Knockdown of P3H4 inhibits proliferation and invasion of bladder cancer

The prolyl 3-hydroxylase family member 4 (P3H4) (alias SC65) is implicated in a variety of physiological and pathological processes. However, little is known about the role of P3H4 in tumors. This study aimed to investigate the role of P3H4 in bladder cancer (BC) and the regulatory mechanisms that influence its expression. P3H4 was highly expressed in BC tissues. Knockdown of P3H4 inhibited BC cell proliferation, cell cycle, migration and invasion in vitro, and inhibited BC growth in vivo. We also found that ETV4 bound directly to the promoter region of P3H4 and activated its transcription. Furthermore, overexpression of ETV4 rescued the inhibition of proliferation and invasion induced by PH4 silencing. ETV4 was significantly overexpressed in the BC tissues. In conclusion, P3H4 functioned an oncogene role in BC progression, and ETV4 bound directly to the P3H4 promoter region to regulate its transcription.


INTRODUCTION
Bladder cancer (BC) is the most common malignancy of the genitourinary system, with more than 430,000 new patients worldwide affected each year [1,2]. BC is also the seventh leading cause of cancer-related deaths worldwide, accounting for 2.8% of these deaths [3]. Surgical resection combined with radiotherapy or chemotherapy has been applied to BC treatment [4]. However, patients receiving aggressive treatment still face the status quo of easy recurrence, poor prognosis, low survival rate and poor quality of life [5,6]. Therefore, finding new and effective therapeutic targets is urgent and important.
The prolyl 3-hydroxylase family member 4 (P3H4) (alias SC65) is originally identified as a protein associated with the association complex (SC) [7]. However, no further evidence supports this point. Subsequent studies detect antibodies against P3H4 in cases of interstitial cystitis [8], membranous nephropathy [9], prostate cancer [10], and meningeal cancer [11]. P3H4 has also been identified as an autoantigen associated with these diseases. Furthermore, distinct functions of P3H4 are reported separately. For example, P3H4 acts as an endoplasmic reticulum (ER) protein that regulates bone mass homeostasis and skin fragility [12]. Studies have shown that P3H4 is expressed at high levels in bone, cartilage and skin. It also forms a stable complex with P3H3 in ER and interacts with lysyl hydroxylase 1 and CYPB [13,14]. Additionally, P3H4 is an adaptor protein that links myelin protein zero (P0) and the activated C kinase 1 (RACK1) receptor [15], a gene that is up-regulated in the hippocampus during sleep [16], and is possibly a PTEN-interacting protein [17]. In general, P3H4 is involved in a variety of physiological AGING and pathological processes. However, little is known about the role of P3H4 in BC.
In the present study, we found that P3H4 was significantly overexpressed in BC tissues, and knockdown of P3H4 inhibits BC cell proliferation, cell cycle, migration and invasion. ETV4 regulated P3H4 transcription by binding directly to its promoter region and was involved in the regulation of BC progression. Overexpression of ETV4 rescued the inhibition of proliferation and invasion induced by PH4 silencing.

P3H4 is highly expressed in BC
To reveal the role of P3H4 in BC, we first examined the P3H4 expression in BC tissues by database analysis and IHC analysis. The GEPIA online analysis website (http://gepia.cancer-pku.cn/) was used to analyze P3H4 mRNA levels in 404 patients with bladder urothelial carcinoma (BLCA) and 28 healthy volunteers. As shown in Figure 1A, the mRNA levels of P3H4 were significantly higher in BLCA patients compared to the healthy volunteers. The disease-free survival and overall survival of BLCA patients with low P3H4 expression was obviously higher than those of BLCA patients with high P3H4 expression ( Figure 1B and 1C). In addition, we collected 32 BC tissues and 26 normal tissues to measure P3H4 expression. As shown in Figure 1D and Table 1, P3H4 was high expressed in BC tissues compared to normal tissues. Furthermore, statistical analysis demonstrated that high expression of P3H4 in BC tissues was related to gender (Table 2). Moreover, the incidence of BC in males is 3-4 times that of females. These results suggest that P3H4 may play an oncogene function in BC. carcinoma (BLCA) tissue and normal bladder tissue, respectively. The data came from the GEPIA database. Disease free survival (B) and overall survival (C) percentage of BLCA patients with high or low P3H4 expression. (D) P3H4 expression in bladder cacner (BC) tissues and adjacent normal tissues was examined by Immunohistochemical (IHC) analysis. After siRNA targeting P3H4 (siP3H4) was transfected into EJ and T24 cells, P3H4 mRNA (E) and protein (F and G) expression were detected by RT-qPCR and western blot, cell proliferation (H and I) were measured by CCK8 assays. *P<0.05.

