Fig. 3

HERH-1 bound with CREB1 and facilitated CREB1-mediated CCNA2 transcription enhancement. a Through bioinformatics method, we found 315 cell cycle associated genes noted by the GSEA database. By combining this gene set with our microarray data, we screened out the top five significantly dysregulated cell cycle-associated genes in the advanced HCC, and part of these genes may be regulated by HERH-1/4. Among them, CCNA2 was the selected gene in the following study. b Correlation between HERH-1/4 and CCNA2 mRNA levels was analyzed in the ten pairs of HCC recurrent and primary tissues by qRT-PCR. c Regulation of CCNA2 by HERH-1/4 in HCC cells was validated by qRT-PCR (n = 2). d Potential TFs that can bind with HERH-1 and the CCNA2 promoter were predicted by bioinformatics. e Schematic presentation of the three predicted CREB1 binding sites within CCNA2 promoter (top) and the components of the plasmids used for promoter activity assessment (bottom). f Activity of the predicted CCNA2 promoter and the effect of CREB1 on transcription intensity was detected by luciferase reporter assay (n = 3). g The interaction between the CREB1 protein and CCNA2 promoter was detected by ChIP assay (n = 2). h, i The interaction between HERH-1 and CREB1 protein was detected by RNA pull-down (h) and RIP (i) assays. Full-length blots and gels are presented in Fig. S8a and Fig. S9a, b. *P < 0.05; **P < 0.01; n.s., not significant