Identification and characterization of long non-coding RNAs in BET inhibitor-treated human hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma (HCC) is the most common cancer worldwide and the third leading cause of cancer-related death. Emerging evidence suggests that genetic and epigenetic alterations play a crucial role in HCC. Since bromodomain-containing 4 (BRD4) is overexpressed in HCC, BRD4 inhibition is considered a new therapeutic approach for HCC. BRD and extraterminal domain (BET) inhibitors have been actively studied as new epigenetic drugs for cancers that showed antitumor effects. However, most studies are focused on the gene networks, and long non-coding RNA (lncRNA) expression patterns and specific molecular events remain mostly unexplored. LncRNA plays a key role in HCC initiation and progression. We obtained the differentially expressed genes and lncRNAs, in response to BET inhibitors in HCC cells. We studied the following aims to investigate lncRNA's function and regulatory mechanisms to obtain novel therapeutic targets for HCC. Firstly, we investigated the comprehensive analysis of mRNA and lncRNAs expression profiles underlying the antitumor functions of BET inhibitors of human HCC cell line HepG2 using RNA sequencing (RNA-Seq). In BET inhibitors (JQ1, OTX015, and ABBV-075)-treated HepG2 cells, 382 differentially expressed (DE) mRNAs (DEmRNAs) and 60 differentially expressed lncRNAs (DElncRNAs) were obtained. Additionally, we analyzed functional annotation based on the expression profiles assessing the relevance of mRNA-lncRNA pairs. We found novel DEmRNAs (GPAM, GPD1, PSMB8) and DElncRNAs (LINC00242, LINC01146, LINC02535, MIR3142HG, PRR7-AS1) involved in proliferation and apoptosis that are commonly reduced by JQ1, OTX015, and ABBV-075 in HCC. Secondly, novel MYC enhancers and the eRNAs targets BET inhibitors were studied in HepG2 cells. The proto-oncogene MYC is vital for development and cell growth. However, its abnormal regulation causes cancer. Recent studies identified distinct enhancers of MYC in various cancers, but any MYC enhancer(s) in HCC remain(s) elusive. The present study identified 6 putative MYC enhancers in HepG2 cells by analyzing various enhancer markers including H3K27ac and enhancer RNA (eRNA) expression in HCC cells. Amongst these, we found two highly active enhancers, located ~800 kb downstream of the MYC gene. We confirmed that cell proliferation was reduced by suppressing MYC expression upon CRISPR/Cas9-based deletion and/or antisense oligonucleotide (ASO)-mediated inhibition. In conclusion, we identified potential MYC enhancers of HCC and proposed that the associated eRNAs may be a novel biomarker for HCC treatment. Thirdly, we identified MALAT1 enhancers and a mechanism of MALAT1 up-regulation by JQ1 treatment. JQ1 treatment significantly down-regulated genes and lncRNAs related to cell proliferation, such as AREG, EREG, IRF5, PVT1, DANCR, and HOXD-AS. However, MALAT1 expression is unexpectedly up-regulated in JQ1-treated HepG2 cells. Recent studies showed that MALAT1 is up-regulated in various cancers. This study was conducted to investigate the cause of the overexpression of oncogenic lncRNA MALAT1 by JQ1 treatment. We analyzed the MALAT1 enhancer regions using chromatin immunoprecipitation (ChIP)-PCR, luciferase reporter assay, chromatin conformation capture (3C) assay, and bioinformatic analysis. The regions of putative enhancer E2 located -20 kb upstream of the MALAT1 transcription start site (TSS) site showed enhancer activity and closely interacted with the promoter region of MALAT1. JQ1 treatment altered chromatin looping on MALAT1 enhancers and gene bodies to increase the expression in HepG2 cells. Furthermore, inhibition of FOXA2 binding to MALAT1 enhancers and promoter by JQ1 increases MALAT1 expression. In conclusion, FOXA2 would be a suitable-target for anti-cancer therapy with BET inhibitors in the approach that inhibits MALAT1 expression. In summary, the present study aimed to develop a new therapeutic biomarker from analyzing lncRNA expression and their mechanism by inhibiting BET proteins that are up-regulated in HCC progression. We characterized the effect of BET inhibitors on HCC cells by extensive transcriptome analysis of gene and lncRNA changes. Among them, we identified lncRNAs that regulate MYC expression; MYC eRNAs. In addition, we characterized enhancers and the transcriptional factor involved in lncRNA MALAT1 expression. From newly identified lncRNAs or the regulatory mechanisms, we suggested the importance of lncRNAs or the regulatory mechanisms as a new paradigm of HCC treatment.