LncRNA ENST00000539653 acts as an oncogenic factor via MAPK signalling in papillary thyroid cancer

Abstract

Background: Papillary thyroid cancer (PTC) is the most frequent type of thyroid malignancy. In this study, we investigated the mechanisms whereby long non-coding RNAs (lncRNAs) are associated with PTC pathogenesis.

Methods: Microarray analysis was used to determine differentially expressed lncRNAs between paired PTC tissues and normal adjacent thyroid tissues. Quantitative RT-PCR was used for validation in 86 PTC cases. Small interfering RNA (siRNA) transfection assays were then performed to assess how a novel lncRNA affected key proliferation and cell death pathways in IHH4 PTC cells.

Results: We identified 1878 differentially expressed lncRNAs versus matched control samples (fold change ≥2.0, P < 0.05), of which 429 were upregulated and 1449 were downregulated. ENST00000539653.1 (ENS-653), one of the top hits in this microarray, was selected for further study. Higher ENS-653 expression was observed in PTC tissue samples versus adjacent normal tissues, and was associated with a larger tumor size and a more advanced clinical stage. In the Cancer Genome Atlas (TCGA) PTC cohort, higher ENS-653 expression was correlated with more frequent BRAF (V600E) mutation and poorer disease-free survival. Furthermore, ENS-653 downregulation reduced the proliferation of PTC cells and led to G1-S arrest, but had no impact on apoptosis. ENS-653 downregulation also inactivated ERK1/2 and ERK5, causing partial MAPK cascade suppression.

Conclusion: ENS-653 exhibits oncogenic properties in PTC, and could be a diagnostic and/or prognostic PTC biomarker, in addition to possibly being a future target for therapy.

Keywords: Papillary thyroid cancer ∙ LncRNA ∙ proliferation ∙ biomarker ∙ MAPK.

Fig. 1

LncRNA microarray analysis in papillary thyroid cancer (PTC) tissues compared with matched adjacent noncancerous thyroid tissues. a Hierarchical clustering analysis of differentially expressed lncRNAs. Red and green colors indicate high and low expression, respectively. In the heat map, columns represent samples and rows represent each lncRNA. b Volcano plot of differentially expressed lncRNAs between PTC and paired noncancerous thyroid tissue. The vertical lines correspond to 2.0-fold upregulation and downregulation, and the horizontal line represents a P value of 0.05

Fig. 2

QRT-PCR validation of selected differentially expressed lncRNAs. a Comparison between microarray and qRT-PCR results. The heights of the columns represent the log-transformed median fold changes (Tumor/Normal tissues) in the expression in 86 paired PTC and noncancerous thyroid tissues. b Relative expression level of ENS-653 in 86 pairs of PTC and noncancerous thyroid tissues. ENS-653 expression was evaluated by qRT-PCR and normalized to GAPDH mRNA expression. Data are expressed as a mean ± SD. c Proportion of cases with positive fold changes of ENS-653 expression in PTC compared with noncancerous thyroid tissues. **P < 0.01

Fig. 3

Association of ENS-653 expression with tumor size and stages in PTC patients. a ENS-653 expression was classified into two groups. Final results are presented as fold change in tumor tissues relative to normal tissues. Fold change is greater than or equal to 2.0 for high expression, and less than 2.0 for low expression. b, c ENS-653 upregulation was associated with larger tumor size and advanced stages. Data are expressed as median, **P < 0.01

Fig. 4

Association of ENS-653 expression with BRAF (V600E) mutation and disease-free survival in TCGA PTC cohort. a Statistical analysis of ENS-653 expression between BRAF (V600E) mutation and wtBRAF PTC patients. Data are expressed with median (interquartile range). **P < 0.01. b Survival was analyzed and compared between patients with high and low levels of ENS-653 expression; n = 457, log-rank test

Fig. 5

ENS-653 downregulation suppresses PTC cell proliferation. a qRT-PCR of IHH4 cells transfected with siRNA silencing ENS-653 (Si-653) or negative control (Si-NC). b CCK-8 assays were performed in Si-653 or Si-NC transfected IHH4 cells. Values represent mean ± SD from three independent experiments in triplicate. c Colony forming assays were performed in Si-653 or Si-NC transfected IHH4 cells. d Relative colony numbers of IHH4 cells transfected with Si-653 or Si-NC. Values represent mean ± SD from three independent experiments in triplicate. *P < 0.05, **P < 0.01

Fig. 6

ENS-653 downregulation induces a G1 cell cycle arrest with no impact on apoptosis. a Flow cytometry was performed in IHH4 cells transfected with Si-653 (siRNA silencing ENS-653) or Si-NC (negative control). The bar chart represents the percentage of IHH4 cells in G0/G1, S or G2/M phase. b Flow cytometry was used to detect the apoptotic cells in IHH4 cells transfected with Si-653 or Si-NC. LR, early apoptotic cells; UR, terminal apoptotic cells. Values represent mean ± SD from three independent experiments. **P < 0.01. c Western blot analysis of PCNA, cyclin D1 and cleaved caspase-3 in Si-653 or Si-NC transfected and IHH4 cells. β-actin was used as a loading control

Fig. 7

Interference with ENS-653 causes partial MAPK cascade suppression. Western blot analysis of total and phosphorylated ERK1/2, ERK5, JNK and p38 in IHH4 cells transfected with Si-653 (siRNA silencing ENS-653) or Si-NC (negative control)