Fig. 4

Triptolide down-regulated the expression of miR-17-92 and miR-106b-25 in a c-Myc-dependent manner. a Treatment of triptolide showed a significant decrease in the transcription of c-Myc, but the transcription of E2F1 was not significantly altered (left panel). Triptolide significantly repressed the transcriptional activity of c-Myc (right panel). pMyc-TA-luc and pRL-TK plasmids were cotransfected into HepG2 cells. b The forced expression of c-Myc counteracted the down-regulation of pri-miR-106b-25 induced by triptolide. HepG2 cells were transiently transfected with pcDNA-c-Myc or pcDNA (mock). After incubation for 48 h, transfected cells were treated with 100 nM triptolide for 24 h. Total RNA were extracted and subjected to qRT-PCR analysis (right panel). c Knockdown of c-Myc using si-RNA induced down-regulation of pri-miR-106b-25 (left panel). Conversely, overexpression of c-Myc up-regulated the pri-miR-106b-25 (right panel). d The schematics diagram of the genomic structure and localization of putative c-Myc binding sites in the promoter region of MCM7. e Chromatin immunoprecipitation assays demonstrated the enrichment of c-Myc s in 5′ flanking region of MCM7 containing putative binding sites. f A schematic of the nucleotide sequences of wild-type and mutant c-Myc binding sites in the promoter region of MCM7 (left panel) Luciferase reporter assays were performed to evaluate the transactivation potential of c-Myc binding sites containing sequences cloned from promoter region of MCM7 (right panel)