LncRNA IGFBP4-1 promotes tumor development by activating Janus kinase-signal transducer and activator of transcription pathway in bladder urothelial carcinoma.

Insulin-like growth factor binding protein 4-1 (IGFBP4-1), a new long noncoding RNA (lncRNA), has been reported to contribute to tumorigenesis and has been suggested to be a poor prognostic marker in human lung cancer. However, there still lacks basic studies that investigated the biological role of IGFBP4-1 in bladder urothelial carcinoma to date. In this study, we investigated the relationship between IGFBP4-1 expression and prognosis in patients with bladder cancer. Cell proliferation, cell cycle and cell apoptosis assays were performed to assess IGFBP4-1 function by up-regulating or down-regulating IGFBP4-1 in bladder cancer cells. A xenograft mice model was used to validate the in vitro results. Blockade of Janus kinase-signal transducer and activator of transcription pathway (JAK/STAT) was used to evaluate JAK/STAT signaling activity. The results showed that IGFBP4-1 was overexpressed in bladder cancer tissues compared with that in normal bladder tissues, and its expression level was positively correlated with poor prognosis in bladder cancer patients. Overexpression of IGFBP4-1 markedly promoted cell proliferation and cell cycle progression, and inhibited cell apoptosis, while knockdown of IGFBP4-1 notably suppressed the proliferation, promoted cell apoptosis, and induced cell cycle arrest at the G0/G1 phase. Mechanistically, we revealed that IGFBP4-1 promotes the activation of the JAK/STAT pathway in bladder cancer cells. Moreover, the JAK/STAT inhibitor dramatically blocked the tumor-promoting activity of IGFBP4-1. Tumor growth in vivo was also suppressed by knocking down of IGFBP4-1. In conclusion, IGFBP4-1 promoted bladder cancer progression by activating the JAK/STAT signaling pathway. These findings suggest that IGFBP4-1 exhibits an oncogenic role in the development of human bladder cancer. © The author(s).

Introduction

As one of the most common malignancies of the urinary tract, bladder cancer is the 11th most frequently diagnosed cancer worldwide 1. Currently, there are approximately 81,190 new cases per year in the United States and 17,240 people die from the disease annually 2. Although there have been significant advancements in the development of surgical techniques and adjunct treatment, the prognosis of 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. Insulin-like growth factor binding proteins (IGFBPs) play significant roles involved in cell proliferation and cell migration in the early processes of embryogenesis and cancers 3-7. Many studies indicate that IGFBPs are highly expressed in a variety of tumor tissues compared to adjacent normal tissues, including lung cancer 8, gastric cancer 9, pancreatic cancer 10, kidney cancer 11, suggesting a tumor promoting role of IGFBPs 12. High IGFBPs expression predicts metastasis formation and poor survival in renal cell carcinoma 13. Moreover, increased IGFBPs expression facilitates the aggression of colorectal cancer cells via regulating epithelial-mesenchymal transition 14. Recently, a study reported that overexpression of IGFBP4-1, a new candidate lncRNA located in the upstream region of IGFBP4 gene by bioinformatics analysis, reprograms energy metabolism to promote lung cancer progression 15.

However, little is known about the function of IGFBP4-1 in bladder cancer. In this study, we explored the expression of IGFBP4-1 in bladder cancer and subsequently investigated the prognostic and molecular function of IGFBP4-1. Our study showed that IGFBP4-1 plays an oncogenic role in the development of human bladder cancer.

Materials and Methods

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- IGFBP4-1 and Bladder Cancer seq data, which contained 267 primary bladder cancer tissues and 19 surrounding non-cancer tissues. All profile data were analyzed using R statistical environment and further calculated. Kaplan-Meier survival analysis was performed to validate the prognostic value of IGFBP4-1 in bladder cancer. To gain further insight into the biological processes/signaling pathway and phenotypes of IGFBP4-1 involved in bladder cancer pathogenesis; a gene set enrichment analysis (GSEA, version 2.2.3) was performed. The canonical pathways gene sets from the Molecular Signatures Database (http://software.broadinstitute.org/gsea/msigdb) were used for enrichment analysis.

