Skip to main content
Fig. 4 | Molecular Cancer

Fig. 4

From: Epigenetic silencing of miR-144/451a cluster contributes to HCC progression via paracrine HGF/MIF-mediated TAM remodeling

Fig. 4

miR-144/miR-451a and EZH2 form a regulatory circuit in HCC cells. a 5′-RACE was performed to identify the full-length transcript from the pri-miR-144/451a cluster. The amplified fragment (664 bp) is indicated by the arrow. b The structural information of pri-miR-144/451a is presented as a diagram, which contains one exon (green box), an enhancer region (E1, orange box) and several typical transcription-related boxes (red stick). c The expression of this primary miRNA was examined in para-tumor and HCC tissues (n = 125). d The potential epigenetic modifications in the promoter and enhancer regions of pri-miR-144/451a were analyzed using the EZH2, H3K27me3, H3K27ac, H3K4me1 and H3K4me3 ChIP-seq data of hepatocytes or HCC cells from the Cistrome Project. e The levels of EZH2 in clinical para-tumor and HCC tissues were measured (n = 125). f The relationship between EZH2 expression and HCC patient prognosis was evaluated using data from TCGA (n = 253). g, h HepG2 cells were modified to overexpress EZH2 (g) or subjected to EZH2 knockdown (h), followed by qRT-PCR assay for the expression of pri-miR-144/451a, miR-144 and miR-451a (n = 6). i Luciferase constructs for truncated fragments of the pri-miR-144/451a promoter were generated, and reporter assays were performed to determine the binding regions of EZH2 on the pri-miR-144/451a promoter (n = 6). 0, vehicle; 1, − 1000 bp to + 1 bp; 2, − 1000 bp to − 300 bp; 3, − 300 bp to + 1 bp. j ChIP assays were performed using HepG2 cell lysates to further determine the pri-miR-144/451a promoter sites involved in EZH2 binding and H3K27me3 modification (n = 4). k, l Bioinformatic prediction identified EZH2 as a target of miR-144 (k), which was validated by an assay showing that miR-144 inhibited luciferase expression of a construct harboring a 3’UTR of EZH2 (l) (n = 6). m The mRNA level of EZH2 was assayed in miR-144-overexpressing or miR-144-inhibited HepG2 cells (n = 6). n HepG2 cells were modified to overexpress miR-144 or to synchronously express miR-144 and EZH2. The expression of pri-miR-144/451a and miR-451a was detected via qRT-PCR (n = 6). Bars, means ± SEMs; *, P < 0.05; **, P < 0.01; ***, P < 0.001

Back to article page