RESUMO
OBJECTIVE: To investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro. METHODS: HepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy. RESULTS: Compared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm. CONCLUSION: DHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.
Assuntos
Aflatoxina B1/farmacologia , Carcinoma Hepatocelular/patologia , Movimento Celular/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Neoplasias Hepáticas/patologia , Ciclo Celular , Complexo de Golgi , Células Hep G2 , Humanos , Mitocôndrias , Invasividade NeoplásicaRESUMO
OBJECTIVE: To construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb'D60K gene, and examine their expression in human liver cancer cells. METHODS: Rheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K. HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles. The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells. The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed. RESULTS: The recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing. Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells. LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells. CONCLUSION: Lentiviral vectors carrying Rheb gene and its mutant has been successfully constructed, which can be useful in further investigation of the role of Rheb gene in cancer cells.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Proteínas Monoméricas de Ligação ao GTP/biossíntese , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Mutantes/genética , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Apoptose/genética , Carcinoma Hepatocelular/metabolismo , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Neoplasias Hepáticas/metabolismo , Células MCF-7 , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , TransfecçãoRESUMO
AIM: To construct a system for selecting reference genes (RGs) and to select the most optimal RGs for gene expression studies in nasopharyngeal carcinoma (NPC). METHODS: The total RNAs from 20 NPC samples were each labeled with Cy5-dUTP. To create a common control, the total RNA from 15 nasopharyngeal phlogistic (NP) tissues was mixed and labeled via reverse transcription with Cy3-dUTP. cDNA microarrays containing 14 112 genes were then performed. A mathematical approach was constructed to screen stably expressed genes from the microarray data. Using this method, three genes (YARS, EIF3S7, and PFDN1) were selected as candidate RGs. Furthermore, 7 commonly used RGs (HPRT1, GAPDH, TBP, ACTB, B2M, G6PDH, and HBB) were selected as additional potential RGs. Real-time PCR was used to detect these 10 candidate genes' expression levels and the geNorm program was used to find the optimal RGs for NPC studies. RESULTS: On the basis of the 10 candidate genes' expression stability level, geNorm analysis identified the optimal single RG (YARS or HPRT1) and the most suitable set of RGs (HPRT1, YARS, and EIF3S7) for NPC gene expression studies. In addition, this analysis determined that B2M, G6PDH, and HBB were not appropriate for use as RGs. Interestingly, ACTB was the least stable RG in our study, even though previous studies had indicated that it was one of the most stable RGs. Three novel candidate genes (YARS, EIF3S7, and PFDN1), which were selected from microarray data, were all identified as suitable RGs for NPC research. A RG-selecting system was then constructed, which combines microarray data analysis, a literature screen, real-time PCR, and bioinformatic analysis. CONCLUSION: We construct a RG-selecting system that helps find the optimal RGs. This process, applied to NPC research, determined the single RG (YARS or HPRT1) and the set of RGs (HPRT1, YARS, and EIF3S7) that are the most suitable internal controls.
