The effect of NPM1 silencing on the tumorigenesis of drug-resistant bladder cancer and related signaling pathway

Background: NPM1 can provide abundant information of bladder cancer changes, but the effect of NPM1 differential expression on tumor and tumor related molecular mechanism has not been elucidated. Methods: NPM1 silencing cell line was established by lentivirus. The tumorigenic ability was judged by wound-healing assay, transwell invasion assay and nude mice tumorigenicity assay. The proteome of NPM1 decient bladder cancer cell line was analyzed by Liquid Chromatography Mass Spectrometry (LC-MS). The results of mass spectrometry are comprehensively analyzed by bioinformatics analysis for tumor related molecules. The signal pathways involved in tumor related molecules will be veried by KEGG and UniProt databases. Results: Compared with the corresponding negative control group, NPM1 silencing cell line T24/DDP Lv-NPM1 showed strong migration ability and high invasive ability. There was no signicant difference in migration ability and the invasive cells proportion between NPM1 overexpressing cell line and related negative control group. The tumorigenesis in nude mice also showed that NPM1 silencing tumor had larger tumor volume. Among all differential proteins analyzed by mass spectrometry, 20 proteins with signal transduction activity showed the most signicant difference (Fold change > 1.5). 6 of them were associated with NF-κB signaling pathway, which may play an important role in the development of tumor. Conclusions: The loss of NPM1 may indicate the poor outcome of bladder cancer. Abnormal expression of NF-κB signaling pathway is an important factor in the progression of bladder cancer. Monitoring NPM1 expression can effectively adjust the treatment strategy of bladder cancer.


Background
Bladder cancer is one of the most common malignancies in urinary system [1], which is prone to drug resistance and relapse [2]. Timely monitoring the drug resistance and progression of the tumor and adjusting the treatment strategy can effectively inhibit the progression of bladder cancer [3]. Therefore, it is urgent to nd a biomarker which can provide abundant clinical information for the treatment of drug resistant bladder cancer.
Nucleophosmin (NPM1) is the nucleolar protein which can shuttle between nucleolus and cytoplasm [4], NPM1 mutation have been demonstrated to be closely associated with malignant tumor. In colon cancer [5], lung adenocarcinoma [6] and hematological tumor [7], NPM1 was found to be able to indicate tumor progression. In addition, NPM1 plays an important role in cell invasion, migration and apoptosis [8].
NPM1 is also involved in drug resistance and re ect the prognosis of tumor, such as leukemia [9]. It is reported that NPM1 can interfere with drug action by histone demethylation or gene rearrangement [10]. This suggests that NPM1 is involved in the speci c mechanism of drug resistance. In salivary gland carcinoma and leukemia cells, tumor proliferation and drug response can be effectively affected by interfering with the expression of NPM1 [8]. Up-regulated NPM1 can inhibit the activity of chemotherapy drugs [11].
Based on the above research, we've paid attention to the clinical information provided by NPM1 in bladder cancer in recent years.
In the research of our laboratory, NPM1 overexpression was found in drug-resistant by proteome analysis [12]. Besides abnormal expression of NPM1 was also found in recurrent patients, which suggested that NPM1 mutation was closely related to the recurrence and drug resistance of bladder cancer [13].
In order to further evaluate the application value of NPM1 gene silencing in drug resistant bladder cancer cells, we used liquid chromatography-mass spectrometry to detect the proteomics of bladder cancer cells after NPM1 gene silencing, 492 differential proteins were detected by mass spectrometry, and their multiple changes were more than 1.5 (P < 0.05). The results of mass spectrometry showed that 57022 polypeptides, 54347 unique polypeptides and 6686 proteomes were identi ed (FDR < 0.01). Compared with the negative control group, 264 functional proteins were down-regulated and 228 were up-regulated.
According to the analysis of Gene Ontology (GO analysis), CD40 is the most signi cant down regulated protein after NPM1 silencing [14]. CD40 is differentially expressed in a variety of tumors, and is considered to be an important biomarker for predicting many cancers. Therefore, the results of proteomic analysis suggest that NPM1 gene silencing may be involved in the development of drug-resistant bladder cancer cells. In this study, we observed the effects of NPM1 silencing on drug resistance, tumor invasion and migration of bladder cancer and performed bioinformatics analysis from the proteomics data in drug resistant bladder cancer from our previous studies to reveal the possible mechanism of NPM1 in drug resistant bladder cancer.

Wound-healing assay
In order to evaluate the migration ability of bladder cancer cells, wound-healing assay was performed.
The migration ability of cells in each group can be judged by the difference of repair ability of cells in different groups for wound area. The plate was irradiated with ultraviolet for 30 minutes before operation.
Approximately 3 × 10 4 infected cells were incubated in 24 holes. Wounds were made according to the instructions. After 24 hours low serum concentration medium was used to incubate the cells. Rinsed the plate gently with PBS for 2 times after 24 or 48 hours. After that the photos were taken under microscopy (CKX41, Olympus, USA). According to the pictures, the rate of cell migration in each group was calculated. The migration rate was the ratio of wound width to 0 hour wound area width.

