Identification of Long Noncoding RNAs Deregulated in Papillary Thyroid Cancer and Correlated with BRAFV600E Mutation by Bioinformatics Integrative Analysis

Abstract

Papillary Thyroid Cancer (PTC) is an endocrine malignancy in which BRAF^V600E oncogenic mutation induces the most aggressive phenotype. In this way, considering that lncRNAs are arising as key players in oncogenesis, it is of high interest the identification of BRAF^V600E-associated long noncoding RNAs, which can provide possible candidates for secondary mechanisms of BRAF-induced malignancy in PTC. In this study, we identified differentially expressed lncRNAs correlated with BRAF^V600E in PTC and, also, extended the cohort of paired normal and PTC samples to more accurately identify differentially expressed lncRNAs between these conditions. Indirectly validated targets of the differentially expressed lncRNAs in PTC compared to matched normal samples demonstrated an involvement in surface receptors responsible for signal transduction and cell adhesion, as well as, regulation of cell death, proliferation and apoptosis. Targets of BRAF^V600E-correlated lncRNAs are mainly involved in calcium signaling pathway, ECM-receptor interaction and MAPK pathway. In summary, our study provides candidate lncRNAs that can be either used for future studies related to diagnosis/prognosis or as targets for PTC management.

Figure 1

Differentially expressed lncRNAs between N × T and WT × BRAF^V600E were identified. (A) Volcano plot of DE lncRNAs between N × T (log2 fold change > 1 or < −1; adj. p-value < 1 × 10⁻⁷). (B) Volcano plot of DE lncRNAs between WT × BRAF^V600E (log2 fold change > 1 or < −1; adj. p-value < 1 × 10⁻⁴). (C) Venn Diagram of common DE lncRNAs between N × T and WT × BRAF^V600E. (D) Heatmap* of DE lncRNAs between N × T (log2 fold change > 3 or < −3; adj. p-value < 1 × 10⁻⁷). (E) Heatmap* of DE lncRNAs between WT × BRAF^V600E (log2 fold change > 2.5 or < −2.5; adj. p-value < 1 × 10⁻⁴). *For hierarchical clustering, one minus Spearman rank correlation was performed.

Figure 2

Experimental validation of DE lncRNAs. Upper part of panel displays the expression levels of the indicated lncRNAs in the TCGA analyses. The nonparametric Mann–Whitney test was applied due to the non-Gaussian expression distribution and p-value was assigned. Lower part of panel displays the experimental validation of these lncRNAs measured by qRT-PCR and calculated with 2^−ΔΔCt method using RPL19 (Ribosomal Protein L19) as endogenous control. Experiments with three biological replicates were performed using two technical replicates for each sample. These results are representative of at least two independent experiments. Values are plotted as expression mean ± Standard Error of Mean (SEM). Unpaired two-tailed t-Test assigned the p-value.

Figure 3

Indirectly validated targets of the DE lncRNAs between N × T are involved in cancer-related processes. (A) GO biological processes and (B) KEGG enriched pathways of the indirectly validated mRNA targets of the DE lncRNAs between N × T. (C) Proteins’ network of genes linked to the pathways in cancer, where black circles are validated targets and grey circles are connective proteins. (D) KEGG Pathways enrichment of the indirectly validated microRNAs targets of the DE lncRNAs between N × T.

Figure 4

Indirectly validated targets of the DE lncRNAs between WT × BRAF^V600E are involved in oncogenic pathways. (A) GO biological processes and (B) KEGG enriched pathways of the indirectly validated mRNA targets of the DE lncRNAs between WT × BRAF^V600E. (C) Proteins’ network of genes linked to calcium signaling pathway, where black circles are validated targets and grey circles are connective proteins. (D) KEGG pathways enrichment of the indirectly validated microRNAs targets of the DE lncRNAs between WT × BRAF^V600E.

Figure 5

Experimentally validated lncRNAs are important to tumor malignancy. (A) Examples of DE lncRNAs between Normal and PTC, which were confirmed in a validation set. (B) Differentially expressed lncRNAs in PTC that alter tumor malignancy.