So alike yet so different. Differential expression of the long non-coding RNAs NORAD and HCG11 in breast cancer subtypes

Abstract

Breast cancer (BC) is a heterogeneous disease, and it is the leading cause of death among women. NORAD and HCG11 are highly similar lncRNAs that present binding sites for PUMILIO proteins. PUMILIO acts on hundreds of mRNA targets, contributing to the modulation of gene expression. We analyzed the expression levels of NORAD and HCG11 in the BC subtypes luminal A (LA) and basal-like (BL), and the regulatory networks associated with these lncRNAs. In the analysis of TCGA cohort (n=329) and Brazilian BC samples (n=44), NORAD was up-regulated in LA while HCG11 was up-regulated in BL subtype. An increased expression of NORAD is associated with reduced disease-free survival in basal-like patients (p = 0.002), which suggests that its prognostic value could be different in specific subtypes. The biological pathways observed for the HCG11 network are linked to the epithelial-to-mesenchymal transition; while NORAD associated pathways appear to be related to luminal epithelial cell transformation. NORAD and HCG11 regulons respectively present 36% and 21.5% of PUMILIO targets, which suggests that these lncRNAs act as a decoy for PUMILIO. These lncRNAs seem to work as players in the differentiation process that drives breast cells to acquire distinct phenotypes related to a specific BC subtype.

Figure 1. NORAD and HCG11 presented different expression patterns in BC subtypes. (A) Analyzing TCGA cohort on luminal A (n = 231) and basal-like (n = 98) subtypes, NORAD was upregulated in Luminal A while HCG11 was upregulated in basal-like subtype. (B) Relative expression measured by RT-qPCR on luminal A (n = 21) and triple-negative (n = 23) Brazilian BC samples, NORAD and HCG11 maintained the pattern of expression observed in TCGA cohort. (C) TCGA cohort data for promoter methylation. HCG11 presented in Luminal A subtype greater promoter methylation. (D) TCGA cohort data for promoter accessibility was performed by assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). HCG11 exhibited a more accessible promoter in Basal-like subtype. * p < 0.05; ** p < 0.01; *** p < 0.001; N.S - Not Significative

Figure 2. Overall survival determined by the Kaplan-Meier plots method and the log-rank test according to NORAD and HCG11 expression (median value). Red and blue lines refer to patients with low and high expression, respectively. Disease-free survival information of breast cancer patients was downloaded from the TCGA cohort.

Figure 3. Ranked dES plot for NORAD and HCG11 using TCGA data. (A) Stratification of TCGA cohort-based on regulon activity and status of the key attributes: ER, PR, and HER2 status. The red section of the plot indicates regulon activation, and the blue section regulon repression. (B) Venn diagram showing the intersection among NORAD and HCG11 regulons and the potential PUMILIO targets; gene list is representing the gene set from both regulons. Genes that present positive or negative mutual information in each regulon are shown in red or green, respectively. Those that are also up or down-regulated in the basal-like signature are shown in blue or pink cells, respectively. (C) Gene Ontology (GO) analysis of the HCG11 and NORAD regulons indicated in (B). (D) Comparison among molecular signatures for the basal-like (up and down-regulated protein-coding genes) with regulons for NORAD and HCG11.