Transwell invasion assay
In order to evaluate the invasion ability of bladder cancer cells, transwell invasion assay was performed. The invasion ability of bladder cancer cells was evaluated by counting the amount of cells which can digest matrigel. Matrigel invasion assay was performed using transwell chambers. 1 × 10 4 Lv-NPM1 cells, 1 × 10 4 Lv5-NPM1 and the cells were seeded in the upper chamber of a 24-well plate, which was coated with growth factor reduced matrigel. The upper chamber was lled with 500 µl serum-free medium. The lower chamber was lled with 500 µl medium containing 10% FBS to induce cell invasion. The chamber was incubated at 37 °C for 24 hours. At the end of incubation, cells in the upper surface of the membrane were removed with a cotton swab. Migrated cells were stained with crystal violet. The images were obtained by microscope and the cells were counted in ten different view elds. The experiment was repeated three times Materials

Xenograft Growth In Nude Mice
To evaluate the tumorigenicity of bladder cancer cells, xenograft model was made by subcutaneous injection of NPM1 silencing cells and the corresponding negative control cells in athymic nude mice.

Preparation of inoculated cells
Lv-NPM1 cell line and Lv-NC cell line labeled by GFP uorescence were cultured in RPMI-1640 medium containing 15% fetal bovine serum at 37 ℃ and 5% CO 2 incubator respectively. After digestion of 0.25% trypsin, they were used for tumorigenicity test.

Tumorigenicity test
The mice were inoculated subcutaneously in the left armpit with 5 × 10 7 NPM1 silencing cells. They were also inoculated subcutaneously in the right armpit with 5 × 10 7 negative control cells (as the negative control). All the mice were kept in standard laboratory conditions and provided with ad libitum food and water. General health of these animals was daily observed and tumor growth at the injection site was monitored by palpation. Tumor volume was measured outside of body by vernier caliper and calculated using the formula: length × (width) 2 × 0.5. 35 days after the experiment, the mice were euthanized. All nude mice were anesthetized with ethyl carbamate (1350 mg/kg, intraperitoneal injection) and sacri ced by cervical dislocation when the mice were unconscious. The experiment was repeated three times.

Proteomic Bioinformatics Analysis
The data came from the proteomic data of our NPM1 gene silencing drug resistant bladder cancer cells [14]. The original data were processed by proteome discovery software. According to the human genome database and UniProt website, mass spectrometry data were searched. The molecular function of proteins were analyzed by gene ontology (GO analysis). The Kyoto Encyclopedia of Genes and Genomes (KEGG) was used to study protein species to map the signaling pathway for systematic functional biology interpretation (http://www.genome.jp/kegg/).

Statistical analysis
All independent experimental data were expressed as mean ± standard deviation (SD). Statistical analysis was carried out by using Graphpad prism 6.0 statistical software Inc (Lajolla, USA). P values were calculated by ANOVA and Bonferroni test (more than two groups) or T test (two groups). When P value < 0.05, the results are considered to be statistical.

Results
The effect of NPM1 silencing and NPM1 overexpressing on the cell mobility of cisplatin resistant bladder cancer cells  We used the nude mouse tumorigenesis experiment to assess the effect of NPM1 silencing on the overall change of tumor in vivo. After 35 days of the experiment, greater masses were observed in animals injected with Lv-NPM1 cells compared with Lv-NC cells. Meanwhile, the tumor mass of the nude mice injected NPM1 silencing cells was more obvious than that of the negative control nude mice at 14-35 days (P < 0.05), as shown in Fig. 3.
The effect of differential proteins of signal transducer activity on NPM1 silencing tumor cells.
After NPM1 silencing, the protein of signal transducer activity on NPM1 silencing tumor cells is of great signi cance to the characteristics of tumor, as shown in Table 1. 12 proteins were up-regulated and 8 proteins were-down regulated (Fold change 1.5, P < 0.05), 6 of the 20 proteins with signal transduction activity are related to NF-κB signaling pathway. Moreover, the up-regulated protein and the affected pathway function have the characteristics of accelerating the tumorigenesis.

