desaminase/genética , Terapia Genética/métodos , Regiões Promotoras Genéticas , Timidina Quinase/genética , Transformação Genética , Cloreto de Cálcio/farmacologia , Recombinação GenéticaRESUMO
To simplify AdEasy system for better efficiency, we attempted some modifications on the original system. Specifically, the Pme -linearized plasmid pAdtrack was transformed into competent AdEasier-1 cells prepared from treatment with CaCl(2). The DNA contained in the identified recombinant plasmid was digested with Pac and transfected into 293 cells to package the adenovirus, followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescence microscope. Chemical transformation of the linearized plasmid into AdEasier-1 cells resulted in very high success rate (20/20) for producing positive recombinant adenovirus clones, confirming the efficiency of the modified AdEasy system in constructing recombinant adenovirus.
Assuntos
Adenoviridae/genética , Vetores Genéticos , Plasmídeos/genética , Recombinação Genética , Humanos , TransfecçãoRESUMO
AIM: To study the effect of TNP-470 on cell growth, proliferation and apoptosis in human colon cancer xenografts in nude mice. METHODS: Human colon cancer xenografts were transplanted into 20 nude mice. Mice were randomly divided into two groups. TNP-470 treated group received TNP-470(30 mg/kg, s.c) every other day and the control group received a sham injection of same volume saline solution. They were sacrificed after 4 weeks and their tumors were processed for histological examination. The expression of proliferating cell nuclear antigen (PCNA) in tumors was detected using immunohistochemical method with image analysis, and apoptosis in tumor cells was measured by TdT-mediated biotinyated-dUTP nick end labeling (TUNEL) staining. RESULTS: Comparing with controls, tumor growth was significantly inhibited in TNP-470 treated group, the inhibitory rate being 54.4 %. Expression of PCNA in tumors of TNP-470 treated group (PI 54.32+/-11.47) was significantly lower than that of control group (PI 88.54+/-12.36), P<0.01. Apoptosis index (AI) of TNP-470 treated group (18.95+/-1.71) was significantly higher than that of control group (7.26+/-1.44), P<0.001, typical morphological change of apoptosis in tumor cells was observed in TNP-470 treated group. CONCLUSION: Besides the anti-angiogenic effects, TNP-470 can inhibit tumor growth by inhibiting the proliferation and inducing apoptosis of tumor cells.