Patients and Immunohistochemistry in Bladder Specimens

A total of 100 primary bladder cancer samples from 2013 to 2019 were enrolled in this study from the Department of Urology, Foshan Maternal and Child Health Hospital, Southern Medical University. The clinicopathological information of these patients is summarized in Table 1. The study was approved by the Ethics Committee of Southern Medical University and informed consent was obtained from all patients. All resected bladder tissues were fixed in 10% formaldehyde for 48 hours and embedded in paraffin. Then paraffin-embedded tissues were cut into 5um thick sections. Slides were deparaffinized with xylene and rehydrated with graded alcohol for pretreatment. Antigen retrieval was performed by microwave heating with citric acid buffer. To block endogenous peroxide, slides were separated with 3% H2O2 for 10 min in a wet box. Then, slides were with primary antibodies anti-IGFBP4 (Abcam, Cambridge, MA) at a dilution of 1: 100 overnight at 4 ° C in a humidified box. After washing with phosphate-buffered saline, slides were maintained with a secondary HRP-labeled anti-rabbit antibody (1:50, Beyotime, Shanghai, China) for 30 min at room temperature. Tissue sections were stained with 3,3'-diaminobenzidine (DAB) and counterstained with hematoxylin. Images were acquired with a Nikon microscope camera (Nikon Americas Inc, NY).

Table 1

Clinicopathological characteristics of patients with bladder cancer

Characteristics	Number of cases
Age, years
≥60	72
<60	28
Gender
Male	64
Female	36
T classification
Ta	14
T1	12
T2	40
T3	25
T4	9
N classification
N0	87
N1	7
N2	5
N3	1
Metastasis
No	99
Yes	1
Tumor grade
PUNLMP	1
Low grade	30
High grade	69
Expression of IGFBP4-1
Low expression	35
High expression	65

PUNLMP, papillary urothelial neoplasm of low malignant potential.

Cell Lines and Culture

A normal human bladder uroepithelium cell line (SV-HUC-1) and four bladder cancer cell lines (5637, J82, T24 and UMUC3) were obtained from The Cell Bank of Chinese Academy of Sciences (Shanghai, China). SV-HUC-1, T24 and UMUC3 were cultured in DMEM (Gibco, Carlsbad, CA). 5637 and J82 were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA). All culture media contained 1% penicillin/streptomycin and 10% fetal bovine serum (FBS, Gibco). The cells were incubated in humidified air at 37 °C with 5% CO2.

Lentivirus Construction and Infection

Three human siRNA sequences (si IGFBP4-1-1, 5'- GCAAAGGATTCGGAAACTT-3', si IGFBP4-1-2, 5'- GGTGAAACTTGAAGCTCAT -3', si IGFBP4-1-3, 5'-CCAGACACCTGATGAGAAT -3') and negative control siRNA (siNC) were cloned in pLKO.1-puro vector to endogenously downregulate IGFBP4-1. The coding sequences (CDS) region of human IGFBP4-1 was synthesized and cloned into pLVX-puro vector to overexpress IGFBP4-1. The synthesized core plasmid was confirmed by DNA sequencing (Majorbio, Shanghai, China). For lentiviral production, 293T cells were transfected with the lentiviral vector along with packaging plasmids using Lipofectamine 2000 (Invitrogen, Shanghai, China). Culture media was collected, pooled and filtered at 48 h and 72 h after transfection. Then the indicated lentivirus was used to infect the bladder cancer cell lines, and the expression of IGFBP4-1 was evaluated by real-time quantitative PCR and western blotting.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted following the manufacturer's protocol with TRIzol reagent (Invitrogen, Carlsbad, CA), dissolved in RNA-free H2O and stored at -80 °C. cDNA synthesis was performed from each 1 µg RNA sample using the Reverse Transcriptase Kit (Thermo, USA). Then, qPCR was performed on a real-time detector (ABI, USA) using a SYBR Green PCR kit (Thermo, USA). Primer sequences of IGFBP4-1 were as follows: primerF, 5'- GGTGGGGATGTTTGATTT -3', primer R, 5'- CCTGTTTTATGGGCTGAT -3'. Expression data were calculated 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 sulfate 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 incubated with primary antibodies against CyclinD1(1:1000, Cell Signaling, Germany), p-STAT3 (1:5000, Abcam, USA), STAT3 (1:2000,Abcam, USA), BAX (1:1000, Abcam, USA), Bcl2 (1:500, Abcam, USA) and GAPDH(1:2000, Cell Signaling, Germany) overnight at 4℃. Following three washes with TBST buffer, the membranes were incubated with secondary goat anti-rabbit antibodies conjugated with HRP for 1 hour at room temperature. Signals were visualized using the enhanced chemiluminescence kit (Millipore, USA) and detected by the Tanon-5200 Imaging system. Integrated relative densities of individual bands were quantified using Image J (National Institutes of Health, Bethesda, MD).

