IGF2BP3 facilitates cell proliferation and tumorigenesis via modulation of JAK/STAT signalling pathway in human bladder cancer

Abstract Insulin‐like growth factor‐2 messenger RNA‐binding protein 3 (IGF2BP3) has been reported to contribute to tumorigenesis in several human cancers. However, the biological functions of IGF2BP3 in bladder cancer are poorly understood. We investigated the relation between IGF2BP3 expression and prognosis of bladder cancer patients. Cell proliferation, cell cycle and cell apoptosis assays were performed to assess IGF2BP3 functions. The results showed that IGF2BP3 was overexpressed in bladder cancer tissues compared with that in normal bladder tissues, and its higher expression was closely correlated with poor prognosis in bladder cancer patients. Overexpression of IGF2BP3 markedly promoted cell proliferation and cell cycle progression and inhibited cell apoptosis, while knockdown of IGF2BP3 notably suppressed the proliferation, promoted cell apoptosis and induced cell cycle arrest at the G0/G1 phase. Mechanistically, we revealed that IGF2BP3 promotes the activation of the JAK/STAT pathway in bladder cancer cells. Moreover, the JAK/STAT inhibitor dramatically blocked the tumour‐promoting activity of IGF2BP3. Tumour growth in vivo was also suppressed by knocking down of IGF2BP3. Hence, IGF2BP3 facilitated bladder cancer cell proliferation by activating the JAK/STAT signalling pathway. These findings suggest that IGF2BP3 exhibits an oncogenic effect in human bladder cancer progression.

bladder cancer remains poor. Therefore, it is essential to understand the molecular mechanism in order to explore effective diagnostic and prognostic markers in bladder cancer.
As a messenger RNA (mRNA) binding protein, the insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) is a member of the IGF2 mRNA-binding protein family and plays significant roles involved regulating the translation of insulin-like growth factor-2. [3][4][5] Previous research reported the oncofoetal protein IGF2BP3 participates in RNA trafficking and stabilization and cell proliferation and cell migration in the early processes of embryogenesis. 6,7 Many studies indicate that IGF2BP3 is highly expressed in a variety of tumour tissues compared to adjacent normal tissues, including lung cancer, 8 gastric cancer, 9 pancreatic cancer, 10 kidney cancer, 11 suggesting a tumour-promoting role of IGF2BP3. 12 High IGF2BP3 expression predicts metastasis formation and poor survival in renal cell carcinoma. 13 Moreover, increased IGF2BP3 expression facilitates the aggression of colorectal cancer cells via regulating epithelial-mesenchymal transition. 14 Recently, a study reported that IGF2BP3 was directly associated with a deubiquitinase named Ubiquitin-specific peptidase 10 and attenuated its function in stabilizing p53 protein in lung cancer. 15 However, little is known about the function of IGF2BP3 in bladder cancer.
In this study, we explored the expression of IGF2BP3 in bladder cancer and subsequently investigated the prognostic and molecular function of IGF2BP3. Our study showed that IGF2BP3 could be a prognostic factor in bladder cancer and serve as a potential target for new therapeutic strategies.

| Data mining
The data for mRNA expressions (mRNA SeqV2) and follow-up data of human bladder cancer were obtained from the Cancer Genome Atlas database (TCGA, https://tcga-data.nci.nih.gov/tcga). The gene expression profile was extracted from TCGA RNA-seq data, which contained 267 primary bladder cancer tissues and 19 surrounding non-cancer tissues. All profile data were analysed using R statistical environment and further calculated. Kaplan-Meier survival analysis was performed to validate the prognostic value of IGF2BP3 in bladder cancer. To gain further insight into the biological processes/signalling pathway and phenotypes of IGF2BP3

| Quantitative real-time polymerase chain reaction (QRT-PCR)
Total RNA was extracted following the manufacturer's protocol with using the 2 -ΔΔCt method and normalized by taking GAPDH as an internal reference to control the relative expression levels.

