Analyses of GO terms

3.8.1 GO analyses of miR-124 predicted targets of human and mouse

GO term analysis of miR-124 predicted targets of human and mouse was done with GOseq (M. D. Young et al., 2010). First, functional sets of targets were defined according to a Context++ Score cutoff corresponding to the median value of Context++ Scores for both human and mouse (-0.17). Good targets were defined as all miR-124 predicted targets <= -0.17 and were used for GOseq analysis. Before the cutoff there were 4,154 filtered predicted targets for human and 4,935 for mouse, after the cutoff there were 2,086 filtered predicted targets for human and 2,520 for mouse.

GOseq analysis was done with filtered predicted targets. The universe of genes was the total number of genes represented on the each species microarray platform (18,154 for human and 17,441 for mouse). GO terms used for the analysis were the ones mentioned before (1,801 GO terms for human and for mouse 1,799, Methods 3.5). The method of analysis was

“Hypergeometric” as mentioned in GOseq vignette for microarray data. FDR of the p-value results from GOseq analyses was calculated with the p.adjust function. Afterwards, the enriched GO terms in human and mouse predicted targets were compared.

Material and methods

3.8.2 GO analyses of human and mouse microarray experiments

After assessing GO analyses methodologies (Appendix 3.1 and 3.2), the camera function was chosen to perform all GO term analyses (D. Wu & Smyth, 2012). The camera function uses the expression matrix of the experiment, the design matrix used in the differential expression analysis, an index of the GO terms with the position of the genes (associated to such GO term) in the expression matrix and the contrast for which the test is desired. GO terms used for the analysis were the ones mentioned before (1,801 GO terms for human and for mouse 1,799, Methods 3.5).

After performing GO terms analyses, results were compared between same organisms experiments and between experiments. Visualisation of such comparisons were done with heatmaps using the pheatmap function in R (Kolde, 2015).

3.8.4 GO term analysis of miR-124 species-specific and shared functional targets

GO term analysis of miR-124 species-specific and shared functional targets between human and mouse was assessed to find common GO terms regulated by species-specific and shared miR-124 functional targets. To do this, sets of species-specific functional and shared functional targets for human and mouse were defined. From the filtered functional sets of predicted targets (Methods 3.8.1)., species-specific and shared targets were defined. Shared targets were defined as all genes that are predicted target for both human and mouse; functional shared targets are shared targets that pass the Context++ Score cutoff (<= -0.17) in both human and mouse.

Species-specific targets were define as all miR-124 functional targets that are not shared targets. After all these processing, three sets were defined:

species-specific functional human miR-124 predicted targets (744 targets), species-specific functional mouse mir-124 predicted targets (1,141 targets) and shared functional predicted targets (868 targets).

Material and methods

GO term analyses of sets of predicted targets was done using two gene universes: differentially expressed genes of human and mouse tissue atlas microarray experiments (6,775 for human and 6,891 for mouse) and downregulated genes of the same experiments (1,965 for human and 3,034 for mouse). Used GO terms were the same as previous mentioned analyses before (1801 GO terms for human and for mouse 1799, Methods 3.5 ) and GOseq method of analysis was “Hypergeometric”. Afterwards, comparison of enriched GO terms between species-specific and shared targets analyses was done.

3.8.5 Enrichment analysis of up and down regulated set of genes in human and mouse data

For Camera analyses "artificial GO terms" were also tested in human and mouse microarray experiments. The “artificial GO terms” consist of the most downregulated and most upregulated genes in each of the microarray experiments of human and mouse. Down and up regulated genes were defined according to Sylamer plot results. Let's remember that genes of Sylamer analysis are order from downregulated to upregulated.

Downregulated gene set consist of the first gene to the gene at the first peak of enrichment of Sylamer plot. Upregulated gene set had the same number as the downregulate gene set but genes were taken from the end of the ordered 3' UTRs, the same way as when comparing camera and fry methods (Appendix 3.1). Then, all the experiments had up and down regulated sets of genes. For human there were 6 gene sets: 3 upregulated and 3 downregulated of each experiment (glioblastoma and time course overexpression, and tissue atlas experiment) and for mouse there were 4 gene sets: 2 upregulated and 2 downregulated of each experiment (overexpression and tissue atlas). The number of genes in the gene sets differed due to the different results of enrichment peaks in Sylamer plots, (Results 4.2). For glioblastoma overexpression experiment each gene set had 550 genes, for time course and overexpression tissue atlas each gene sets had 1,000. For mouse overexpression each gene set had 550 genes and the tissue atlas gene sets had 1,000.

Material and methods

The reason of this analysis was to find if up and down regulated genes were enriched in the different experiments, which would aid us in understanding if, for example, miR-124 direct effect (overexpression experiments) was also found in tissue atlas experiments and vice versa.

3.8.6 Analyses of X. tropicalis tissue atlas

The X. tropicalis GO term camera analysis needed a prior data modification.

The camera function uses a numeric matrix of expression values or log-ratios of expression, to use it in RNA-seq data. Due to this X. tropicalis counts matrix was transformed to a microarray data format using the voom function from the limma R package which transforms the counts matrix to log-expression values (Ritchie et al., 2015). The parameters used for voom were:

DGEList and the design matrix. Camera analysis was done with the voom output, the GO terms index, the design matrix and the desired contrast.

Results