Knockdown of P3H4 inhibits BC cell proliferation by impeding cell cycle progression
To expose the action of P3H4 in BC cells' biological function, expression of P3H4 was down-regulated with siRNA ( Figure 1E, 1F), and siRNA-4 targeting P3H4 (siP3H4) was used in subsequent experiments. As shown in Figure 1H and 1I, CCK8 assay showed that proliferation of EJ and T24 cells was inhibited when siP3H4 was transfected into cells. Similarly, colony formation assay revealed that knockdown of P3H4 inhibited the colony forming ability of EJ and T24 cells (Figure 2A). Furthermore, we detected the action of P3H4 knockdown on the cell cycle and apoptosis of BC cells. As shown in Figure 2B and 2C, the proportion of cells in G0/G1 phase increased while the proportion of cells in S and G2/M phase decreased, when siP3H4 was transfected into cells.
These results indicate that knockdown of P3H4 impedes cell cycle in the G0/G1 phase. In addition, transfection of siP3H4 reduced the protein levels of Cyclin D1, Nusap1 and p70 ( Figure 2D). Cyclin D1 is a key protein that regulates the G1 phase of the cell cycle [18]. Nusap1 is an indispensable microtubule and chromatin binding protein, which regulates the dynamics of kinetochore microtubules, governs chromosome oscillations, and ensures the normal progress of cell cycle [19,20]. Finally, flow cytometry detection found that knockdown of P3H4 had no effect on the apoptosis of BC cells ( Figure 2E). These results demonstrate that the knockdown of P3H4 can inhibit BC cell proliferation by arresting cell cycle progression, but that it has no effect on the apoptosis of BC cells.

Knockdown of P3H4 inhibits BC cell migration and invasion by affecting EMT progression
In addition to the effects on BC cell proliferation, we also examined the effects of P3H4 knockdown on BC cell motility. Transwell assay showed that compared to NC, fewer EJ and T24 cells could pass through the matrigel and invade the lower surface of the chamber membrane ( Figure 3A). Additionally, wound healing assay revealed that transfection of siP3H4 caused wound healing of EJ and T24 cells to be slower ( Figure  3B and 3C). Moreover, the knockdown of P3H4 reduced protein levels of N-cadherin and Vimentin, as AGING well as increased the protein levels of E-cadherin ( Figure 3D-3F). Decreased E-cadherin expression, increased N-cadherin and increased Vimentin were the most important sign change of the Epithelial-Mesenchymal transition (EMT) [21]. EMT plays a pivotal role in the primary infiltration and secondary metastasis of a variety of solid tumors [22].

Knockdown of P3H4 inhibits BC growth in vivo
In addition to cells in vitro, a subcutaneous xenograft model was used to further assess the biological role of P3H4 in BC progression. As shown in Figure 3G, 3H, knockdown of P3H4 in EJ and T24 cells significantly inhibited BC growth. Western blot and IHC analysis also demonstrated that the expression of P3H4 was downregulated by the shP3H4 transfection ( Figure 3I, 3J).
Given that ETV4 may be an important transcription factor for P3H4, we further examined the expression of ETV4 in BC tissues. As shown in Figure 5A and Tables  3, 4, ETV4 was significantly high expressed in BC tissues compared to normal tissues, and ETV4 expression was related to clinical stage of BC. Furthermore, there was a significant positive correlation between the expression of P3H4 and ETV4 ( Figure 5B, P < 0.001, R = 0.7876). In addition, ETV4 overexpression partially restored the inhibitory of siP3H4 transfection on proliferation, colony formation and invasion ability of EJ and T24 cells ( Figure 5C-5E).

