Long non-coding RNA dysregulation is a frequent event in non-small cell lung carcinoma pathogenesis

Abstract

Background: Long non-coding RNAs compose an important level of epigenetic regulation in normal physiology and disease. Despite the plethora of publications of lncRNAs in human cancer, the landscape is still unclear.

Methods: Microarray analysis in 44 NSCLC paired specimens was followed by qPCR-based validation in 29 (technical) and 38 (independent) tissue pairs. Cross-validation of the selected targets was achieved in 850 NSCLC tumours from TCGA datasets.

Results: Twelve targets were successfully validated by qPCR (upregulated: FEZF1-AS1, LINC01214, LINC00673, PCAT6, NUTM2A-AS1, LINC01929; downregulated: PCAT19, FENDRR, SVIL-AS1, LANCL1-AS1, ADAMTS9-AS2 and LINC00968). All of them were successfully cross validated in the TCGA datasets. Abnormal DNA methylation was observed in the promoters of FENDRR, FEZF1-AS1 and SVIL-AS1. FEZF1-AS1 and LINC01929 were associated with survival in the TCGA set.

Conclusions: Our study provides through multiple levels of internal and external validation, a comprehensive list of dysregulated lncRNAs in NSCLC. We therefore envisage this dataset to serve as an important source for the lung cancer research community assisting future investigations on the involvement of lncRNAs in the pathogenesis of the disease and providing novel biomarkers for diagnosis, prognosis and therapeutic stratification.

Fig. 1. Overexpressed lncRNAs in NSCLC.

Scatterplot diagrams demonstrating the expression levels of lncRNAs FEZF1-AS1 (a), LINC01214 (b), LINC00673 (c), PCAT6 (d), LINC01929 (e) and NUTM2A-AS1 (f), which are overexpressed in lung tumours compared to adjacent normal lung tissue. Means are also indicated along with the 95% confidence intervals. Technical validation values are given by circles, while biological independent validation values are given as triangles. p-values are derived from Wilcoxon ranked tests. RQ relative quantity.

Fig. 2. Downregulated lncRNAs in NSCLC.

Scatterplot diagrams demonstrating the expression levels of lncRNAs PCAT19 (a), SVIL-AS1 (b), LANCL1-AS1 (c), FENDRR (d), LINC00968 (e) and ADAMTS9-AS2 (f), which are downregulated in lung tumours compared to adjacent normal lung tissue. Means are also indicated along with the 95% confidence intervals. Technical validation values are given by circles, while biological independent validation values are given as triangles. p-values are derived from Wilcoxon ranked tests. RQ relative quantity.

Fig. 3. Bar chart diagrams showing the expression levels of the validated lncRNAs in NSCLC cell lines and a normal lung fibroblast cell line (IMR90), which was used as a calibrator (RQ = 1) for the analysis, with the exception of FEZF1-AS1 and LINC01929, which are not expressed in IMR90.

In this case Calu-1 was used as calibrator. All downregulated lncRNAs in lung cancer tissue were reduced (SVIL-AS1 (a), FENDRR (b), ADAMTS9-AS2 (c)) or completely undetected (PCAT19, LANCL1-AS1 and LINC00968, data not shown). Upregulated in lung cancer tissue lncRNAs did not necessarily follow the same pattern in cell lines. FEZF1-AS1 (d), LIN00673 (e) and LINC01214 (f) were higher in NSCLC cell lines, PCAT6 (g) and LINC01929 (h) were lower in NSCLC cell lines compared to IMR90, while NUTM2A-AS1 (i) was highly variable. Error bars represent standard error of the mean. RQ relative quantity.

Fig. 4. DNA methylation of lncRNA promoters in NSCLC.

Scatterplot diagrams demonstrating the DNA methylation levels of lncRNAs FENDRR (a), FEZF1-AS1 (b) and SVIL-AS1 (c) in lung tumours compared to adjacent normal lung tissue. The horizontal dotted line in each diagram defines the 95% reference range (= mean + 2 × Standard deviation) of the normal tissues. Tumour samples with values over this line are classified as hypermethylated.