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1.
Gene ; 725: 144159, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31629818

RESUMO

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide due to its frequent metastasis, tumor recurrence, and lack of curative treatment. However, the underlying molecular mechanisms involved in HCC progression remain unclear. Here, we analyzed the global gene expression of spontaneous liver tumor tissue from CBA/CaJ mice by RNA-Seq and identified 10,706 and 10,374 genes in the normal and liver tumor groups, respectively. Only 9793 genes were expressed in both, 913 genes were identified in only the liver tumor group, and 581 genes were found in normal liver tissues. There were 2054 differentially expressed genes (DEGs), with 975 down-regulated genes and 1079 up-regulated genes. Gene ontology (GO) term enrichment analysis showed that 43 up-regulated genes were significantly associated with cell cycle regulation and hundreds of up-regulated genes were related to cell migration, adhesion, or metabolic processes. KEGG pathway enrichment also demonstrated that some DEGs were tightly associated with the cell cycle, extracellular matrix (ECM)-receptor interactions, as well as protein digestion and absorption pathways, indicating that the activation of these oncogenic cascades was closely related to tumor liver progression in CBA/CaJ mice. Ninety-three genes with elevated expression levels preferentially localized in microtubules, kinetochores, and spindles play an important role during mitosis and meiosis and are associated with the reorganization of the cytoskeleton in cancer cells during migration and invasion. Some ECM-related genes were significantly different in the tumor group, including collagen types I, III, IV, V, and VI, non-collagenous glycoproteins, laminin, and fibronectin. We further validated the functions of upregulated genes, such as cyclin-dependent kinase 1 (CDK1) and polo-like kinase 1 (PLK1), with regards to cell cycle regulation, apoptosis, and proliferation in normal human liver or liver tumor-derived cell lines. Our results indicated that the cell cycle dysregulation, ECM-receptor interaction, and cytoskeleton-associated genes in mouse livers may promote HCC progression and deciphering the function of the genes will help investigators understand the underlying molecular mechanism of HCC.


Assuntos
Neoplasias Hepáticas Experimentais/genética , Animais , Apoptose/genética , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos CBA , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transcrição Genética , Transcriptoma
2.
J Gastroenterol Hepatol ; 34(12): 2196-2205, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30995348

RESUMO

BACKGROUND AND AIM: The high mortality and poor prognosis of hepatocellular carcinoma (HCC) have raised the public attention. Gene therapy is considered as a promising treatment option for cancer; thus, finding a new therapeutic target for HCC is urgently needed. GATA4 is a tumor suppressor gene in multiple cancers, but its role in HCC is unclear. In this study, we explored the function of GATA4 in HCC. METHODS: Reverse transcription-polymerase chain reaction and quantitative polymerase chain reaction were used to detect the mRNA expression of GATA4 in HCC cells and tissues. Cell viability, transwell, colony formation, and flow cytometry assays were applied to examine different aspects of biological effects of GATA4 in vitro. Xenografts, immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling assays were performed to evaluate the effect of GATA4 on tumorigenicity in vivo. Western blotting, immunofluorescence, and ß-galactosidase staining were used to investigate the mechanism underlying the function of GATA4. RESULTS: We found that GATA4 was silenced in 15/19 (79%) HCC tissues. Restoring the expression of GATA4 induced G0 /G1 phase arrest, promoted apoptosis, suppressed HCC proliferation in vitro, and inhibited HCC tumor growth in vivo. Our data further showed that the ectopic expression of GATA4 induced cellular senescence through regulating nuclear factor-κB and inducing mesenchymal-to-epithelial transition. CONCLUSIONS: Our data demonstrated that by inducing cellular senescence and mesenchymal-to-epithelial transition, GATA4 plays a crucial role as a tumor suppressor in HCC. It may thus be a potential cancer therapeutic target for HCC.

