Targeting MYC-inducing enhancer-associated noncoding (MYC-IEANC) RNAs inhibits the proliferation of HCC cells

MYC, a critical oncogene, encodes the c-MYC transcription factor (TF) and plays an essential role in hepatocellular carcinoma (HCC) development. Recent studies have identied numerous tissue-specic enhancers of MYC in various cancers, but an HCC-specic enhancer of MYC remains elusive. We analyzed enhancer markers, including H3K27ac enrichment and enhancer RNA (eRNA) expression, to determine putative enhancer regions of MYC in HCC cells. Enhancer activity was detected using a dual-luciferase reporter assay. We used the CRISPR-Cas9 system to edit the enhancer regions and performed antisense oligonucleotide (ASO) to inhibit eRNA. The functions of enhancers and eRNAs on HCC cells were conrmed by cell proliferation assay and sphere formation assay. choose analyzing regulated HCC


Conclusion
In this study, we present MYC enhancers in HCC and elucidate the molecular functions of MYC-inducing enhancer-associated noncoding (MYC-IEANC) RNAs in the proliferation of HCC cells. Furthermore, our results suggest that MYC-IEANC RNAs, which play an oncogenic role in HCC cells, can be a target for HCC treatment.

Background
Enhancers, as epigenetic regulators, are short genomic elements that provide speci city to target gene expression. Enhancer activity is regulated by various factors associated with epigenetic modi cation [1].
Therefore, the activity and location of enhancers are known to be cell type-speci c and are bound by tissue-or cell type-speci c transcription factors (TFs) [2]. Enhancers consist of DNA enriched with H3K27ac and H3K4me marks and are closely related to critical oncogenic drivers [3]. Accordingly, studies have been actively conducted to elucidate the regulation of cancer by identifying and regulating enhancers that function differently depending on the cancer type [4][5][6]. Perturbing enhancer activity has revolutionized cancer treatment [7,8]. These therapies have been developed based on the ability of cancers to undergo aberrant transcription through the dysregulation of enhancers. eRNA, a noncoding transcript generated by active enhancers, plays an essential role in regulating gene expression along with enhancers [9]. eRNAs are expressed cell type-speci c to control cell fate, and in cancer, they can be used as new cancer diagnostic markers and drugs [10,11]. There are many studies on various methods for eRNA control and their effects; among them, inhibition of eRNA and functional regulation using antisense oligonucleotide (ASO) has signi cant results that can be utilized to treat various diseases including cancer [12][13][14].
MYC is essential for various cellular processes, including cell growth, proliferation, differentiation, and apoptosis [15]. MYC acts as an oncogene and tightly regulates the normal state. In contrast, dysregulation of MYC is prevalent in cancer. And upregulation of MYC expression is observed in 50-60% of all cancers [16,17]. Accordingly, studies on the enhancer of MYC were also conducted, and it was found that the location of enhancers is cancer-speci c [18]. As a representative example, the enhancer is located at approximately 0.7 Mb downstream of MYC in prostate cancer and relatively close to 70 kb upstream in pancreatic cancer. In contrast, it is located 1.9 Mb upstream of MYC in glioma and regulates MYC expression [19][20][21]. However, studies on MYC enhancers in liver cancer have been insu cient.
In this study, we identi ed regions presumed to be enhancers of MYC that regulate oncogene MYC expression. We con rmed the activity of eRNA as well as the enhancer. In addition, this study suggests that ASO may be a therapeutic agent by e ciently decreasing enhancer activity and inhibiting eRNA expression, resulting in the inhibition of cell proliferation and sphere formation in HCC.