Cell Proliferation

Cell proliferation was tested using the Cell Counting Kit-8 (CCK-8, Beyotime, Shanghai, China). Each well of a 96-well culture plate was seeded with approximately 3×103 target cells and maintained at 37 °C overnight. At 0, 12, 24, 48, and 72 hours after transfection, cells were treated with CCK-8 (10ul/well) and incubated for 1 hour. 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 analyzed 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 ul Annexin V- FITC at 4 °C for 15 min and 5 µl PI at 4 °C for 5 min in a dark place. After incubation, the apoptotic cells were quantified by flow cytometry (BD Biosciences), and data were analyzed with BD AccuriTM C6 software.

Xenograft Tumor Model

Male BALB/c nude mice (four to six weeks old), weighting 18-20g, were purchased from Shanghai Experimental Animal Center (Shanghai, China). All mice were kept in strict pathogen-free conditions. The Ethics Committee for Animal Experiments approved the animal experiments. We divided the twelve mice into two groups: a control group (siNC transfected cells) and a si IGFBP4-1 group. To establish the xenograft model, a total of 4×106 tumor cells were subcutaneously injected into the right flank of the nude mice. Every three days, we measured the tumor length and width with caliper. Tumor volume was calculated using the formula: (length* width2)/2. At the end point, the mice were euthanized, and tumor tissues were weighted.

Statistical Analysis

All data were represented as means ± standard deviations (SD). All differences between two independent groups were analyzed using a two-tailed Student's t-test. All differences between multiple groups were analyzed using ANOVA tests. Survival data were analyzed by univariate and multivariate Cox regression analyses. Survival curves were analyzed using the Kaplan-Meier method and compared using the log-rank test. SPSS version 16.0 software (IBM Corporation, USA) was used for statistical analysis. A p-value< 0.05 was considered statistically significant.

Results

Upregulation of IGFBP4-1 in Bladder Cancer

To explore the tumor promoting or suppressing effect of IGFBP4-1 on bladder cancer, the expression of IGFBP4-1 was evaluated in bladder cancer tissues and normal tissues from TCGA datasets. As shown in Figure 1A, the expression of IGFBP4-1 in bladder cancer tissues was significantly higher than that in normal tissues (P= 0.0004); the means ±SD for IGFBP4-1 expression in normal tissues and bladder cancer were 3.687217± 0.258 and 6.195 ± 0.187, respectively. Real-time PCR and western blotting showed higher IGFBP4-1 in all four bladder cancer cell lines (5637, J82, T24 and UMUC3) compared to normal human bladder uroepithelium cell line (SV-HUC-1) (Figure 1B, 1C). Furthermore, we assessed the expression of IGFBP4 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 IGFBP4 were higher in bladder cancer tissues (Figure 1D). In addition, we also evaluated the expression of IGFBP4 by immunohistochemical analysis of 100 patients who underwent transurethral resection of the bladder or radical cystectomy. High expression of IGFBP4 was observed in the tumor cells of 65% of the patients (Figure 1E, 1F).

Figure 1

IGFBP4-1 is upregulated in bladder cancer tissues, and this upregulation is associated with poor prognosis. A. Comparison of IGFBP4-1 expression of bladder cancer patients in the TCGA profile. B. qRT-PCR analysis of IGFBP4-1 expression in SV-HUC-1 and bladder cancer cell lines. C. Western blotting of IGFBP4 expression in SV-HUC-1 and bladder cancer cell lines. D. IGFBP4 protein expression level in five paired bladder cancer tissues. E. Representative images of IGFBP4 high expression in bladder cancer. F. Representative images of IGFBP4 low expression in bladder cancer. G. Patients with high IGFBP4-1 expression level had a shorter overall survival compared with that of the low IGFBP4-1 expression level group. H. Kaplan- Meier curves of overall survival of bladder cancer patients in the TCGA profile. *, p<0.05, **, p<0.01, ****, p<0.0001.

High IGFBP4-1 Expression in Bladder Cancer Correlates with Poor Survival

The association between IGFBP4-1 expression and the clinicopathological features of bladder cancer was shown in Table 2. Overexpression of IGFBP4-1 was observed to be significantly associated with T classification (p=0.026) and tumor grade (p=0.001). Yet, the IGFBP4-1 level was not associated with gender, age, N classification and metastasis. As shown in Table 3, Univariate Cox regression analysis confirmed that higher IGFBP4-1 expression (p< 0.001), T classification (p< 0.001) and tumor grade (p= 0.002) were associated with an increased risk of death. Multivariate Cox regression analysis revealed that IGFBP4-1 level (HR= 4.583, 95% CI 2.242-8.662, p< 0.001) and T classification (HR= 1.39, 95% CI 1.051-1.837, p= 0.021) could be independent prognostic factors for overall survival (OS). The OS rate of patients with bladder cancer with higher IGFBP4-1 expression in tumors was significantly poorer than that of patients with lower IGFBP4-1 expression in tumors (P=0.0277, Figure 1G). That was consistent with the Kaplan-Meier analysis of TCGA which revealed a negative correlation of IGFBP4-1 expression with the OS (P= 0.0319, Figure 1H). Hence, these findings suggest that IGFBP4-1 is highly expressed in bladder cancer, and that IGFBP4-1 is a factor for predicting poor survival in patients with bladder cancer.