3.
Biochem Biophys Res Commun ; 496(2): 502-507, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29339154

RESUMO

Infection with hepatitis B virus (HBV) e-antigen (HBeAg)-negative strains is increasingly prevalent. Currently, detailed information of the obtained natural HBV strain is not available except for the B genotype and HBeAg-negative. The aim of the present study was to characterize the natural genetic variation of the HBeAg-negative strain and investigate its function. The genic sequence was determined using Sanger sequencing, and compared to related sequences using alignment and phylogenetic analysis. In vivo, virus-specific serum markers were investigated in CBA/CaJ mice. The sequence had a full genome length of 3215 nucleotides. Sites 122, 125, 127, and 160 in S regions were identified as lysine, threonine, proline, and lysine respectively. The main four point variants including A1762T, G1764A, G1896A, and G1899A were detected in the full-length genome. The genotype of the sequence was B, with sub-genotype B2 and serological subtype adw2. The characterize of the natural genetic variation strain showed no reported drug-resistant variant in P region and no reported immune escape site in S region. The strain will increase viral replication and infection for mutations A1762T and G1764A in the basal core promoter region, and mutations G1896A and G1899A in the pre-core region. The G1896A variant resulted in a premature stop codon and abolished HBeAg expression. HBsAg persisted for 26 weeks and HBeAg was still negative in CBA/CaJ mice. The present sequence is representative of the HBeAg-negative genome and may serve as a valuable reference for studying HBeAg-negative strains. The present findings were successfully verified in CBA/CaJ mice, demonstrating good applicability of the sequence.


Assuntos
DNA Viral/genética , Genoma Viral , Antígenos E da Hepatite B/genética , Vírus da Hepatite B/genética , Hepatite B/virologia , Animais , DNA Circular/genética , DNA Circular/imunologia , DNA Viral/imunologia , Modelos Animais de Doenças , Variação Genética , Genótipo , Hepatite B/imunologia , Antígenos E da Hepatite B/imunologia , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus da Hepatite B/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos CBA , Mutação , Regiões Promotoras Genéticas , Padrões de Referência , Análise de Sequência de DNA , Replicação Viral
4.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 31(6): 758-62, 2015 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-26062417

RESUMO

OBJECTIVE: To construct a eukaryotic expression vector containing human cannabinoid receptor 2 (hCB2R) gene and investigate its expression, location and the influence on the apoptosis of cervical cancer Caski cells. METHODS: The eukaryotic expression vector GV230-hCB2R was constructed and identified by double enzyme digestion and DNA sequencing analysis. Then it was transfected into HEK293 cells and Caski cells by Lipofectamine(TM) 2000. The expression and cellular localization of hCB2R protein were detected by Western blotting and immunofluorescent cytochemistry combined with laser scanning confocal microscopy, the apoptosis rate was tested by flow cytometry. The mRNA and protein expressions of hCB2R, Bcl-2, Bax and Bad were examined by real-time fluorescent quantitative PCR (qRT-PCR) and Western blotting, respectively. RESULTS: The gene fragment of 1128 bp was obtained by double enzyme digestion, it had 99% homology with human hCB2R gene nucleic acid sequence reported (NM_001841.2). After transfected into HEK293 cells, hCB2R protein, with the relative molecular mass (Mr) being 40 000, was expressed in both cytoplasm and cellular membrane. The over-expression of hCB2R promoted apoptosis of Caski cells via up-regulating the Bax, Bad expressions and down-regulating the Bcl-2 expression. CONCLUSION: The up-regulated expression of hCB2R could induce cell apoptosis by enhancing the expressions of Bax, Bad and suppressing the expression of Bcl-2 in Caski cells.


Assuntos
Apoptose/genética , Receptor CB2 de Canabinoide/genética , Neoplasias do Colo do Útero/patologia , Feminino , Expressão Gênica , Humanos , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Transfecção , Proteína X Associada a bcl-2/genética , Proteína de Morte Celular Associada a bcl/genética
5.
Int J Mol Sci ; 16(3): 5141-60, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25751726

