Targeting of non-coding RNAs encoded by novel MYC enhancers inhibits the proliferation of human hepatic carcinoma cells in vitro

Hae In Choi,Eunyoung Yoo,Mina Baek,Kyoung Hwa Jung,Young Seek Lee,Jin Choul Chai,Young Gyu Chai,Bert Binas,Ga Yeong An

doi:10.1038/s41598-022-04869-w

Hae In Choi, Eunyoung Yoo + Show 7 more

Open Access

https://doi.org/10.1038/s41598-022-04869-w

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Abstract

The proto-oncogene MYC is important 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 hepatocellular carcinoma (HCC) remain(s) elusive. By analyzing H3K27ac enrichment and enhancer RNA (eRNA) expression in cultured HCC cells, we identified six putative MYC enhancer regions. Amongst these, two highly active enhancers, located ~ 800 kb downstream of the MYC gene, were identified by qRT-PCR and reporter assays. We functionally confirmed these enhancers by demonstrating a significantly reduced MYC expression and cell proliferation upon CRISPR/Cas9-based deletion and/or antisense oligonucleotide (ASO)-mediated inhibition. In conclusion, we identified potential MYC enhancers of HCC and propose that the associated eRNAs may be suitable targets for HCC treatment.

Highlights

The proto-oncogene MYC is important for development and cell growth, its abnormal regulation causes cancer
Bromodomain and Extra-Terminal motif (BET) inhibitors interfere with BRD4-associated enhancer RNA (eRNA) e longation[19]
The expression of VEGF is elevated in various cancers, including hepatocellular carcinoma (HCC), and is one of the targets of BET inhibitors[20,21,22]

Summary

Introduction

The proto-oncogene MYC is important for development and cell growth, its abnormal regulation causes cancer. Two highly active enhancers, located ~ 800 kb downstream of the MYC gene, were identified by qRT-PCR and reporter assays We functionally confirmed these enhancers by demonstrating a significantly reduced MYC expression and cell proliferation upon CRISPR/Cas9-based deletion and/or antisense oligonucleotide (ASO)-mediated inhibition. Abbreviations HCC Hepatocellular carcinoma eRNA Enhancer RNA ASO Antisense oligonucleotide SE Super-enhancer GRO-seq Global run-on sequencing H2K27ac Acetylated H3 lysine 27 Chr Chromosome TAD Topologically associated domains CSC Cancer stem cell qRT-PCR Quantitative reverse transcription-polymerase chain reaction. The dysregulation of MYC plays a vital role in proliferation and invasion, including tumor initiation and progression in H CC11,12 Both genetic and epigenetic changes could lead to altered MYC expression[13]. Based on published genome maps featuring various epigenetic parameters, we identified novel MYC enhancers in HCC cell lines and confirmed them with functional, genetic, and gene expression analyses. We demonstrate that, at least in part, these novel enhancers operate through the expression of eRNAs

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