Effects of BET inhibition on HCC cell proliferation and MYC expression
To study the effects of enhancer activity inhibition on HCC cells, we incubated them with BET inhibitors (JQ1 or OTX-015) for different durations (4 h, 24 h, 48 h, and 72 h). Cell proliferation significantly reduced Huh7 cells after 48 h and HepG2 cells after 24 h of BET inhibitor treatment (Fig. 1A). Moreover, we performed a 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay of HCC cell lines treated with a BET inhibitor (5 µM) for 24 h. We found that BET inhibition decreased the proportion of EdU-positive cells for 24 h, indicating that BET inhibition reduced the proliferation of HCC cell lines (Fig. 1B). JQ1 and OTX-015 are well-known small-molecule inhibitors that prevent BRD4, a member of the BET protein family, which is required to maintain SE activation [24]. BRD4 is involved in transcription by interacting with TFs and chromatin remodeling proteins under active SE [25]. HepG2 cells were treated for 24 h with BET inhibitors at 5 µM to determine the change in MYC and vascular endothelial growth factor A (VEGFA) mRNA expression levels. As MYC mRNA expression was reduced to approximately 70%, the mRNA expression of the target genes of MYC and VEGFA [26] was also reduced by JQ1 and OTX-015 treatment (Fig. 1C). Furthermore, we analyzed the gene expression pattern in response to RNA transcription inhibition, such as p300/cAMP response element-binding (CREB)-binding protein (CBP) inhibitor (C646, 50 µM, 24 h) and RNAPⅡ transcription elongation inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB, 50 µM, 24 h). C646 treatment significantly reduced the expression of MYC but not that of VEGFA. DRB treatment significantly reduced the expression of MYC and VEGFA (Fig. 1D).
These results suggest that the proliferation of HCC cells and the expression of oncogenes and tumor suppressor genes were inhibited in BET inhibitor-treated cells, as expected. Furthermore, the effect of BET inhibitors on reducing MYC expression is closely related to RNA transcription inhibition.
Identification of a distal MYC enhancer in HCC cells
We examined ENCODE ChIP-seq data and GRO-seq data of the HepG2 cell line on the MYC locus (Fig. 2). We found that the downstream regions of MYC were more enriched for H3K27ac, a histone mark for an active enhancer in HCC cells. Using the GRO-seq peak (GSE92375) (Liivrand et al., 2017; Benhammou et al., 2019), H3K27ac ChIP-seq peak (GSE29611) [2], and p300 ChIP-seq (GSE32465) [27] at the UCSC Genome Browser (http://genome.ucsc.edu), six putative enhancer loci of the MYC gene were identified (R1-6) (Fig. 2A, Additional file 1: Table 4). More specifically, we set the H3K27ac enrichment level above 50 and the GRO-seq level, meaning eRNA expression, above 20 to reflect the effect on eRNA. LUAD-R3 is a superenhancer (SE) of MYC that actively regulates MYC expression in lung adenocarcinoma cells, and LUAD-R4 has low activity [6]. To seek direct functional evidence for the suspected enhancer activities in HCC cells, we examined the activity of a luciferase reporter in transiently transfected HCC cells. Each enhancer region was cloned into the minimal promoter vector pGL4.26 immediately upstream of the luciferase gene for this assay. As expected, transfection of R2 and R3 increased luciferase activity by approximately 10-fold. However, enhanced activity was not observed when LUAD-R3 and LUAD-R4 were transfected. Furthermore, we cloned a few constructs containing 500 bp fragments in the R2 and R3 regions and analyzed them for luciferase activity. The R2-3-containing plasmid showed the highest enhancer activity in HCC cells, and the R3-2- and R3-3-containing plasmids were most highly expressed in HCC cells. Our results suggest that R2 (R2-3) and R3 (R3-2 and R3-3) can be considered candidate regulators of the transcriptional activation of MYC in HCC cells.
eRNA of putative MYC enhancers in HCC cells
Next, we analyzed R2 and R3 regions of enhancer RNA (eRNA) using qRT-PCR (Additional file 1: Table 1). Using analysis of newly synthesized RNA with GRO-seq peaks, six different sets of primers were designed to analyze eRNA expression at putative enhancer regions (Fig. 3A). qRT-PCR detected a significant reduction in sense eRNA expression in the RNAPⅡ transcription elongation inhibitor DRB in regions R2, R3, R4, and R6 but not R1 and R5 (Fig. 3B). R2 and R3 were, therefore, further studied for expression changes through treatment with BET inhibitors. As expected, eRNA expression in regions R2 and R3 was significantly decreased in HCC cells treated with BET inhibitors (Fig. 3C). Additionally, R1, R4, R5, and R6 eRNAs were decreased in BET inhibitor-treated HCC cells (Fig. S1). Together, these results indicate that there is a correlation between the activity of enhancers and eRNA transcription. From this, it can be inferred that BET inhibitors suppress MYC expression through the regulation of eRNA expression.
