PNO1 promotes cell proliferation in prostate cancer

Prostate cancer (PCa) is one of the most commonly diagnosed cancers. The functions of PNO1 in yeasts were involved in regulating ribosome and proteasome biogenesis. However, its roles in PCa remained largely unclear. The present study for the rst time demonstrated PNO1 was up-regulated in PCa samples compared to normal tissues. ShRNA mediated knockdown of PNO1 signicantly suppressed PCa proliferation and clone formation, however, induced PCa apoptosis. Microarray analysis and bioinformatics analysis revealed PNO1 was involved in regulating multiple cancer related biological processes, such as regulation of DNA repair, single organismal cell-cell adhesion, translational initiation, RNA splicing, transcription, and positive regulation of mRNA catabolic process. OF note, in vivo results showed PNO1 knockdown remarkably reduced the PCa growth rate. Despite more in-depth research is still required, this study showed PNO1 could serve as a potential biomarker for PCa.

In vivo tumorigenicity shPNO1-infected or control PC-3 cells were suspended in phosphate-buffered saline (PBS) and injected subcutaneously into the backs of six-week-old male BALB/c nude mice (Shanghai SLAC Laboratory Animals Co., Ltd. Shanghai, China). A luciferase-expressing PNO1 knockdown or control DU145 cell line were constructed to race tumors in live animals. Bioluminescence imaging (BLI) was conducted to monitor in vivo tumor growth using an in vivo imaging system-Lumina II (Perkin Elmer). Each mouse was intraperitoneally injected with 150 mg/kg luciferin potassium salt (115144-35-9; Fanbo). Ten minutes later, the mice were anesthetized using 1.5% iso urane. To maintain body temperature, mice were placed on a thermostatically controlled heating pad (37°C) during imaging. Acquisition binning and duration were set according to tumor activity. Signal intensity was quanti ed as the total ux (photons/s) within regions of interest drawn manually around the tumor area using Living Image 4.0 software (Perkin Elmer). Tumor growth was measured using calipers over the course of 49 days. Tumor volume was calculated according to the formula: volume = 0.5 × length × width2. The mice were euthanized with CO2 and sacri ced on day 49. Tumor weight was measured and compared between two groups. All in vivo studies protocols were approved by the Shanghai Medical Experimental Animal Care Commission (Approval ID: ShCI-14-008).

Western blot analysis
In accordance with standard Western-blot protocols, proteins were separated in 10% SDS-PAGE and transferred to PVDF membrane through Bio-Rad systems (Bio-Rad, Hercules CA, USA). Rabbit anti-PNO1, and mouse anti-GAPDH antibodies were used in this study. The Quantity One software package (Bio-Rad, USA) was used for the quantitation of signal intensities.

Statistical analysis
The SPSS 19.0 software (IBM Corp, Armonk, NY, USA) was used to perform statistical analysis. Each experiment was performed for three times. Differences between 2 groups was calculated using student' Ttest. For more groups, one-way ANOVA followed by Newman-Keuls posthoc test was used. P<0.05 was considered to indicate a statistically signi cant difference.

Results
PNO1 was overexpressed in PCa samples.
Three independent GEO datasets, GSE45016 [19], GSE55945 [20] and GSE17951 [21], were used to determine the expression levels of PNO1 in PCa samples. Our results revealed PNO1 was markedly upregulated 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 78percent 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 signi cantly 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, DNAtemplated, 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 modi cation, and response to oxidative stress ( Figure 5C).
Identi cation 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 identi ed as key targets of PNO1 in PCa.
These genes interacted with more than 10 nodes in PPI network.
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 coexpression 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 sacri ced 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 signi cantly lower than that of shNC group ( Figure 9C-D, P<0.05).