Table 2

Relationship between IGFBP4-1 expression and clinicopathological features in patients with bladder cancer

	IGFBP4-1
Characteristics	Low expression no.	High expression no.	P value
Age, years			0.135
≥60	22	50
<60	13	15
Gender			0.793
Male	23	41
Female	12	24
T classification			0.026
Ta	9	5
T1	6	6
T2	14	26
T3	4	21
T4	2	7
N classification			0.476
N0	31	56
N1	2	5
N2	1	4
N3	1	0
Metastasis			0.461
No	35	64
Yes	0	1
Tumor grade			0.001
PUNLMP	1	0
Low grade	18	12
High grade	16	53

Table 3

IGFBP4-1 regression analysis for predicting cancer specific survival of bladder cancer

		Univariate analysis			Multivariate analysis
	P value	Hazard Ratio	95% confidence interval	P value	Hazard Ratio	95% confidence Interval
IGFBP4-1	<0.001	4.942	2.851-8.567	<0.001	4.583	2.242-8.662
T classification	<0.001	1.599	1.286-1.987	0.021	1.39	1.051-1.837
Tumor grade	0.002	2.358	1.38-4.028	0.981	0.991	0.467-2.102

IGFBP4-1 Enhanced Proliferation of Bladder Cancer Cells

GSEA analysis was performed in the TCGA database and the results showed that higher levels of IGFBP4-1 are positively associated with an enrichment of cell cycle gene signatures (Figure 2A). To further explore the biological function of IGFBP4-1 in bladder cancer, three IGFBP4-1-specific siRNAs were used to construct IGFBP4-1 knockdown cells in T24 and UMUC3 cell lines, which had relatively higher IGFBP4-1 expression, and IGFBP4-1 was ectopically overexpressed in 5637 and J82 cell lines, which displayed relatively lower IGFBP4-1 expression. The expression of IGFBP4-1 and IGFBP4 were significantly decreased in T24 and UMUC3 cells and overexpressed in 5637 and J82 cells (Figure 2B and 2C). CCK-8 assays showed silencing of IGFBP4-1 using si IGFBP4-1-1 and si IGFBP4-1-2 significantly reduced cell viability of T24 and UMUC3 cells (Figure 2D). Furthermore, overexpression of IGFBP4-1 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 IGFBP4-1 promotes the proliferative ability of bladder cancer cells.

Figure 2

IGFBP4-1 enhances proliferation of bladder cancer cells. A. GSEA plot showing that IGFBP4-1 expression positively correlated with cell cycle activated gene signatures. B, C. IGFBP4-1 and IGFBP4 were stably overexpressed in 5637 cells and J82 cells, silenced in T24 cells and UMUC3 cells by transfection and selection, respectively. IGFBP4-1 expression was confirmed by real-time PCR and IGFBP4 expression was detected by western blotting analysis. D. CCK-8 assays revealed that downregulation of IGFBP4-1 inhibited the growth rate of T24 and UMUC3 cells and overexpression of IGFBP4-1 promoted the growth rate of 5637 and J82 cells. ****, p<0.0001.

IGFBP4-1 is involved in Cell Cycle G1 to S Phase Transition in Bladder Cancer Cells

The role of IGFBP4-1 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 IGFBP4-1. The converse was true after overexpression of IGFBP4-1 in the cell lines. As shown in Figure 3A, cells in the G0/G1 phase were increased in IGFBP4-1 knockdown UMUC3 and T24 cells compared to the control groups. IGFBP4-1 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 downregulated after IGFBP4-1 was silenced (Figure 3C). Meanwhile IGFBP4-1 overexpressed cells showed the opposite trend (Figure 3D).

