RESUMEN
OBJECTIVES: We investigated the effects of xeroderma pigmentosum D (XPD) on the growth of hepatoma cells and the expressions of P21, Bax, Bcl-2 and Hepatitis B virus X protein (HBx). In addition, we examined whether XPD affected the aforementioned genes via the P53 pathway. METHODS: Human hepatoma cells (HepG2.2.15) were transfected with XPD expression vector, followed by incubation with Pifithrin-α (P53 inhibitor). By using RT-PCR and Western blotting, the expression levels of XPD, P53, phospho-P53 (ser-15), P21, Bax, Bcl-2 and HBx were detected. The cell cycle and the apoptosis rate were examined with flow cytometry, and the cell viability was detected by MTT. RESULTS: Over-expression of XPD up-regulated the expressions of P53, phospho-P53 (ser-15), P21 and Bax but down-regulated the expressions of Bcl-2 and HBx. XPD inhibited the viability of HepG2.2.15 and exacerbated the apoptosis. However, the inhibition of P53 by Pifithrin-α abolished the above-mentioned effects of XPD. CONCLUSION: XPD could suppress growth of hepatoma cells, up-regulate the expressions of P21 and Bax, and down-regulate the expressions of Bcl-2 and HBx through the P53 pathway. There may be mutual influences among XPD, P53 and HBx that co-regulate hepatocarcinogenesis.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Transactivadores/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo D/metabolismo , Benzotiazoles/farmacología , Carcinoma Hepatocelular/metabolismo , Proliferación Celular , Supervivencia Celular , Regulación hacia Abajo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacología , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteínas Reguladoras y Accesorias Virales , Proteína de la Xerodermia Pigmentosa del Grupo D/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
OBJECTIVE: To investigate the inhibitory effects of antisense oligonucleotides to different sequences on VEGF gene expression by human hepatoma cells. METHODS: SMMC7721 cells were cultured under normoxic or hypoxic conditions for 24 h, followed by being transfected with different antisense oligonucleotides (A06513 to cap structure, A06514 to translation initiation, A06515 to Exon-3 and A06516 to translation terminal). The total RNAs from the cells were extracted and the VEGF expression were examined with RT-PCR. The relative concentrations of VEGF transcripts in SMMC772 cells from different groups were determined using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA as internal standard. RESULTS: In response to the hypoxic challenge, SMMC7721 cells upregulated VEGF mRNA; Comparative to the control (no oligonucleotides), A06513, A06514, A06515, and A06516 had obvious sequence-specific inhibitory effect on VEGF gene expression, with the ratio of VEGF over GAPDH of 0.49+/-0.08, 0.71+/-0.12, 0.72+/-0.11 and 0.86+/-0.12, respectively (F=12.21, P< 0.05). A06513 showed the strongest inhibitory effect (P<0.01). CONCLUSION: The antisense oligonucleotides complementary to VEGF cap structure, may become a potential alternative for antisense gene therapy of HCC.