RESUMO
BACKGROUND AND AIM: MicroRNAs are a class of small non-coding RNAs that negatively regulate the expression of their target genes. The aim of the present study was to explore the effects of microRNA on biological behaviors of HepG2 cells and further analyze its characteristics. METHODS: We detected different expression profiles of miRNAs in HepG2 and L02 cell lines by microRNA microarray. Northern blot, quantitative real-time polymerase chain reaction, methylthiazolyl tetrazolium, fluorescence-activated cell sorting, scratch wound, transwell migration and Matrigel invasion assays and western blot were carried out to determine whether or not microRNA-224 (miR-224) can influence the biological behaviors of HepG2 cells. RESULTS: MiR-224 was significantly upregulated in HepG2 cells. Cell proliferation, migration and invasion, but not cell cycles, were altered after changing the expression of miR-224. Taking invasion and migration as a breakthrough, a close relationship between the expression of miR-224 and its proteins such as PAK4 and MMP9, which were involved in the invasion of tumor, was found. CONCLUSIONS: Overexpression of miR-224 was involved in the malignant phenotype of HepG2 cells, and it may be an important factor in regulating the migration and invasion of HepG2 cells.
Assuntos
Carcinoma Hepatocelular/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , MicroRNAs/metabolismo , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/patologia , Ciclo Celular , Proliferação de Células , Separação Celular/métodos , Citometria de Fluxo , Perfilação da Expressão Gênica/métodos , Genótipo , Células Hep G2 , Humanos , Neoplasias Hepáticas/patologia , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
OBJECTIVE: To investigate the expression and role of B-cell translocation gene 2(BTG2) in the carcinogenesis of hepatocellular carcinoma (HCC). METHODS: Modified Diethylnitrosamine (DEN)-induced primary hepatocellular carcinoma rat model was established. The expression of BTG2, p53 and cyclinD1 was detected by RT-PCR, western blot and immunohistochemistry. RESULTS: The BTG2 protein was predominantly localized in the nucleus, with faint cytoplasmic staining in normal liver cells; however, it is mainly a cytoplasmic protein in HCC cells. BTG2 was over-expressed during the early stage after DEN treatment, the expression level peaked at 5 weeks and then it gradually decreased to the normal level after 16 weeks. The expression of cyclin D1 and cyclin E was increased gradually after DEN treatment, and peaked at 16 weeks and 5 weeks respectively. A significant increase in p53 was not observed until 5 weeks after DEN treatment, and it gradually decreased after 16 weeks. CONCLUSIONS: Decreased expression of BTG2 may be an important step in carcinogenesis of HCC. BTG2 may positively regulate p53 expression and negatively regulate cyclin D1 expression in the carcinogenesis of HCC.
Assuntos
Carcinoma Hepatocelular , Dietilnitrosamina , Animais , Linfócitos B , Hepatócitos/metabolismo , Neoplasias Hepáticas , RatosRESUMO
AIM: To prepare a rabbit anti-RAI16 polyclonal antibody and then to identify and apply the rabbit polyclonal antibody against NH2-terminal peptides of RAI16. METHODS: The peptide (aa.44 to 55)of human RAI16 NH2-terminus was synthesized by standard Fmoc.The synthesized peptide was purified by reversed phase high-performance liquid chromatography (RP-HPLC) and cross-linked with keyhole limpet hemocyanin (KLH)by sodium metaperiodate. The rabbits were immunized with the conjugated peptide 4 times(400 mug/rabbit).The polyclonal antibody was purified by Protein G from the collected antiserum. Then it was identified and employed for ELISA, immunohistochemistry and Western blot et cetera. RESULTS: The NH2-terminal peptide of human RAI16 with the purity of 96% was prepared.The titer of the purified polyclonal antibody was 1:125 000. Western blot results showed that the antibody could recognize the protein with molecular weight of 55 000 in the total lysates of human spleen. CONCLUSION: The polyclonal antibody against NH2-terminal peptide generated and identified in the present research can correspond specifically to natural RAI16 protein and therefore it can be employed in ELISA, immunohistochemistry, immunoprecipitation and immunoblotting. The successful preparation of the polyclonal antibody is helpful not only for the study of the tissue distribution and subcellular localization of RAI16, but lso for the research into the function and mechanism of human RAI16.