Cell culture of HCCs
The HepG2 and Huh7 HCC cell lines were purchased from Korean Cell Line Bank (KCLB) and maintained in minimum essential medium or RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin (100 units/ml)/streptomycin (100 mg/ml) (Thermo Fisher Scienti c). The medium was replaced every 3-4 days. The cells were maintained in a humidi ed incubator with 95% air and a 5% CO 2 atmosphere at 37°C. JQ1, OTX-015, C646, and 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside (DRB) were purchased from Tocris Bioscience (Minneapolis). JQ1, OTX-015, C646, and DRB were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM. The cells were treated with different concentrations of JQ1 and DRB for different durations.
Cell proliferation assay (WST-1 assay) The cell proliferation assay was performed using a premixed water-soluble tetrazolium salt (WST-1) cell viability test (Takara) according to the manufacturer's instructions. The cells were seeded at a density of 1 x 10 4 cells per well. WST-1 was added to each well, and the absorbance of the microplate at 450 nm was measured after an additional 4 h incubation. The data represent three independent experiments (n = 3).
Ethynyl deoxyuridine (EdU) analysis was performed using an EdU cell proliferation assay kit (Invitrogen) following the manufacturer's instructions. Then, the cells were washed with phosphate-buffered saline, mounted with a 4',6-diamidino-2-phenylindole (DAPI)-containing mounting solution (VECTASHIELD, Vector Laboratories), and imaged by microscopy (Nikon Eclipse 80i). The percentage of EdU-positive cells was examined in HCC cell lines using ImageJ (Bethesda) software. The data represent three independent experiments (n = 3).
Gene expression analysis using quantitative PCR (qRT-PCR) Total RNA was extracted from HepG2 cells using RNAiso Plus (Takara) according to the manufacturer's instructions. cDNA was synthesized by PrimeScript reverse transcriptase (Takara) and ampli ed using gene-speci c primers. The primers used for qRT-PCR are listed in Additional le 1: Table 1. The primers were designed by Primer Bank (https://pga.mgh.harvard.edu/primerbank/). qRT-PCR was performed with TBGreen Premix Ex Taq II (Takara). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The data represent three independent experiments (n = 3). After performing qRT-PCR, the results were analyzed using the critical threshold (△C T ) and the comparative critical threshold (△△C T ) methods in ABI 7500 (Applied Biosystems) software with the NormFinder and geNorm PLUS algorithms.
Enhancer regions were ampli ed using forward and reverse primers to generate NheI of SacI and XhoI sites, respectively. These constructs were cloned into the pGL4.26 construct (Promega). The primers used for cloning are listed in Additional le 1: Table 2. The cells were seeded into 24-well plates and transfected with Lipofectamine 3000 (Thermo Fisher Scienti c). According to the manufacturer's instructions, luciferase activity was measured using the Dual-Glo Luciferase Assay kit (Promega). PRL-TK (Renilla luciferase expression construct; Promega) was used as an internal control. Luciferase activity was normalized to Renilla luciferase and the control (empty vector).
Construction of R3 region KO HCCs using the CRISPR-Cas9 system Forward and reverse oligomers for gRNA synthesis against target sites were designed according to the manufacturer's instructions. The oligomers were extended into 100-mer insert DNA using Phusion High-Fidelity PCR Master Mix (M0531, Biolabs) with the following setup: 2 min at 98°C, 4 cycles of ampli cation (10 sec at 98°C, 20 sec at 53°C, 30 sec at 72°C), and 5 min at 72°C. Then, the insert DNA was puri ed and combined with gRNA_Cloning Vector (#41824, Addgene) using Gibson Assembly Master Mix (E2611, New England Biolabs) at 50°C for 1 h, followed by transformation and colony PCR. The cloned vectors were then puri ed and ordered to be sequenced (Macrogen) to con rm the recombination.
HCCs were transfected with the recombinant gRNA plasmid vector and pCas9_GFP plasmid (#44719, Addgene) in a 95:5 ratio using Lipofectamine 3000 (L3000-001, Life Technologies) according to the manufacturer's instructions. The Cas9 sgRNA vector was a gift from Su-Chun Zhang, and pCas9_GFP was a gift from Kiran Musunuru [22,23]. Then, transfected cells were seeded into 96-well plates at a ratio of less than 1 cell per well to ensure that every well contained a single unique cell. The cells were grown for ~2 weeks and moved into 24-well plates separately. After the cells were su ciently grown, genomic DNA (gDNA) was extracted using a Wizard Genomic DNA Puri cation Kit (A1125, Promega). Then, gDNA was ampli ed by PCR with target-speci c primers and sequenced to check properly generated deletions.
Con rmed cells were moved and grown in 100 mm dishes, and the KO of HCC gene expression was con rmed by qRT-PCR.
Knockdown of eRNA using ASO Locked nucleic acid (LNA)-modi ed ASOs complementary to eRNA of MYC were designed from Antisense LNA GapmeRs (Qiagen). The ASOs were purchased from Qiagen. The sequences are listed in Additional le 1: Table 3. For the transfection of Huh7 cells, ASOs were mixed with RNAiMAX in serum-free Opti-MEM (Gibco). At varying concentrations of ASOs, dissolved Opti-MEM was added, and the cells were incubated in a growth medium for 4 h at 37°C and 5% CO 2 . For total RNA extraction, the cells were harvested 48 h posttransfection.
Colony formation assay R3-deleted and WT Huh7 cells were seeded on 6-well plates (SPL) in growth media at a density of 2500 cells/well and incubated in a CO 2 incubator for 10 days. Then, the cells were washed with PBS, xed with 4% paraformaldehyde for 20 min and washed once with PBS. The cells were stained with 1% crystal violet (Sigma) for 30 min. After crystal violet was removed, the plates were washed with DW for 5 min and dried. The stained cells were analyzed for colony formation rates using ImageJ (Bethesda).

