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 (Figure 1A-C). Moreover, we detected PNO1 expression in PCa cell lines, including LNCaP, PC-3 and DU145 cells, and found PNO1 was highly expressed in metastatic cell lines PC-3 and DU145 than that in LNCaP (Figure 1D).
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 PC-3 (Figure 2A) and DU145 (Figure 2C) cells, respectively. western blot assay also showed the protein levels of PNO1 in PC-3 (Figure 2B) and DU145 (Figure 2D) 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 PC-3 (Figure 2E-F) and DU145 (Figure 2G-H) cells. Similar with Celigo analysis results, the CCK-8 assay also demonstrated that PNO1 knockdown suppressed proliferation compared to control (P<0.01; Fig. 2I-J).
In addition, we found that knockdown of PNO1 suppressed PC-3 (Figure 3A and B) and DU145 (Figure 3C and D) 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 (Figure 4A and B) and DU145 (Figure 4C and D) 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 regulated 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 (Figure 5A). The top 10 up- and down-regulated genes were shown in Table1. Bioinformatics analysis revealed PNO1 induced genes was 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 (Figure 5B). Meanwhile, the PNO1 reduced genes were 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 (Figure 5C).
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 and 7, we showed the top 3 PNO1 up-regulated (Figure 6A-C) and down-regulated (Figure 7A-C) genes mediated hub networks. Five PNO1 up-regulated genes (PRPF8, CDC5L, RPL36, RPL23, RPL28) and 10 PNO1 down-regulated 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.
Furthermore, cytoscape plugin clueGO was used for functional enrichment analysis of these key genes in PCa. These results suggested that PNO1 correlates to translational silencing of Ceruloplasmin expression and GTP hydrolysis and joining of the 60S ribosomal subunit through suppressing EIF4A2, EIF2S1, EIF3M, and RPSA (Figure 6D). PNO1 was involved in regulating Spliceosome via down-regulating GCFC2, PRPF8, PRPF3, RBM22, SART1, RBM25, SNRNP200, CDC5L, and SMNDC1 (Figure 6D). PNO1 was involved in regulating Glycosylphosphatidylinositol (GPI)-anchor biosynthesis through enhancing PIGN, GPAA1 and PIGP, involved in regulating ubiquitin conjugating enzyme activity through promoting UBE2Z, UBE2N, UBE2J1 and UBE2G1, involved in regulating Clathrin-mediated endocytosis via promoting AP2B1, CLTCL1, AGFG1, STAM2 and EGFR (Figure 7D).
Ubiquitin conjugating enzymes played an important role in regulating degradation of unfolded protein. In order to provide several clues to validate the effects of PNO1 on these proteins in PCa, we conduction co-expression analysis between PNO1 and these ubiquitin conjugating enzymes using GEPIA database. The results showed PNO1 was positively correlated to the expression of UBE2Z (Figure 8A), UBE2N (Figure 8B), UBE2J1 (Figure 8C) and UBE2G1 (Figure 8D).
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 PNO1 stable knockdown or control DU145 cell line 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 (Figure 9A). 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 (Figure 9B and E). 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 9C-D, P<0.05).