DISCUSSION
In the present study, we discovered the role of P3H4 in BC, and found that ETV4 can act as a transcription factor to influence BC progression by regulating P3H4 transcription.
Database analysis and IHC results showed abnormally high expression of P3H4 mRNA and proteins in BC tissues. Li et al also detected P3H4 mRNA expressions in 44 pairs of BC tissues and adjacent normal tissues by qRT-PCR, as well as discovered that P3H4 mRNA were highly expressed in BC tissues [23]. Their findings are consistent with our results.
Knockdown of P3H4 arrested the EJ and T24 cell cycle in the G1 phase, thus inhibiting cell proliferation. However, this no effect on cell apoptosis. In addition, knockdown of P3H4 also impeded migration and invasion of both EJ and T24 cells by inhibiting the EMT process. The xenograft nude mouse model also showed that knockdown of P3H4 inhibited the growth of subcutaneous BC tumors. These results indicate that P3H4 may play an oncogene function in BC   progression. Although studies have displayed that P3H4 may be associated with several tumors (prostate cancer [10] and meningeal cancer [11]), this study was the first to conduct experiments in vivo and in vitro to determine the role of P3H4 in BC.

AGING
Furthermore, ETV4 could regulated the P3H4 transcription in EJ and T24 cells. ETV4 (ETS Variant 4) is a transcription factor belonging to the EST family and is specifically involved in the carcinogenesis of various tissues, including pancreatic cancer, breast cancer and intestinal cancer [24][25][26]. However, little is known about the role of ETV4 in BC. ETV4 is frequently up-regulated in tumors and acts as an oncogenic factor via different mechanisms. IHC analysis also found a significant upregulation of ETV4 expression in BC. By fusion with other genes, ETV4 regulates genome transcriptional pattern and carcinogenesis [27]. For example, TMPRSS2-ETV4, KLK2-ETV4 and CANTT-ETV4 [27,28]. ETV4-related gene fusion may be an early event in prostate cancer and could define a new molecular subtype of prostate cancer [28]. On the other hand, ETV4 can directly bind to the promoter region of target genes and regulate their transcriptional activity. In pancreatic cancer and breast cancer, ETV4 directly upregulates the transcription of cyclin proteins, such as cyclin D1 [26] and cyclin D3 [29] to promote cell proliferation. MMPs, such as MMP13 and urokinase plasminogen activator (uPA) are also transcriptionally regulated by ETV4, which are involved in the ETV4-driven malignant phenotypes in prostate cancer [30] and breast cancer [25].
In the present study, our results of western blot and Luciferase activity assay demonstrated that ETV4 can directly bind to the promoter of P3H4 and activate transcription of P3H4. Moreover, three binding sites of the ETV4 and P3H4 promoter regions were found.
Overexpression of ETV4 rescued the inhibition of proliferation and invasion induced by PH4 silencing.
These results indicate that ETV4 can participate in the regulation of BC progression by transcriptionally regulating the expression of P3H4.
In conclusion, P3H4 regulated proliferation, cell cycle, migration and invasion, playing an oncogene role in BC progression. In BC cells, ETV4 bound directly to the P3H4 promoter region to regulate its transcription.

Tissues specimens
32 fresh BC tissues and 26 normal adjacent tissues were obtained from BC patients who underwent a urethral cystectomy (TURB) at Xuzhou Central Hospital in 2018. Normal tissues were excised from 5 to 7 cm away from the tumor. The specimens were immediately frozen in liquid nitrogen and stored at -80 °C. The pathological samples were reassessed by an experienced urinary tract AGING division (ALB) to confirm the diagnosis. The hospital ethics committee approved the study and all patients signed written informed consent.

