# 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
We showed that all genes increased significantly with respect to the single dataset (Figure 3A) (Figure S1). Figure 2: Network and GO analysis by GeneMania. (**A**) The 43 key genes up-regulated in PCa biopsies and down-regulated in the LNCaPP cell line treated with Vitamin D (100 nM for 24 h) were loaded into GeneMania, in order to generate a GO and a Regulatory molecular network. Red circle genes represent the six most significantly up-regulated in PCa and down-regulated by the action of Vitamin D in our LNCaPP cell line (**B**). The analysis showed that 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. In order to verify a possible correlation between the selected genes and the PCa tumor progression, we correlate the z-score intensity expression levels of all six genes highlighted with the genes expression of _kallikrein related peptidase 3_ (KLK3). The KLK3 is a serine protease produced by epithelial cells in the prostate and its protein is also known as _PSA_ (prostate _specific antigen_). It is largely demonstrated that the serum levels of PSA increase in the presence of cancer [36].We showed a significantly positive correlation between all genes and KLK3 gene expression (Figure 3B). SOX4 was an exception (Table S1). Interesting data were obtained when we decided to analyze the correlation between the z-score expression levels of the six genes and the histological parameter of tumor percent, available in the datasets. We showed that all genes were significantly positively correlated to the tumor % (\(p<0.0001\) with r = 0.3246 for PRSS8; \(p=0.0003\) with r = 0.2449 for MCCC2; \(p=0.0045\) with r = 0.1922 for SOX4; \(p<0.0001\) with r = 0.3734 for CCNG2; \(p<0.0001\) with r = 0.3030 for SMYD2; \(p<0.0001\) with r = 0.3162  
Figure 3: Selection of six key genes modulated in PCa. (**A**) In order to allow the comparison of microarray datasets independent of the original hybridization intensities, we decided to apply a Z score transformation. The normalization allowed us to applicate a Z test for the six genes most significantly up-regulated in PCa and down-regulated by the action of Vitamin D in the LNCaP cell line. We obtained 251 healthy subjects and 548 PCa samples. All genes selected (KLK3, PRSS8, SOX4, SMYD2, MCCC2, CCNG2 and CD2 AP) resulted significantly up-regulated in PCa biopsies compared to prostate biopsies of healthy subject (\(p<0.0001\)) (**B**). A significantly positive correlation was observed between all six key genes selected and KLK3 expression levels. (**C**) Similar results were obtained when we analyze the correlation between the six genes’ z-score expression levels and the tumor percent. Data are expressed as z-score intensity expression levels and presented as vertical scatter dot plots. \(p\) values \(<0.05\) were considered to be statistically significant **** \(p<0.00005\)). for CD2 AP) (Figure 3C) (Table S1), particularly, we identified that CCNG2 was the most correlated to the tumor %. Furthermore, we also hypothesized that the genes involved in the production and inactivation of Vitamin D could be modulated in PCa patients. The expression levels analysis of the _cytochrome P450 family 27 subfamily B member 1_ (CYP27B1), the _cytochrome P450 family 24 subfamily A member 1_ (CYP24A1), the _cathelicidin antimicrobial peptide_ (CAMP) and the _vitamin D receptor_ (VDR) showed interesting results (Figure 4). All genes, except CYP27B1 (\(p=\) ns), were downregulated in PCa patients compared to healthy controls subjects (CYP24A1 \(p<0.0001\); CAMP \(p<0.0001\); VDR p\(<0.0001\)) (Figure 4A). In addition, we showed that the expression levels of VDR and CYP24A1 were negatively correlated with KLK3 in healthy (CYP24A1 with r = \(-0.1513\) and \(p=0.0164\); VDR with r = \(-0.1616\) and \(p=0.0103\)) (Figure 4B) and in PCa patients (CYP24A1 with r = \(-0.1975\) and \(p<0.0001\); VDR with r = \(-0.2651\) and \(p<0.0001\)) (Figure 4C).