CAPÍTULO IV Análisis y síntesis
4.3 Triangulación de datos específica por cada técnica
In the validation cohort of EO-PCa patients (validation set, RNA-seq, EO-PCa - Table 3-1), DLX6- AS1 along with DLX6 are highly and significantly overexpressed compared to normal prostate (p- value<0.0001) (Figure 4-3A). In line with the hypothesis that the pair of transcripts is coordinately expressed, expression levels of DLX6 and DLX6-AS1 are associated by a positive, linear and significant Spearman correlation (r=0.8256, p-value<0.0001) (Figure 4-3B).
Figure 4-3 Coordinated upregulation of DLX6 and DLX6-AS1 in EO-PCA cohort
(A) DLX6 and DLX6-AS1 RNA expression levels measured by RNA sequencing in tumors compared to normal tissue (NORMAL=10, TUMOR=91) from ICGC EO-PCa cohort. The horizontal bar represents the mean ± SEM. Mann-Whitney U test ****p<0.0001 (B) Corresponding Spearman correlation plot of DLX6-
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An independent cohort of patient-matched tumor/normal samples of late-onset PCa patients from the TCGA was used to validate the result from the ICGC cohort (Validation cohort, RNA-seq, TCGA matched - Table 3-1). Similar to the ICGC cohort, DLX6-AS1 expression is highly and significantly overexpressed (p<0.001), whereas DLX6 expression is not significantly different compared to their matched normal tissues (Figure 4-4A). Indeed, DLX6 expression separates into two groups with expression values from 0 to 5.7 RPKM and thus seemed to be upregulated in a subset of prostate tumors. We therefore divided the TCGA dataset in “LOW” and “HIGH” expression groups according to the median expression value of DLX6-AS1 (median= 4.11). When comparing “TUMOR-HIGH” PCa cases with their matched normal prostate tissues (“NORMAL- HIGH”), DLX6-AS1 along with DLX6 were significantly overexpressed (p<0.0001). DLX6-AS1 expression in the “TUMOR-LOW” subgroup was not significantly different compared to matched normal tissue (“NORMAL-LOW”), whereas DLX6 expression in the same subgroup was significantly downregulated in comparison to its matched normal counterpart (Figure 4-4A). Similarly to the ICGC dataset, the expression of DLX6-AS1 and DLX6 was significantly and positively correlated (Spearman r=0.7848, p-value<0.0001) (Figure 4-4B).
Figure 4-4 Coordinated upregulation of DLX6 and DLX6-AS1 in TCGA PCa cohort
(A) DLX6-AS1 and DLX6 RNA expression levels measured by RNA sequencing separated into LOW and HIGH tumor subgroups compared to matched normal tissue (NORMAL-LOW=26 , TUMOR-LOW=26, NORMAL- HIGH=26 and TUMOR-HIGH=26) from TCGA late-onset PCa cohort. Patients were divided into LOW and HIGH expression groups according to the median expression value of DLX6-AS1 in tumor tissue. The horizontal bar represents the mean ± SEM. Wilcoxon signed-rank test ****p<0.0001, ***p<0.001, **p<0.01, ns= non-significant. (B) Spearman correlation plot of DLX6-AS1 and DLX6 expression measured by RNA sequencing in the TCGA PCa cohort.
63 Overall, DLX6/DLX6-AS1 pair is overexpressed in a subset of PCa patients and the expression of both genes is positively associated independently of the age at diagnosis of patients.
To examine whether DLX6 expression is regulated by DNA methylation in both datasets, corresponding 450k data were used. Direct comparison of the normal versus tumor samples from the ICGC cohort did not reveal a difference at the DNA level (Figure 4-5B). Since DLX6 overexpression was restricted to a subgroup in the TCGA dataset, we also divided the cancerous patients of the ICGC cohort into “LOW” and “HIGH” subgroups according to DLX6-AS1 median expression value (median= 0.89) (Appendix Figure 7-1). In agreement with elevated DLX6 expression in the “HIGH” PCa samples (Appendix Figure 7-1) the DLX6 promoter region is significantly hypomethylated in the “HIGH” versus “LOW” patients in the ICGC cohort (Figure 4- 5B). DNA methylation changes are even more pronounced when comparing the “TUMOR-HIGH” and “TUMOR-LOW” subgroup in the TCGA cohort. Indeed, DNA methylation in samples of patients belonging to the “TUMOR-HIGH” subgroup shows a similar profile at the DLX6 promoter as the matched (“NORMAL-HIGH”) and non-matched (“NORMAL-LOW”) normal samples. In contrast, in the subset of patients associated with low DLX6-AS1 expression (“TUMOR-LOW”) the DLX6 promoter is hypermethylated compared to matched “NORMAL-LOW” or to the “TUMOR- HIGH” subgroup of samples (Figure 4-5C).
Noteworthy, in both cohort the most pronounced DNA methylation differences are observed in eight CpG probes (cg09327602, cg01229860, cg05663341, cg04599026, cg02898094, cg20479774, cg07598549 and cg20703729) (Figure 4-5A probes highlighted with blue), suggesting that the regulatory elements controlling DLX6 expression overlap with these regions.
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Figure 4-5 Hypomethylation of DLX6 promoter in “HIGH” subset of PCa patients
(A) Representation of HumanMethylation450 beadchip probes and Mass-Array amplicons overlapping with DLX6 promoter region and CGIs. (B and C) DNA methylation levels are displayed on the y-axis as a percentage ranging from 0% to 100%. (B) Each point represents mean methylation levels of single CpG sites in normal prostate, tumors as well as tumors separated into the HIGH or LOW subgroups of EO-PCa patients from the ICGC cohort. (C) Each point represents mean methylation levels of single CpG sites in matched normal/tumor samples subdivided into HIGH and LOW subgroups of late-onset patients from TCGA. (B) Mann-Whitney U test or (C) Wilcoxon signed-rank test was used to calculate statistical
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significances between each group by comparing the mean DNA methylation levels across the eight CpG sites highlighted in light blue. ****p<0.0001, ***p<0.001, ns= non-significant.
We further tested whether DLX6 expression levels are inversely proportional to DLX6 promoter methylation levels. Based on the combined average DNA methylation of the eight CpG probes correlated with DLX6 expression, we could show that DLX6 expression and DLX6 promoter methylation are significantly and negatively correlated in both ICGC (r= -0.7720, p-value<0.0001) (Figure 4-6A) and TCGA datasets (r= -0.5337, p-value<0. 0001) (Figure 4-6B).
Figure 4-6 DLX6 expression is inversely correlated with DLX6 promoter DNA methylation
Spearman correlation plot of DLX6 expression measured by RNA-sequencing and the average DNA methylation across the eight CpG sites for each sample in (A) ICGC or (B) TCGA datasets.
Collectively, these results show that elevated DLX6/DLX6-AS1 expressions along with DLX6 promoter hypomethylation are linked to a subset of cancer patients. The positive correlation between DLX6 and DLX6-AS1 suggests that the expression of these two genes is linked by a common mechanism. Our hypothesis is that DLX6-AS1 expression regulates DLX6 mRNA expression through hypomethylation of the DLX6 promoter region in a mechanism that might involve DNA demethylation.
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