RESUMO

Studies on molecular mechanisms of the persist infection of hepatitis B virus have been hampered by a lack of a robust animal model. We successfully established a simple, versatile, and reproducible HBV persist infection model in vitro and in vivo with the circularized HBV DNA. The cells and mice were transfected or injected with circularized HBV DNA and pAAV/HBV1.2, respectively. At the indicated time, the cells, supernatants, serum samples, and liver tissues were collected for virological and serological detection. Both in vitro and in vivo, the circularized HBV DNA and pAAV/HBV1.2 could replicate and transcribe efficiently, but the infection effect of the former was superior to the latter (p < 0.05). The injection of circularized HBV genome DNA into the mice robustly supported HBV infection and approximately 80% of HBV infected mice established persistent infection for at least 10 weeks. This study demonstrated that the infection efficiency and replication ability of the circularized structure of HBV DNA overmatched that of the expression plasmid containing the linear structure of HBV DNA in vitro and in vivo. Meanwhile, this research results could provide useful tools and methodology for further study of pathogenic mechanisms and potential antiviral treatments of human chronic HBV infection in vitro and in vivo.


Assuntos
DNA Viral/metabolismo , Vírus da Hepatite B/genética , Alanina Transaminase/metabolismo , Animais , Linhagem Celular , DNA Viral/química , Modelos Animais de Doenças , Vetores Genéticos/metabolismo , Hepatite B/metabolismo , Hepatite B/patologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Replicação Viral
6.
Zhonghua Gan Zang Bing Za Zhi ; 22(4): 260-5, 2014 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-25173223

RESUMO

OBJECTIVE: To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA. METHODS: Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis. RESULTS: HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice). CONCLUSION: The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.


Assuntos
DNA Circular/administração & dosagem , DNA Viral/administração & dosagem , Modelos Animais de Doenças , Hepatite B Crônica/virologia , Animais , Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Masculino , Camundongos , Camundongos Nus , Transdução Genética , Replicação Viral
7.
Mol Med Rep ; 10(3): 1245-51, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25018054

RESUMO

A simple technique for the identification of common genotypes of the hepatitis B virus (HBV) remains to be identified. The present study was conducted to establish such a methodology. Four plasmids of genotypes A­D and 123 clinical serum specimens of HBV­infected patients were genotyped. HBV genotypes would be detected successfully when the HBV genotype reached a viral load of 1 x 103 copies/ml or the BC genotype mixed samples reached a 5% level. The lower limit of detection of HBV DNA in serum specimens was determined to be 2.14x102 IU/ml. The assay sensitivity and specificity were 100% and the consistency was demonstrated to reach as high as 90.24 and 100% compared with that of the DNA sequencing and cloning. The frequencies of the genotypes B, C, BC, BD and BCD were found to be 65.0, 23.6, 7.3, 3.3 and 0.8%, respectively. The accuracy of detection of the mixed infections was also higher using the rapid and simple SNaPshot method compared with that achieved with the DNA sequencing methods. The results of the present study indicated that the SNaPshot technique accurately distinguishes the HBV genotypes A­D and is able to be readily applied as a monitoring tool in HBV prognosis and treatment.


Assuntos
DNA Viral/isolamento & purificação , Vírus da Hepatite B/genética , Clonagem Molecular , Primers do DNA/genética , DNA Viral/sangue , Genótipo , Técnicas de Genotipagem , Vírus da Hepatite B/isolamento & purificação , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Análise de Sequência de DNA , Carga Viral
8.
Int J Mol Med ; 30(5): 1041-7, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22960954

RESUMO

The objective of this study was to screen for antigens of the hepatitis C virus (HCV) to establish a new double antibody sandwich-lateral flow immunoassay (DAS-LFIA) method for testing the presence of anti-HCV antibodies in human serum or plasma. A series of different recombinant HCV proteins in Escherichia coli cells were constructed, expressed, purified and the new DAS-LFIA strip was developed. The sensitivity and specificity of new the DAS-LFIA strip were evaluated by detecting 23 HCV-positive sera, a set of quality control references for anti-HCV detection that contain known amounts of anti-HCV antibodies, and 8 HCV-negative sera. A total of 300 clinical serum samples was examined by both the new DAS-LFIA strip and enzyme-linked immunosorbent assay (ELISA). Data were analyzed using SPSS 11.5 software. The sensitivity and specificity of the new DAS-LFIA strip were 100%. The lowest test line of the HCV DAS-LFIA strips was 2 NCU/ml. Additionally, the concordance between the new DAS-LFIA strip and ELISA methods was 94.33%. In conclusion, our new testing method is rapid, simple, sensitive and specifically detects the presence of anti-HCV antibodies in human serum or plasma. Therefore, it may be used for monitoring HCV.


