AIM: To investigate the effects of hepatitis B virus X (HBx) gene on apoptosis and cell cycle in hepatocyte line HL-7702 and to discuss the possible mechanisms in the pathway. METHODS: The recombinant plasmid pcDNA3-X and vector pcDNA3 were transfected into HL-7702 cells and selected by G418 to construct two new cell lines, which were named HL-7702-HBx and HL-7702-con, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to confirm that HBx gene was expressed steadily in the HL-7702-HBx cells. Then apoptosis and cell cycle of the two cells were detected by DNA ladder, flow cytometric analysis, and electronic microscope observation. Apoptosis and cell cycle gene expressions in the two cells were subsequently evaluated by using gene arrays. Some of results were further confirmed by real-time PCR and western blot analysis. RESULTS: RT-PCR and the western blot analysis showed that HL-7702-HBx expressed the HBx gene steadily. Comparedwith the HL-7702-con cells, there was increased apoptosis and accumulation of the S phase in the HL-7702-HBx cells. The gene array analysis indicated that some DNA repair genes (XRCC1, DDB1, etc.) and DNA damage checkpoint-related genes (Cdc47, RAD17, etc.) played roles in the HBx-mediated imbalance of apoptosis and cell cycle. Both cDNA array analysis and real-time RT-PCR showed that mRNA of XRCC1, Cdc47 and RAD17 were upregulated by HBx. Unexpectedly, the western blot analysis revealed that HBx inhibited their protein expression. CONCLUSION: The expression of HBx in HL-7702 cells promoted apoptosis and accumulation of the S phase through the inhibition of DNA repair and checkpoints via post-transcriptional mechanisms.
AIM: To investigate the effects of hepatitis B virus X (HBx) gene on apoptosis and cell cycle in hepatocyte line HL-7702 and to discuss the possible mechanisms in the pathway. METHODS: The recombinant plasmid pcDNA3-X and vector pcDNA3 were transfected into HL-7702 cells and selected by G418 to construct two new cell lines, which were named HL-7702-HBx and HL-7702-con, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to confirm that HBx gene was expressed steadily in the HL-7702-HBx cells. Then apoptosis and cell cycle of the two cells were detected by DNA ladder, flow cytometric analysis, and electronic microscope observation. Apoptosis and cell cycle gene expressions in the two cells were subsequently evaluated by using gene arrays. Some of results were further confirmed by real-time PCR and western blot analysis. RESULTS: RT-PCR and the western blot analysis showed that HL-7702-HBx expressed the HBx gene steadily. Comparedwith the HL-7702-con cells, there was increased apoptosis and accumulation of the S phase in the HL-7702-HBx cells. The gene array analysis indicated that some DNA repair genes (XRCC1, DDB1, etc.) and DNA damage checkpoint-related genes (Cdc47, RAD17, etc.) played roles in the HBx-mediated imbalance of apoptosis and cell cycle. Both cDNA array analysis and real-time RT-PCR showed that mRNA of XRCC1, Cdc47 and RAD17 were upregulated by HBx. Unexpectedly, the western blot analysis revealed that HBx inhibited their protein expression. CONCLUSION: The expression of HBx in HL-7702 cells promoted apoptosis and accumulation of the S phase through the inhibition of DNA repair and checkpoints via post-transcriptional mechanisms.
Authors: Samantha Therezinha Almeida Pereira Leite; Marilene Borges da Silva; Marco Andrey Pepato; Francisco José Dutra Souto; Raquel Alves dos Santos; Carmen Lucia Bassi-Branco Journal: Mem Inst Oswaldo Cruz Date: 2014-02 Impact factor: 2.743