| Western blotting
For Western blotting, cells were lysed with RIPA lysis buffer kit (Jrdun Biotechnology, CA), supernatants were collected after spin and total proteins were measured using the BCA protein quantification kit (Thermo Scientific, USA). Total protein samples were separated by 10% or 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the samples were transferred onto nitrocellulose membranes. After blocking with 5% fat-free milk for 1 hour at room temperature, the membranes were incu-

| Cell proliferation
Cell proliferation was tested using the Cell Counting Kit-8 (CCK-8 Subsequently, the optical density of each well was read at 450 nm.
Every sample was assayed three times.

| Cell cycle assay
Cells were collected, washed and fixed in 70% ethanol. The fixed cells were subsequently washed in PBS, incubated with RNAase and stained with 50 μg/ml propidium iodide at 37°C for 30 min. Flow cytometric analysis was performed using a FACSCalibur instrument (Becton-Dickinson, San Jose, CA), and the results were analysed using FlowJo7.6 software.

| Cell apoptosis assay
Apoptotic cells were detected using Annexin V-FITC/PI apoptosis detection kit (Beyotime, Shanghai, China). Briefly, cells were collected with trypsin/EDTA and washed by ice-cold PBS and then resuspended in binding buffer. Next, cells were incubated with 5 µl Annexin V-FITC at 4°C for 15 minutes and 5 µl PI at 4°C for 5 minutes in a dark place. After incubation, the apoptotic cells were quantified by flow cytometry (BD Biosciences), and data were analysed with BD AccuriTM C6 software.

| Transwell migration and invasion assay
Transwell migration and invasion assays were performed using Transwell chambers (Costar Corporation, MA, USA) without or with Matrigel (Corning, Kennebunk, USA). 0.7 ml of 10% FBS-containing medium was placed to the lower chamber, in which 3 × 10 4 cells suspended in 300 µl of serum-free medium were seeded in the upper chamber for 24 hours. Cells were fixed with 4% formaldehyde for 10 minutes, and then, we removed the adhering cells, followed by washing once with PBS. Subsequently, 1 ml/well 0.5% crystal violet was used to stain the cells for 30 minutes, followed by washing with PBS three times. The migrating or invading cell numbers were counted by a 200 × microscope.

| Xenograft tumour model
Male BALB/c nude mice (four to six weeks old), weighting 18-20 g, were purchased from Shanghai Experimental Animal Center (Shanghai, China). All mice were kept in a strict pathogen-free con- experiments. We divided the twelve mice into two groups: a control group (siNC transfected cells) and a siIGF2BP3 group. To establish the xenograft model, a total of 4 × 10 6 tumour cells were subcutaneously injected into the right flank of the nude mice. Every three days, we measured the tumour length and width with caliper. Tumour volume was calculated using the formula: (length* width 2 )/2. At the endpoint, the mice were euthanized, and tumour tissues were weighted.

| Statistical analysis
All data were represented as means ± standard deviations (SD). All differences between two independent groups were analysed using a

| Up-regulation of IGF2BP3 in bladder cancer
To explore the tumour-promoting or tumour-suppressing effect of IGF2BP3 on bladder cancer, the expression of IGF2BP3 was evaluated in bladder cancer tissues and normal tissues from TCGA datasets. As shown in Figure 1A, the expression of IGF2BP3 in bladder cancer tissues was significantly higher than that in normal tissues (P = 0.0004); the means ± SD for IGF2BP3 expression in normal tissues and bladder Furthermore, we assessed the expression of IGF2BP3 with Western blotting in 5 pairs of bladder cancer and matched normal adjacent tissues. Compared with adjacent noncancerous tissues, we found that the protein levels of IGF2BP3 were higher in bladder cancer tissues ( Figure 1D). In addition, we also evaluated the expression of IGF2BP3 by immunohistochemical analysis of 100 patients who underwent transurethral resection of the bladder or radical cystectomy. High expression of IGF2BP3 was observed in the tumour cells of 65% of the patients. As shown in Figure 1E and F, IGF2BP3 was expressed at higher levels in advanced stage tumours (T2-T4) ( Figure 1E) and at lower levels in early stage tumours (Ta-T1) ( Figure 1F).