Assuntos
Cromatografia de Afinidade/métodos , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/sangue , Hepatite C/diagnóstico , Antígenos Virais/biossíntese , Antígenos Virais/genética , Antígenos Virais/imunologia , Clonagem Molecular , Escherichia coli , Hepacivirus/genética , Hepatite C/sangue , Hepatite C/imunologia , Humanos , Testes Imunológicos , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/imunologia
9.
Zhonghua Gan Zang Bing Za Zhi ; 19(12): 884-9, 2011 Dec.
Artigo em Chinês | MEDLINE | ID: mdl-22525498

RESUMO

OBJECTIVE: To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system. METHOD: 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively. RESULT: Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/µl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test. CONCLUSION: Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.


Assuntos
Farmacorresistência Viral/genética , Vírus da Hepatite B/genética , Hibridização de Ácido Nucleico/métodos , DNA Viral/genética , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Hibridização Genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Sensibilidade e Especificidade
10.
Zhonghua Gan Zang Bing Za Zhi ; 18(6): 414-8, 2010 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-20587309

RESUMO

OBJECTIVES: To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues. METHODS: According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively. RESULTS: The specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing. CONCLUSION: Reverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.


Assuntos
Farmacorresistência Viral/genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Mutação , Substituição de Aminoácidos , Antivirais/farmacologia , DNA Viral/genética , Farmacorresistência Viral/efeitos dos fármacos , Vírus da Hepatite B/efeitos dos fármacos , Humanos , Hibridização de Ácido Nucleico/métodos
11.
Bing Du Xue Bao ; 25(1): 35-40, 2009 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-19437884

RESUMO

The recombined adenovirus DNA was transfected into 293 cells for packing and amplification of replication-deficient Ad-CMV-E6/E7, Ad-K14 -E6/E7 virus was purified by CsCl density gradient centrifugation , recombined adenovirus Ad-CMV-E6/E7, Ad-K14 -E6/E7 were used as experimental group, while pAd-CMV and pAdtrack-K14 were used as control group. Four of them were injected through one main vein of nude mice tail respectively. These mice were then treated with 0.05 mg 17beta-estradiol over 12 weeks. Mice were anaesthesiaed with 2.5% Avertint and the vagina, mammary gland, ovaries and uterus were dissected and fixed in 3.75% paraformaldehyde overnight at 4 degrees C. Paraffin-embedded sections, HE staining and identification of P53 and Bcl-2 protein via immunohistochemistry were performed. The expression of E6/E7 was verified by RT-PCR in different tissue of nude mice. HE staining showed evident hyperplasy in cervix-uterus transformation zone of experimental group 2. The expression of mutant P53 and Bcl-2 were higher than control group via immunohistochemical S-P method in uterus stroma-cell. Western blotting also showed that E6 protein was expressed. The expression of E6/E7 was higher than control group by human cytokeratin promoter 14 and hyperlasy changes were detected in epithelial tissue of cervix-uterus transformation zone.


Assuntos
Doenças dos Genitais Femininos/patologia , Doenças dos Genitais Femininos/virologia , Genitália Feminina/patologia , Papillomaviridae/fisiologia , Adenoviridae/genética , Animais , Western Blotting , Linhagem Celular , Feminino , Genitália Feminina/virologia , Humanos , Imuno-Histoquímica , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Nus , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Ovário/metabolismo , Ovário/patologia , Papillomaviridae/metabolismo , Proteínas E7 de Papillomavirus , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Supressora de Tumor p53/metabolismo , Útero/metabolismo , Útero/patologia , Vagina/metabolismo , Vagina/patologia
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