| ObjectiveTo further reveal the molecular mechanisms of hepatitis B virusregulated Raf1expression in HepG2.2.15cells.Methods1. A series of Raf1promoter luciferase report plasmids and sequencedeletion luciferase report plasmids were constructed and transfected intoHepG2cells to identify the main target sequence in Raf1promoter region.The activity of Raf1promoter was detected with Dual-Luciferase ReporterAssay System.2. Deletion mutants of Raf1promoter plasmids were co-transfected intoHepG2cells with either pCMV-HBx (HBx expression plasmid) orpCMV-sport6(as a control), and HBx-siRNAs were used to block theexpression of HBx in HepG2.2.15cells to investigate the relationshipbetween HBx and main target sequence in Raf1promoter.3. The AP-2α expression plasmid and5’ deletion mutants of Raf1promoter plasmid were co-transfected into HepG2.2.15cells. Luciferaseactivity was measured48h after. AP-2α interfering RNA plasmids were co-transfected into HepG2.2.15cells with5’ deletion mutants of Raf1promoter plasmid, respectively, in order to investigate the role of AP-2α inregulating Raf1expression.4. RT-PCR, Real-Time PCR and Western Blot analysis were used tocompare the expressions of AP-2α in HepG2and HepG2.2.15cells.Results1. Luciferase activity assay showed that-209bp~-133bp region was themain target sequence in Raf1promoter region.2. The sequence-209bp~-133bp was critical for activation of Raf1promoter by HBx.3. Transcription factor AP-2α could up-regulate the activity of Raf1promoter. After blocking the expression of AP-2α in HepG2.2.15cells, theactivity of Raf1promoter was markedly decreased.4. RT-PCR, Real-Time PCR and Western Blot analysis showed mRNAand Western levels of AP-2α in HepG2.2.15cells were obviously higher thanthat in HepG2cells.ConclusionHepatitis B virus enhanced Raf1promoter activity may throughactivation of transcription factor AP-2α. These findings provided a possiblemechanism for the occurrence and development of HBV-related HCC. |
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