# Identification of Novel Markers of Prostate Cancer Progression, Potentially Modulated by Vitamin D
## 3 Results  
### The Vitamin D Is Able to Modulate 43 Key Genes in PCa  
The comparison of the three human PCa datasets (GSE70770, GSE62872 and GSE6919) produced 276 common significantly up-regulated genes (458 down-regulated genes) (Figure 1A) (Table S1). These common regulated key genes were compared to significantly down-regulated genes in GSE64657, composed by the transcriptome of the LNCaP cell line, treated for 24 h with 100 nM of 1,25D3 (Figure 1B). The data overlapping produced 43 genes significantly in common between the dataset (chi-square \(p<0.0001\)), namely meaning being virtually modulated by the action of Vitamin D in PCa. Gene Ontology (GO) analysis of the 43 genes belonging to the common group modulated by Vitamin  
Figure 1: Venn diagram analysis of PCa transcriptome. (**A**) The three PCa datasets (GSE70770, GSE62872 and GSE6919) overlapping produced 276 common significantly up-regulated key genes. The overlapping between GSE70770 (2279 SUG) vs. GSE62872 (2312 SUG) showed 1010 genes in common (the sum of 276 and 734). As regard GSE6919 (1747 SUG) vs. GSE70770 overlapping, we identified 411 genes. Similar results were obtained when we compared GSE6919 vs. GSE62872 (444 genes in common). (**B**) The analysis of the intersection between the transcriptome of genes common up-regulated in the three PCa datasets (276) and the transcriptome of the down-regulated genes in LNCaP cell line treated with Vitamin D for 24 h (2196 SUG), showed 43 (almost 15%) significantly common regulated (chi-squared, \(p<0.0001\)). (**C**) When we loaded the 43 genes in the GATHER browser, we showed that the biological process most significantly regulated by these genes were organogenesis, organ development, skeletal development, chromatin assembly, morphogenesis and cell migration. D identified six main molecular functions as statistically significant: _organogenesis_ (GO:0009887)(neg ln(_p_ value) = 6.37); _organ development_ (GO:0048513)(neg ln(_p_ value) = 6.37); _skeletal development_ (GO:0001501)(neg ln(_p_ value) = 5.43); _regulation of chromatin assembly or disassembly_ (GO:0001501)(neg ln(_p_ value) = 5.06); _morphogenesis_ (GO:0009653)(neg ln(_p_ value) = 4.95); _cell migration_ (GO:0016477)(neg ln(_p_ value) = 4.41) (Figure 1C). Complete annotation, significance and genes involved are available in Table S1, section Gather Analysis. We also overlapped the common down-regulated key genes in PCas obtained by the Venn analysis of the three datasets, with the significantly up-regulated genes in GSE64657. The overlap produced 41 genes significantly in common between the dataset (chi-square p \(<\) 0.0001), also modulated by Vitamin D (Table S1). In this study, we decided to focus our analysis only to the 43 key genes in common obtained overlapping the up-regulated genes in cancer and the genes down-regulated by the Vitamin D. We used these 43 genes to generate a network analysis by GeneMania web utility software (Figure 2A). The 92.30% of genes resulted co-expressed, 4.61% belonging to the same pathways, 1.97% present a genetic interaction, 0.82% were co-localized and 0.30% shared the same protein domains (Figure 2B). Complete data report is available in Table S2. Belonging to among the 43 key genes, we selected the six most significantly up-regulated in PCa and down-regulated by the action of Vitamin D in LNCaPP cell line: _serine protease 8_ (PRSS8); _SRY-box 4_ (SOX4); _SET and MYND domain containing 2_ (SMYD2); _methylcrotonoyl-CoA carboxylase 2_ (MCCC2); _cyclin G_ (CCNG2); _CD2 associated protein_ (CD2 AP). We decided to perform a Z-score transformation in order to allow the comparison of microarray data independent of the original hybridization intensities. The three microarray dataset clustering (GSE/0770, GSE62872 and GSE6919) allowed us to obtain a large number of samples (251 healthy control and 548 PCa samples). Complete demographic data of patients and healthy controls can be retrieved from the relative publication and in the Table S1. The samples were homogeneous for the age (Table S1).