An epigenomic approach to identifying differential overlapping and cis-acting lncRNAs in cisplatin-resistant cancer cells [BiSulfite-seq]

Long noncoding RNAs are critical regulators of cell biology whose alteration can lead to the development of diseases such as cancer. The potential role of lncRNAs and their epigenetic regulation in response to platinum treatment is largely unknown. We analyzed four paired cisplatin-sensitive/resistant non-small cell lung cancer and ovarian cancer cell lines. The epigenetic landscape of overlapping and cis-acting lncRNAs was determined by combining human microarray data on 30,586 lncRNAs and 20,109 protein coding mRNAs with whole genome bisulfite sequencing. Selected candidate lncRNAs were further characterized by PCR, Gene-Ontology analysis and targeted bisulfite sequencing. Differential expression in response to therapy was observed more frequently in cis-acting compared to overlapping lncRNAs (78% vs 22%, fold change ≥1.5), while significantly altered methylation profiles were more commonly associated with overlapping lncRNAs (29% vs. 8%; p<0.001). Moreover, overlapping lncRNAs contain more CpG islands (CGIs) (25% vs. 17%) and the majority of CGI-containing overlapping lncRNAs share these CGIs with their associated coding genes (84%). The differences in expression between sensitive and resistant cell lines were replicated in 87% of the selected candidates (p<0.05), while our bioinformatics approach identifying differential methylation was confirmed in all of the selected lncRNAs (100%). Five lncRNAs under epigenetic regulation appear to be involved in cisplatin resistance (AC091814.2, AC141928.1, RP11-65J3.1-002, BX641110 and AF198444). These novel findings provide new insights into epigenetic mechanisms and acquired resistance to cisplatin that highlight specific lncRNAs, some with unknown function that may signal strategies in epigenetic therapies. A total of four cell lines were purchased from ATCC and ECACC (Sigma-Aldrich) and cultured as recommended. To analyze the changes in the transcriptome as a result of CDDP treatment, we established the CDDP-resistant variants of H23-R, H460-R, A2780-R and OVCAR3-R from the parental-sensitive variants H23, H460, A2780 and OVCAR3, after exposure to increasing doses of CDDP treatment in a time period of 6–18 months.