PNO1 was overexpressed in PCa samples.
Three independent GEO datasets, GSE45016 , GSE55945  and GSE17951 , were used to determine the expression levels of PNO1 in PCa samples. Our results revealed PNO1 was markedly up-regulated in PCa compared to normal tissues. Moreover, we detected FAM64A expression in PCa cell lines, including LNCaP, PC-3 and DU145 cells, and found FAM64A was highly expressed in metastatic cell lines PC-3 and DU145 than that in LNCaP.
Silencing of PNO1 suppressed PCa cell proliferation
In present study, we used lentiviral vectors mediated knockdown to suppress PNO1 expression in PC-3 and DU145 cells. By using RT- qPCR, we found that 80% and 65% PNO1 endogenous expression was knockdown in DU145 and PC-3 cells, respectively. western blot assay also showed the protein levels of PNO1 in DU145 and PC-3 cells were also successfully knockdown using a special shRNA.
Celigo analysis was used to detect the effect of PNO1 knockdown on PCa proliferation. Five days’ post transfection, we found the cell numbers in PNO1 knockdown groups decreased by 88.3% and 65% compared to control group in both DU145 and PC-3 cells. Similar with Celigo analysis results, the CCK-8 assay also demonstrated that PNO1 knockdown suppressed proliferation compared to control (P<0.01; Fig. 3D and E).
In addition, we found that knockdown of PNO1 suppressed DU145 and PC-3 cell colony formation. The relative colony number in PNO1 knockdown decreased by 85- and 78- percent in DU145 and PC-3 cells, respectively.
PNO1 knockdown induced apoptosis in PCa cells.
To investigate the effect of PNO1 knockdown on PCa cell apoptosis, cells transfected with shPNO1 and shCtrl were subjected to FACS analysis. We revealed the apoptosis of PC-3 and DU145 cells was significantly increased after PNO1 knockdown compared with control groups. These results suggested that PNO1 suppressed PCa cell apoptosis.
Bioinformatics analysis revealed the targets regualted by PNO1 in PCa.
Microarray analysis was conducted to identify PNO1 targets in PCa. Totally, 291 genes were found to be up-regulated and 498 genes were found to be suppressed after PNO1 knockdown in PC-3 cells. The top 10 up- and down-regulated genes were shown in Table1. Bioinformatics analysis revealed PNO1 induced genes was involved in regulating positive regulation of DNA repair, single organismal cell-cell adhesion, cellular amino acid metabolic process, response to stress, preassembly of GPI anchor in ER membrane, membrane to membrane docking, regulation of angiogenesis, viral process, post-translational protein modification, and response to oxidative stress. Meanwhile, the PNO1 reduced genes were involved in regulating translational initiation, RNA splicing, transcription, DNA-templated, positive regulation of mRNA catabolic process, regulation of energy homeostasis, cellular response to hypoxia, rRNA processing, mRNA processing, energy reserve metabolic process, and response to unfolded protein.
Identification of key targets of PNO1 in PCa using PPI network analysis
Furthermore, PPI networks were constructed to reveal the protein-protein interaction among PNO1 induced and reduced genes using String database. A Mcode plugin in Cytoscape was used to identify hub genes in these networks. As shown in Figure 6, we showed the top 3 PNO1 up-regaulted and down-regualted genes mediated hub networks.
Five PNO1 down-regaulted genes (PRPF8, CDC5L, RPL36, RPL23, RPL28) and 10 PNO1 up-regaulted genes (TNF, EGFR, RNF213, CLTCL1, AP2B1, CXCL1, KLHL5, UBE2J1, CXCL8, PLAU) were identified as key targets of PNO1 in PCa. These genes interacted with more than 10 nodes in PPI network.
Knockdown of PNO1 suppressed prostate cancer growth in vivo
We further conducted in vivo tumor growth assay to determine the effect of PNO1 on tumor growth. A luciferase-expressing DU145 cell line transfected with shPNO1 or shNC were constructed to race tumors in live animals. In vivo tumor growth were detected using caliper measurements. The tumor growth curve analysis showed the PNO1 knockdown tumor xenografts had an obvious reduction of tumor volume relative to that in control groups. On day 41, the luciferase expression in all mice were detected and the results showed the luciferase levels in shPNO1 group was down-regulated compared to normal group. Then mice were sacrificed and the tumor xenografts were excised and weighed according to the manufacturer’s instruction. It was observed that the weight of tumor xenografts in shPNO1 group was significantly lower than that of shNC group (Figure 7C, P<0.05).