Discussion
The functions of PNO1 in human cancers remained unclear. Previous studies showed PNO1 was involved in regulating ribosome and proteasome biogenesis [8]. NOB1, a PNO1 cofactor, was overexpressed in multiple cancer types, including laryngeal cancer [11], and ovarian cancer [22]. These reports suggested PNO1 may be also involved in regulating progression of human cancers. In present study, we for the rst time demonstrated PNO1 was up-regulated in PCa samples compared to normal tissues. ShRNA mediated knockdown of PNO1 signi cantly suppressed PCa proliferation and clone formation. Moreover, our results showed PNO1 knockdown induced PCa apoptosis. OF note, in vivo results showed PNO1 knockdown remarkably reduced the PCa growth rate. Until to now, this is the rst study revealed PNO1 played as an oncogene in PCa. Very interestingly, we found knockdown of PNO1 had no signi cantly effects on cell cycle regulators by analyzing microarray data. Moreover, we found that knockdown of PNO1 did not affect the cell cycle progression using ow cytometer (data not shown). Based on these ndings, we thought the regulation of PNO1 on PCa cell proliferation may not depend on cell cycle process. Despite we validated that PNO1 could suppress cell apoptosis, the mechanisms of PNO1 regulating PCa proliferation remained to be further investigated.
Due to that emerging studies demonstrated that the functions of PNO1 were involved in regulating ribosome and proteasome biogenesis in yeast and the site 3 cleavage at the 3 -end of 18S pre-rRNA in human, we thought PNO1 may play its functions in PCa cells through affecting a series of targets. With the development of high-through methods, microarray and RNA-sequence were widely used to identify functions and molecular mechanism of novel genes in human cancers. For example, Yao Li et al conducted microarray to reveal downstream targets of Androgen Receptor (AR) [23]. Sunkel et al identi ed CREB1/FoxA1 transcriptional targets using ChIP-seq and RNA-seq method [24]. Despite a previous study showed EBF1-mediated upregulation of PNO1 contributes to cancer progression by negatively regulating the p53 signaling pathway. The mechanism of PNO1 in PCa remained to be further investigated [16]. Thus, in present study, we investigated the mechanisms of PNO1 in PCa using highthroughput method and bioinformatics analysis. In this study, we identi ed 291 up-regulated and 498 down-regulated targets of PNO1 in PCa. Bioinformatics analysis showed PNO1 was involved in regulating multiple cancer related pathways, such as DNA repair [25], regulation of angiogenesis [26,27], translational initiation [28,29], RNA splicing [30,31], and cellular response to hypoxia [32,33]. Emerging studies showed these pathways played crucial roles in PCa progression and treatment. Defective DNA repair induced tumour evolution and progression. DNA repair pathway was widely observed to be mutated in primary and advanced-stage PCa [34]. Angiogenesis was considered one of the hallmarks of tumor initiation, growth and development. Emerging studies had demonstrated angiogenesis also plays a fundamental role for PCa growth [35]. Changes in mRNA splicing patterns have been associated with key pathological mechanisms in prostate cancer progression [36].
TNF was involved in regulating cancer apoptosis through multiple kinases. Including ASK1 and MEK4 [38]. EGFR played crucial roles in regulating PCa proliferation and apoptosis [39,40]. Targeting EGFR was considered as a potential therapeutic target. CXCL1 was increased in high-grade PCa. In PCa, CXCL1 was reported to induce cell growth and metastasis through activating a secretory network [41]. KLHL5 knockdown was able to increase the sensitivity of cancer cells to anticancer drugs [42]. clueGO analysis showed that PNO1 was involved in regulating multiple cancer related pathways via these targets. For example, we found that PNO1 was involved in regulating Spliceosome via down-regulating GCFC2, PRPF8, PRPF3, RBM22, SART1, RBM25, SNRNP200, CDC5L, and SMNDC1, 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. Ubiquitin conjugating enzymes were reported to participate in regulating cancer proliferation, metastasis [43][44][45]. In present study, microarray analysis showed UBE2Z, UBE2N, UBE2J1 and UBE2G1 was down-regulated after PNO1 knockdown in PCa cells. Furthermore, GEPIA database analysis showed PNO1 was positively correlated to the expression of UBE2Z, UBE2N, UBE2J1 and UBE2G1. UBE2Z functions as an E2 enzyme downstream of UBA6 in the conjugation of either ubiquitin or FAT10 onto target proteins [46]. A previous study showed UBE2Z up-regulation is associated with human hepatocellular carcinoma [47]. UBE2N was also found to be up-regulated in glioblastoma, colorectal cancers, and Melanoma [48]. For instance, UBE2N Promotes Melanoma Growth via MEK/FRA1/SOX10 Signaling [48]. These reports and our nding showed PNO1 played key roles in PCa through regulating these important cancer regulators.
In this study, there also existed some limitations. Firstly, we evaluated the potential mechanisms of PNO1 in PCa based on microarray analysis and bioinformatics analysis. These ndings should be further validated using experimental assays. Secondly, the bioinformatics analysis demonstrated that PNO1 was involved in regulating angiogenesis. Exploring the roles of PNO1 in regulating angiogenesis could broad our understanding about the functional importance of this gene in PCa. Thirdly, despite bioinformatics analysis and functional validation showed PNO1 was related to angiogenesis, RNA splicing and apoptosis. However, whether PNO1 affect PCa proliferation through these pathways remained to be further investigated in the near future.
In conclusion, this study for the rst time showed PNO1 was up-regulated in PCa. PNO1 played its oncogenetic role in PCa progression through inducing cell proliferation and reducing cell apoptosis. Despite more in-depth research is still required, this study showed PNO1 could serve as a potential biomarker for PCa.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Competing interests
The authors declare that they have no con ict of interest.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.  Table   Table 1. The top 10 up-and down-regulated genes after knockdown of PNO1.   Differences between 2 groups was calculated using student' T-test. For more groups, one-way ANOVA followed by Newman-Keuls posthoc test was used. p < 0.05; **, p < 0.01; ***, p < 0.001).  analysis results presented as mean ± SD (n = 3). Differences between 2 groups was calculated using student' T-test. Signi cance was de ned as p<0.05 (*, p < 0.05; **, p < 0.01; ***, p < 0.001).     The luciferase signaling in PNO1 knockdown group was signi cantly lower than that in control groups. (C) The growth curve revealed PNO1 knockdown signi cantly inhibits PCa growth in vivo. Differences between 2 groups was calculated using student' T-test. Data are presented as the mean ± SD (n = 3) *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Supplementary Files
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