Statistical analysis
The data are presented as the mean ± standard deviation (SD) of the mean. All statistical analyses were performed using the IBM SPSS Statistics 26.0 program (IBM). We used one-way analysis of variance followed by Tukey's honestly signi cant difference post hoc test. p values < 0.05 were considered signi cant.

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 signi cantly 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 RNAP transcription elongation inhibitor 5,6-dichloro-1-β-D -ribofuranosylbenzimidazole (DRB, 50 µM, 24 h). C646 treatment signi cantly reduced the expression of MYC but not that of VEGFA. DRB treatment signi cantly 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.

Identi cation 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 identi ed (R1-6) ( Fig. 2A, Additional le 1: Table 4). More speci cally, we set the H3K27ac enrichment level above 50 and the GRO-seq level, meaning eRNA expression, above 20 to re ect 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 le 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 signi cant reduction in sense eRNA expression in the RNAP 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 signi cantly 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 le 1: Table 3). Huh7 cells were transfected with each target gRNA plasmid and the Cas9 plasmid and sorted into single unique cells. After su cient 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 veri cation di cult. 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 signi cantly decreased in the R3-deleted cells (Fig. 4D) (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 con rm that enhancer deletion in uences 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 con rm 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 le: 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 speci cally decreased in HCC-transfected ASO-R2 cells (Fig. 5B). The eRNA expression of MYC-R3 was speci cally decreased in HCC cells transfected with ASO-R3 (Fig. 5C). Both ASO-R2-and ASO-R3-transfected cells had signi cantly reduced MYC gene expression relative to the ASO-NC-transfected cells (Fig. 5D). As a result of con rming the eRNA inhibitory effect, when MYC-R2 was inhibited, the expression of lncRNA was con rmed 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 signi cantly 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][31][32]. Our results indicate that MYC regulation affects the stemness of HCC cells.