| High igf2bp3 expression in bladder cancer correlates with poor survival
The association between IGF2BP3 expression and the clinicopathological features of bladder cancer was shown in Table 2.
Overexpression of IGF2BP3 was observed to be significantly associated with T classification (P = 0.026) and tumour grade (P = 0.001).
Yet, the IGF2BP3 level was not associated with gender, age, N classification and metastasis. As shown in Table 3 Figure 1G). That was consistent with the Kaplan-Meier analysis of TCGA which revealed a negative correlation of IGF2BP3 expression with the OS (P = 0.0319, Figure 1H). Hence, these findings suggest that IGF2BP3 is highly expressed in bladder cancer and that IGF2BP3 is a factor for predicting poor survival in patients with bladder cancer.

| IGF2BP3 enhanced proliferation of bladder cancer cells
GSEA analysis was performed in the TCGA database and the results showed that higher levels of IGF2BP3 are positively associated with an enrichment of cell cycle gene signatures (Figure 2A).
To further explore the biological function of IGF2BP3 in bladder cancer, three IGF2BP3-specific shRNAs were used to construct IGF2BP3 knockdown cells in T24 and UMUC3 cell lines, which had relatively higher IGF2BP3 expression, and IGF2BP3 was ectopically overexpressed in 5637 and J82 cell lines, which displayed relatively lower IGF2BP3 expression. The expression of IGF2BP3 was significantly decreased in T24 and UMUC3 cells and overexpressed in 5637 and J82 cells both at mRNA and protein level ( Figure 2B and C). CCK-8 assays showed silencing of IGF2BP3 using siIGF2BP3-1 and siIGF2BP3-2 significantly reduced cell viability of T24 and UMUC3 cells ( Figure 2D). Furthermore, overexpression of IGF2BP3 significantly promoted cell viability of 5637 and J82 cells, which were approximately 1.0-fold higher than that of vector control cells at 72 hours after transfection ( Figure 2D).
Collectively, these results indicate that IGF2BP3 promotes the proliferative ability of bladder cancer cells.

| IGF2BP3 is involved in cell cycle G1 to S phase transition in bladder cancer cells
The role of IGF2BP3 in the cell cycle of bladder cancer cells was explored using flow cytometry assay. The flow cytometry assay showed a significant increase in the percentage of cells in the G0/G1 phase and a significant decreased that in the S phase after silencing of IGF2BP3. The converse was true after overexpression of IGF2BP3 in the cell lines. As shown in Figure 3A, cells in the G0/G1 phase were increased in IGF2BP3 knockdown UMUC3 and T24 cells compared to the control groups. IGF2BP3 overexpression promoted the cell cycle progression by raising the proportion of cells in the S phase in 5637 and J82 cells, compared to the vector control cells ( Figure 3B). Moreover, Western blotting analysis revealed that cell cycle promotor cyclin D1 was down-regulated after IGF2BP3 was silenced ( Figure 3E).

| IGF2BP3 inhibits apoptotic activity in bladder cancer cells
As decreasing the IGF2BP3 expression greatly inhibited cell viability, it was hypothesized that IGF2BP3 may affect cell apoptosis in blad- anti-apoptotic factor, were significantly decreased whereas Bax, a pro-apoptotic factor, was significantly increased after IGF2BP3 was silenced ( Figure 3E). Meanwhile IGF2BP3 overexpressed cells showed the opposite trend ( Figure 3F).

IGF2BP3 Promotes the Migration and Invasion in Bladder Cancer
Cells.
Next, a transwell assay was performed to determine whether IGF2BP3 affected the migratory and invasive potential of bladder cancer cells. Firstly, the number of bladder cancer cells passed through the transwell membrane was observed to be reduced by siRNA-mediated knockdown of IGF2BP3 in T24, suggesting that silencing IGF2BP3 could inhibit cell migration in bladder cancer ( Figure S1A). Moreover, the transwell invasion assay also showed that silencing IGF2BP3 could significantly restrain the invasive ability of T24 cells ( Figure S1A). At the same time, we found that overexpression IGF2BP3 significantly increased cell migration and invasion of 5637 cells ( Figure S1B). These data showed that IGF2BP3 could promote the migration and invasion of bladder cancer cells.