Immunohistochemistry (IHC)
Collected clinical samples were immersed in formalin for 24 h at 4 °C, followed by paraffin embedding. Paraffin blocks were cut into 4 μm sections by a cryo-cutting machine and IHC was performed to evaluate P3H4 or ETV4 protein expression. IHC was performed using nonbiotinylated rabbit polyclonal anti-human P3H4 ( Proteintech , Manchester, UK, 15288-1-AP, 1:100) and rabbit polyclonal anti-human ETV4 (Proteintech, Manchester, UK, 10684-1-AP, 1:200). Van-Clear (Hongci., Shanghai, China) and concentration gradient ethanol were used for the dewaxing of paraffin sections. Antigen retrieval was performed by microwave pretreatment in 0.01 M citrate buffer for 10 min. After being blocked with 5% sheep serum at room temperature for 1 h, the sections were incubated with the primary antibody, and incubated with Enzyme-labeled goat anti-mouse/rabbit IgG polymer (160101405L, Maixin., Shanghai, China). The immune response was visualized by a Reinforced DAB Chromogenic Kit (1705252031, Maixin., Shanghai, China), and hematoxylin was used for counterstaining. Finally, images were obtained using an upright microscope system (Nikon, JAPAN).

Cell culture
BC cell lines EJ and T24, as well as 293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in DMEM medium (Gibco, NY, USA) containing 10% fetal bovine serum (FBS; Gibco, Australia) and 1% penicillin-streptomycin with 5% CO 2 at 37 °C in a humidified incubator.
SiRNA targeting P3H4 sequence was transformed into short hairpin RNA (shRNA), which was cloned into a pLVX-shRNA2 green fluorescent protein (GFP) vector.

Flow cytometry detection for cell apoptosis
After 48 h of transfection, cells were trypsinized and collected by the Annexin V binding buffer. Subsequently, cells were double stained with V-FITC and propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA), then measured by BD FACS Canto II (BD Biosciences). Data was analyzed using FlowJo software.

Transwell assay
Transwell chambers (BD Biosciences) were used to evaluate the invasion ability of EJ and T24 cells. The upper surface of chambers was coated with Matrigel. 200 μl of cell suspension with a concentration of 1x10 6 cells/ml was added into the upper chamber. The lower chambers were filled with 600 μl of DMEM containing 10% FBS. After incubation at 37 °C for 24 h, cells on the upper chambers were removed, and invaded cells on the lower surface of the chambers were fixed and stained with 0.1% crystal violet. Invaded cells were counted and photographed under a microscope (200x magnification, Nikon TE2000).

Wound healing assay
Wound healing assay was performed to measure the migration ability of GC cells. Cells were grown to confluence in 6-well plates. A sterile plastic tip was used to create wounds. After washing with PBS, cells were cultured in serum-free medium for 48 h. Images were taken using a microscope. The average of five random widths of each wound was measured for quantification.

Xenografts in mice
All experimental procedures were approved by the Ethics Committee. Approximately 1x10 7 cells were injected subcutaneously into the armpits of female athymic BALB/C nude mice (4-6 weeks old, 18-22 g, 5 mice per group). Tumor growth was monitored weekly. After 4 weeks, the mice were euthanized and photographed. The subcutaneous tumor mass removed, and a picture was taken. A portion of the tumor mass was fixed and embedded for IHC, and a portion of the tumor mass was used for western blot.

Luciferase activity assay
The pGL3.0 recombinant plasmid and pcDNA3.1-ETV4 plasmid were co-transfected into EJ and T24 cells using Lipofectamine 2000. After 48 h, cells were harvested and luciferase activity was detected using a luciferase assay system (Promega, Madison, Wisconsin, USA).

Statistical analysis
Statistical analysis was performed as the mean ± SD and conducted using SPSS 22.0 (SPSS, IBM, Beijing, AGING China) and GraphPad Prism 6 (GraphPad, San Diego, CA). Differences were calculated with the Student's ttest between two groups or with one-way ANOVA among multiple groups. P < 0.05 was considered statistically significant.

AUTHOR CONTRIBUTIONS
Lin Hao, Kun Pang and Hui Pang generated the hypothesis, designed and performed the experiments, and analyzed the data. All author performed the experiments, analyzed the data, provided conceptual advice and technical expertise, and edited the manuscript. Zhenduo Shi and Conghui Han conceived and supervised the study. All authors have reviewed and approved the final version of the manuscript.