Discussion
Bladder cancer is a common urologic neoplasm which is easy to relapse and drug resistance. The existing monitoring methods for bladder cancer have high side effects and heavy costs, which are di cult for patients to accept [15,16]. Proteomic analysis of tumor progression can reduce the burden of patients and provide valuable clinical information [17]. The accurate judgment of tumor characteristics of bladder cancer can effectively guide the treatment of bladder cancer and improve the treatment effect.
Nucleophosmin (NPM1) is a nucleolar protein which can provide clinical information for the development of cancers [18]. NPM1 plays important roles in p53, MDM2 and other signal pathways [19], which means NPM1 can affect tumor characteristics and provide important information for drug resistance. The upregulation of NPM1 expression signi cantly affects the relapse rate and the sensitivity of chemotherapy drugs of leukemia [20].
In this experiment, NPM1 silencing and NPM1 overexpressing bladder cancer cell lines were constructed by lentivirus infection and limited dilution method to evaluate the value of NPM1 in drug resistant bladder cancer. In this study, the results showed that the loss of NPM1 was correlated with high cell mobility in bladder cancer cells. Compared with negative control group, NPM1 silencing cell line T24/DDP Lv-NPM1 showed strong migration ability in 24 hours (P < 0.05) and 48 hours (P < 0.05). Besides, NPM1 silencing could make cisplatin resistant bladder cancer aggressive. Compared with the negative control, the invasive cells proportion of T24/DDP Lv-NPM1 cells was higher than Lv-NC cells (P < 0.05). Compared with the above results in vitro, tumorigenesis experiment also showed similar results in vivo. In this animal experiments, we found that NPM1 silencing increased tumor growth in vivo, indicating that downregulated NPM1 may bring poor prognosis to patients. Besides, the speci c mechanism of NPM1 on bladder cancer has not been elucidated. In previous study, we used mass spectrometry to analyze proteins in bladder cancer. A total of 57022 peptides, 54347 unique peptides and 6686 protein groups were identi ed in all proteins of NPM1 silencing drug resistant bladder cancer cells (FDR < 0.01) [14]. In this study we analyzed these functional proteins from the previous study and focused on the proteins that play a role in the signaling pathway. Among the 20 proteins with the most obvious difference in signal transducer activity (Fold change 1.5), 6 proteins were associated with NF-κB signaling pathway.
Most of the functions of these proteins were positive regulation of NF-κB signaling pathway. NF-κB signaling pathway was activated abnormally in many tumors, hence it is suspected to be involved in malignant transformation of tissues, carcinoma cells invasion and metastasis [21]. Combined with the results of our animal experiments, the low expression level of NPM1 may re ect the acceleration of tumor growth. Moreover, tumor deterioration mainly points to the abnormal changes of NF-κB signal pathway. It has been reported that the abnormal activation of NF-κB signal pathway can accelerate the development of tumors [22], and has an impact on the drug resistance of chemotherapy drugs [23]. The change of NF-κB signaling pathway was consistent with the characteristic of bladder cancer that is easy to relapse and drug resistance. In our previous study, we found that NPM1 has an increasing expression in drug-resistant bladder cancer [13]. This result may indicate that NPM1 can protect gene stability in bladder cancer cells. Under the attack of chemotherapy drugs, NPM1 with high expression can prevent genome damage [24].
However, the low expression of NPM1 does not re ect the improvement of tumor therapy. In gastric cancer and breast cancer, NPM1 is associated with poor prognosis [25,26]. In the researches of bladder cancer, NPM1 can regulates c-Myc protein stability [27] to in uence β-catenin/c-Myc signaling pathway [28] and AFF4/NF-κB/Myc signaling pathway [29] increase the ability of cell migration and proliferation.
In this study, we discovered the relationship of NPM1 and NF-κB signaling pathway in the proteomic study of bladder cancer. It was suggested that the low expression of NPM1 could re ect the enhancement of NF-κB signaling pathway and accelerate the development of tumor. NF-κB signaling pathway, as an important tumor related signaling pathway, was able to accelerate tumor progression and change the drug-resistant activity in a variety of non-urinary tumors [30]. It has also been reported that the blocking of NF-kB signaling pathway can effectively alleviate the tumor [31], which may provide a new strategy for the treatment of bladder cancer. These results indicated that NPM1 can provide important information for the development of bladder cancer. Monitoring the expression of NPM1, which might be able to effectively detect the malignant change of bladder.

Conclusions
Down-regulated expression of NPM1 could indicate the poor outcome of bladder cancer, which meant that the tumor may be more malignant. Abnormal expression of NF-κB signaling pathway is an important factor in the progression of bladder cancer. Monitoring the changes of NPM1 in time could effectively adjust the treatment strategy of bladder cancer and treat bladder cancer.

Declarations
Ethics approval and consent to participate All applicable international, national, and institutional guidelines for the care and use of animals were followed.
All procedures performed in studies involving animals were in accordance with the ethical standards of the Pecking University Ethics Committee. (Permit number: LA2017287)

Consent for publish
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its supplementary information les].

Competing interests
The authors declare that they have no competing interests. The effect of NPM1 silencing and NPM1 overexpressing on cisplatin resistant bladder cancer cell migration. With experimental times lasting (0-48 hours), the cell migration rate of NPM1 silencing cisplatin resistant bladder cancer cells was higher than that of negative control and NPM1 overexpressing bladder cancer cells.

Figure 2
The effect of NPM1 silencing and NPM1 overexpressing on the cell invasion of cisplatin resistant bladder cancer cells. 48 hours after the experiment, NPM1 silencing cisplatin resistant bladder cancer cells were more invasive than negative control cells.