| IGF2BP3 regulates the tumorigenesis in vivo
To verify the effects of IGF2BP3 on the tumorigenicity in vivo, xenograft models were established by injecting stable knockdown

IGF2BP3 T24 cells and vector-transfected T24 cells into subcutane-
ous tissues of nude mice. All nude mice developed xenogeneic tumours at the injection site ( Figure 4A). Tumour growth of IGF2BP3 silenced cells was slower than that of the vector-transfected cells

TA B L E 3 IGF2BP3 regression analysis
for predicting cancer specific survival of bladder cancer ( Figure 4B). As shown in Figure 4C and D, down-regulation of IGF2BP3 significantly decreased the xenograft tumour volume and tumour weight compared to the control group. qRT-PCR and Western blotting confirmed that IGF2BP3 expression levels were lower in the tumours injected with IGF2BP3 depleted cells than that in the vectortransfected cells ( Figure 4E and F). As shown in Figure 4G, Western blotting also showed that p-STAT3 expression levels were lower in the tumours injected with IGF2BP3 depleted cells than that in the vector-transfected cells. Thus, JAK/STAT3 pathway was activated in control group xenograft tumours but attenuated in siIGF2BP3 xenografts. Taken together, these results indicate that IGF2BP3 plays a vital role in the tumorigenicity of bladder cancer in vivo.

| IGF2BP3 promoted the proliferation by regulating the JAK/STAT signalling pathway
By performing GSEA analysis in the TCGA database, we found that IGF2BP3 expression was strongly associated with the JAK/STAT signalling pathway, which plays an essential role in cell proliferation. 16 The results suggested that the JAK/STAT signalling pathway may be involved in the function of IGF2BP3 ( Figure 5A). As shown in Figure 5B, phosphorylation of STAT3 was increased in IGF2BP3

| D ISCUSS I ON
Previous studies indicated that IGF2BP3 involved in the growth, chemo-resistance and progression in many cancers. [17][18][19][20] has been identified as a positive regulator of cell proliferation and metastasis. A study by Xu et al revealed that IGF2BP3 was upregulated in colorectal cancer and associated with worse clinical outcome, which implies that IGF2BP3 harbours prognostic significance (14). IGF2BP3 overexpression has been linked to advanced disease stage and adverse clinical outcome in several cancers. [21][22][23] Furthermore, IGF2BP3 was found to act as an oncogenic factor promoting proliferation and invasion of glioblastoma via activating PI3K/MAPK pathway. 24 However, the role of IGF2BP3 in driving proliferation of bladder cancer has yet be elucidated.
In our study, we found IGF2BP3 could promote tumorigenesis of bladder cancer by JAK/STAT signalling. Thus, we have identified Additionally, we further discovered IGF2BP3 promote cell growth of bladder cancer cells via JAK/STAT signalling. JAK/STAT signalling plays a crucial role in regulating cell growth, apoptosis and differentiation and is activated in many tumours. 25,26 The continuous activation of JAK/STAT could promote tumorigenesis. 27 A previous study reported that lncRNA PART1 knocking down could inhibit proliferation, migration, and invasion via inactivating JAK/STAT signalling in non-small cell lung cancer. 28 Inhibition of JAK/STAT signalling suppresses cell growth and induces apoptosis, cell cycle arrest and inhibits cell invasion in colorectal cancer. 29 Moreover, aberrant activated STAT3 was found in prostate cancer tissues but not in the normal tissues. 30 Interleukin-6 induces cell growth of prostate cancer by activating STAT3 signalling pathway. 31 Our results showed that up-regulation of IGF2BP3 could increase the expression of phosphorylation of STAT3 and IGF2BP3 In summary, our study found IGF2BP3 up-regulation exerted the positive biological role to promote the cell proliferation ability of bladder cancer cells in vitro and in vivo by modulating the JAK/STAT pathway. IGF2BP3 is a potential target for gene therapy of bladder cancer to make a better prognosis in the future.

ACK N OWLED G EM ENTS
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.  The data used to support the findings of this study are available from the corresponding author upon request.