An epigenomic approach to identifying differential overlapping and cis-acting lncRNAs

Long noncoding RNAs (lncRNAs) 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 are largely unknown. Researchers at La Paz University Hospital 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 than in 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 value <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.

Experimental design and general overview of expression changes

(A) Pipeline of the followed steps for this study. Arrays combining lncRNAs and mRNAs probes were performed for four paired sensitive/resistant cell lines from lung and ovarian cancer. The threshold for selection was fold change ≥1.5. Inclusion of WGBS data was used to identify lncRNAs under epigenetic regulation by DNA methylation. Selection and further validation of lncRNAs was performed to confirm observed changes in expression and methylome analysis. (B and C) Venn diagram of shared lncRNAs (B) and mRNAs (C) that change in resistance in both lung cancer cell lines (top), both ovarian cancer cells (middle) or when comparing lung and ovarian cells (bottom).