Fig. 5

HERH-4 acted as a miR-29b/c sponge to stabilize CCNA2 mRNA and assisted its translation. a Potential ceRNA that bore the same MRE with HERH-4 was predicted by bioinformatics. We screened out the genes that were annotated as cell cycle associated genes by the GSEA database, showed upregulation in our microarray data, and were predicted as potential miR-29 targets by the TargetScan database. b The miR-29c response element within HERH-4 was cloned into a GFP reporter vector. The regulation of miR-29b/c on HERH-4 MRE function was detected by GFP reporter assay in HCC cells (n = 3). c To confirm the negative regulation of miR-29b/c on their target HERH-4, miR-29b/c levels were altered in HCC cells and the endogenous HERH-4 level was detected by qRT-PCR (n = 2). d To confirm the regulation of miR-29b/c on their target CCNA2, the miR-29c response element within CCNA2 mRNA was cloned into GFP reporter vector. The effects of miR-29b/c on the CCNA2 MRE function was detected by GFP reporter assay in HCC cells (n = 3). e The negative regulation of miR-29b/c on the endogenous CCNA2 level was detected by qRT-PCR (n = 2) and Western blot assays in HCC cells. Full-length blots are presented in Fig. S8c, d. f, g The positive regulation of HERH-4 MRE on CCNA2 expression and the role of miR-29b/c within the ceRNA regulation were detected by GFP reporter (f, n = 3), qRT-PCR (g, n = 2), and Western blot (g) assays in HCC cells. Full-length blots are presented in Fig. S8e-g. h The absolute level of HERH-4, CCNA2 mRNA, and miR-29b/c in HCC cells were detected by qRT-PCR (n = 2) in order to evaluate the molecular environment of the ceRNA regulation among these factors. i The recruitment of miR-29b/c, HERH-4 and CCNA2 mRNA to AGO2 protein was confirmed by RIP assay in order to support the fact that miR-29b/c enrolled their targets into RISC to exercise miRNAs’ function. Full-length gels are presented in Fig. S9c, d. ASO, antisense oligonucleotide; MRE, miRNA response element; NC, negative control; wt, wild-type; mut, mutant; *P < 0.05; **P < 0.01; n.s., not significant