Discussion
HCC is the most common type of primary liver cancer. HCC is prevalent cancer globally and a leading cause of cancer-related death [33,34]. Signi cant genetic and epigenetic alterations exist in HCC. Its accumulation in key genes involved in cell survival, proliferation, apoptosis, and metastasis leads to carcinogenesis [35]. The dysregulation of MYC plays a vital role in proliferation and invasion, including tumor initiation and progression in HCC [36,37].
The antitumor effect of BET inhibitors such as JQ1 has been proven in various cancer cells, including HCC cells [38][39][40]. The antitumor effect is due to the reduction of oncogenes, including MYC. Several studies have already con rmed the inhibitory effect of tumor growth by inhibiting MYC expression through BRD4 inhibitors [19,39,41,42]. Despite the effects of BET inhibitors on MYC downregulation, they are not appropriate or effective for use in patients due to various side effects, such as toxicity and resistance.
Recent studies have shown that RNA transcripts from SEs, large clusters of enhancers, are directly or indirectly involved in oncogene expression, cell growth, metastasis, drug resistance, etc. [43]. The enhancer is a DNA region in which the E1A-binding proteins p300 (p300), RNAP , and TFs are enriched through increased accessibility caused by histone modi cation. SEs help the transcriptional activation of the promoter directly through looping or indirectly affect transcription by expressing the eRNA [3,44].
Previous studies have revealed that the presence of SEs, including cancer-speci c enhancers and the expression of eRNA in cancer cells; accordingly, the roles of SEs in the abnormal expression of oncogenes have been elucidated [5,45]. In addition, since the binding of lineage-speci c TFs affects the tumorigenesis function of the enhancer, it can be seen that the enhancer activity is also cancer typespeci c [46]. Therefore, we used ChIP-seq data with HCC cells and used global run-on sequencing (GROseq) data that included nascent transcripts to identify eRNA expression, an active enhancer marker [4,47]. Considering these data, we found that speci c MYC enhancers R2 and R3 in HCC cells are signi cantly different from those in other cancers in terms of their location and activity [5,6,48]. In acute myeloid leukemia (AML), MYC SE, consisting of ve distinct small enhancers located 1.7 Mb downstream of the MYC promoter regulates MYC expression [45]. In colorectal cancer (CRC) and prostate cancer, the MYC enhancer located 335 kb upstream of MYC regulates MYC expression by interacting with its promoter [19]. In addition, the Zhang group deleted approximately 1.5 kb of the MYC enhancer located 450 kb toward the 3′ end in lung adenocarcinoma cells. MYC expression was reduced by 70%, and clonogenic growth was inhibited by approximately 50%. In HCC, we con rmed the reduction of MYC expression and inhibition of cell growth by more than one-half upon deletion of a 400 bp HCC-speci c MYC enhancer located 800 kb downstream of MYC. eRNAs are generally upregulated in various cancers compared to normal tissues, and they can be used as pan-cancer diagnostic markers [49]. In addition, tissue-speci c highly expressed eRNAs, such as CCAT1 in colorectal cancer and androgen receptor (AR)-induced Kallikrein-related peptidase 3 (KLK3) eRNA (KLK3e) in prostate cancer, are considered new targets for treating various cancers [9,50,51]. Although the function of eRNAs has not been fully elucidated, eRNA depletion reduces the transcription of target genes by affecting alterations in chromatin structure and contributing to transcriptional initiation of target genes [52]. eRNA transcription can be regulated by inhibiting enhancer activity or effectively targeting ASO to control target gene expression and cancer cell progression [13,53].
Inhibition of MYC-IEANC RNAs using ASO effectively perturbs enhancers to inhibit HCC cell progression, suggesting that eRNAs can be helpful as therapeutic targets. Several studies have reported the therapeutic effect of eRNA depletion on cancer [53-55]. In addition, Epstein-Barr virus (EBV) superenhancer (ESE) RNAs facilitated the expression of the MYC oncogene in lymphoma and showed a therapeutic effect on EBV-related malignancies by targeting ESE eRNA [55]. Similarly, in our results, targeting MYC-IEANC RNAs transcribed from MYC enhancers R2 and R3 in HCC cells con rmed an effective decrease in MYC expression along with proliferation and stemness reduction in HCC cells, suggesting a therapeutic effect on HCC. The study that downregulation of MYC, a marker of stemness, suppressed spheroid growth of colon CSCs and tumor growth in vivo can support our suggestion [32].

Conclusion
In this study, we identi ed the putative MYC enhancer in HCC cells. Enhancer activity and eRNA transcription were analyzed to determine the region involved in MYC expression, and it was con rmed that inhibiting enhancers and eRNAs suppressed the proliferation and reduced sphere formation of HCC cells. These results implied that the inhibition of eRNA by ASO treatment had corresponding effects on the deletion of MYC enhancers. Thus, our study suggests that for HCC, a strategy for reducing MYC expression through speci c targeting with ASO has therapeutic potential without the side effects of gene editing.    The values are the mean ± SD from triplicate well measurements. *, p < 0.05 and **, p < 0.01.

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