Disruption of MYC enhancers affects MYC-related gene expression and cell growth in HCC cells
Since our eRNA expression experiments showed enhancer activity of R3, we tested whether deletion of the R3 region regulates MYC gene expression. We established the deletion of the R3 region in the Huh7 cell line using the CRISPR-Cas9 system. The targeted sequences are located in R3 (Chr 8: 128,556,059–128,557,653) of the MYC gene downstream (Additional file 1: Table 3). Huh7 cells were transfected with each target gRNA plasmid and the Cas9 plasmid and sorted into single unique cells. After sufficient growth of the selected cells, PCR using genomic DNA revealed a deletion of the R3 region. Genomic DNA sequencing revealed a 400 bp deletion on Chr 8:128,556,578 − 128,556,952 (Fig. 4A). Huh7 cells transfected with CRISPR-Cas9 constructs showed R3 regions deletion of approximately 350 bp in length in the R3 region (Fig. 4B). Most MYC enhancer-knockout cells died, which made experimental verification difficult. Therefore, we used MYC enhancer knockout cells that were coexistent with wild-type cells within 10 passages. R3-deleted cells had reduced MYC gene expression relative to wild-type cells (Fig. 4C). Using qRT-PCR analysis, we revealed that R2 eRNA and R3 eRNA expression was significantly decreased in the R3-deleted cells (Fig. 4D). To assess the effect of R3 deletion on lncRNA expression of MYC located downstream within Chr 8: 127,735,502 − 130,060,473, the expression levels of PVT1, CCAT1, and FAM49B were analyzed by qRT-PCR. LncRNA CCAT1 (Located in Chr 8: 127,207,382 − 127,219,268), and FAM49B (located in Chr 8: 129,841,470 − 130,016,672) were reduced in R3-deleted cells, whereas the expression level of PVT1 (located on Chr 8: 127,795,799 − 128,101,256) were almost identical in R3-deleted cells and the wild-type cells (Fig. 4E). We further analyzed the expression of genes regulated by MYC, such as IRF2 and TERT. We found that the expression of IRF2 was upregulated in R3-deleted cells, whereas the expression of TERT was downregulated (Fig. 4F). Furthermore, we performed a cell proliferation assay and colony formation assay of R3-deleted Huh7 cells. We found that the R3-deleted cells had decreased proliferation and colony formation ability (Fig. 4G, H). The results confirm that enhancer deletion influences cancer cell growth by reducing MYC expression [28]. Deletion in the R3 region reduced the sphere formation of Huh7 cells (Fig. 4I).
Inhibition of MYC eRNA represents an effect equivalent to MYC enhancer disruption
To further confirm the functional role of MYC eRNA, antisense oligonucleotides (ASOs) were designed to bind an eRNA at R2 and R3 (MYC-R2 and MYC-R3, respectively) (Additional file: Table 1). To validate the predicted eRNA reduction, we delivered 125 pmol ASO to HCC cells by transfection and analyzed the expression levels of eRNAs and MYC by qRT-PCR. The eRNA expression of R2 was specifically decreased in HCC-transfected ASO-R2 cells (Fig. 5B). The eRNA expression of MYC-R3 was specifically decreased in HCC cells transfected with ASO-R3 (Fig. 5C). Both ASO-R2- and ASO-R3-transfected cells had significantly reduced MYC gene expression relative to the ASO-NC-transfected cells (Fig. 5D). As a result of confirming the eRNA inhibitory effect, when MYC-R2 was inhibited, the expression of lncRNA was confirmed as in MYC-R3 deletion, as shown in Fig. 4, and PVT1 expression increased when MYC-R3 was inhibited (Fig. 5E). In addition, the expression of ICAM1 and IRF2, which MYC regulates, was decreased (Fig. 5F) [29]. We further analyzed the proliferation and sphere-forming ability of HCC cells after treatment with ASO. As eRNA expression of MYC-R2 and R3 decreased, it significantly reduced cell proliferation compared to NC ASO-treated HCC cells (Fig. 5G). In addition, colony formation assays showed that ASO-R3 treatment negatively affected cell adhesion and growth initiation (Fig. S2). The sphere-forming ability was also decreased in ASO-treated HCC cells, especially in ASO-R2 cells (Fig. 5H). The results shown in Fig. 5 show that inhibition of eRNA reduces MYC expression without direct deletion of DNA and suppresses cell proliferation and stemness of HCC cells. Additionally, MYC is involved in regulating the self-renewal and survival of glioma cancer stem cells (CSCs) and colon CSCs as a key regulator of stem cell biology [30–32]. Our results indicate that MYC regulation affects the stemness of HCC cells.