Figure 3

IGFBP4-1 is involved in cell cycle progression. A. Downregulation of IGFBP4-1 induced G0/G1 phase arrest in T24 and UMUC3 cells. B. Upregulation of IGFBP4-1 promoted cell cycle G1/S phase transition in 5637 and J82 cells. C. Western blotting analysis revealed that Cyclin D1 was decreased after IGFBP4-1 silenced. D. Western blotting analysis revealed that Cyclin D1 was increased after IGFBP4-1 overexpressed. ****, p<0.0001.

IGFBP4-1Inhibits Apoptotic Activity in Bladder Cancer Cells

As decreasing the IGFBP4-1 expression greatly inhibited cell viability, it was hypothesized that IGFBP4-1 may affect cell apoptosis in bladder cancer cells. To further investigate the mechanisms underlying the regulatory function of IGFBP4-1 in the proliferation of bladder cancer cells, we performed flow cytometry assays with Annexin V and PI double staining in UMUC3, T24, 5637 and J82 cell lines. Flow cytometry disclosed that IGFBP4-1 silencing significantly increased the percentage of apoptotic cells compared with the control group in T24 and UMUC3 cells, while the overexpression of IGFBP4-1 yielded inverse results in 5637 and J82 cells. (Figure 4A, 4B). Furthermore, the expression level of cell apoptosis related biomarkers in the aforementioned cell lines was also examined. We found the Bcl2, an anti-apoptotic factor, were significantly decreased whereas Bax, a pro-apoptotic factor, was significantly increased after IGFBP4-1 was silenced (Figure 4C). Meanwhile IGFBP4-1 overexpressed cells showed the opposite trend (Figure 4D).

Figure 4

IGFBP4-1 interfered with bladder cancer cell apoptosis. A. Downregulation of IGFBP4-1 promoted the T24 and UMUC3 cells apoptosis. B. Upregulation of IGFBP4-1 decreased the percentage of 5637 and J82 apoptotic cells. C. Western blotting analysis of IGFBP4-1, Bax and Bcl2 expression in indicated cells. ****, p<0.0001.

IGFBP4-1 Regulates the Tumorigenesis in Vivo

To verify the effects of IGFBP4-1 on the tumorigenicity in vivo, xenograft models were established by injecting stable knockdown IGFBP4-1 T24 cells and vector transfected T24 cells into subcutaneous tissues of nude mice. All nude mice developed xenogeneic tumors at the injection site (Figure 5A). Tumor growth of IGFBP4-1 silenced cells was slower than that of the vector transfected cells (Figure 5B). As shown in Figure 5C and 5D, downregulation of IGFBP4-1 significantly decreased the xenograft tumor volume and tumor weight compared to the control group. qRT-PCR and western blotting confirmed that IGFBP4-1 and IGFBP4 expression levels were lower in the tumors injected with IGFBP4-1 depleted cells than that in the vector transfected cells (Figure 5E and 5F). Taken together, these results indicate that IGFBP4-1 plays a vital role in the tumorigenicity of bladder cancer in vivo.

Figure 5

Overexpression of IGFBP4-1 promoted the tumorigenesis of bladder cancer Representative images of xenograft models. B. Xenograft tumors from respective groups were shown after injection with IGFBP4-1 stable knockdown T24 cells and vector transfected T24 cells. C. Tumor growth curves were measured every three days. D. Average weight of excised tumors. E, F. RT-qPCR and western blotting analysis of IGFBP4 expression in excised xenograft tumors. ***, p<0.001, ****, p<0.0001.

IGFBP4-1 Promoted the Proliferation by activating the JAK/STAT Signaling Pathway

By performing GSEA analysis in the TCGA database, we found that IGFBP4-1 expression was strongly associated with the JAK/STAT signaling pathway, which plays an essential role in cell proliferation 16. The results suggested that the JAK/STAT signaling pathway may be involved in the function of IGFBP4-1 (Figure 6A). As shown in Figure 6B, phosphorylation of STAT3 was increased in IGFBP4-1 overexpressed J82 and 5637 cells compared with the vector group but decreased in IGFBP4-1 silenced T24 and UMUC3 cells. To confirm the role of the JAK/STAT pathway in the process of IGFBP4-1 mediated cell proliferation, IGFBP4-1 overexpressed 5637 cells were treated with 10 µmol/L AG490, a JAK/STAT pathway inhibitor, or vehicle (DMSO), for 24 hours. As shown in Figure 6C, 6D and 6E, AG490 could inhibit the effect of IGFBP4-1 on cell proliferation, cell cycle and cell apoptosis. Moreover, the inhibition of JAK/STAT rescued the effects of IGFBP4-1 on cyclin D1, Bax, Bcl2, and phosphorylation of STAT3 (Figure 6F). These results suggest that IGFBP4-1 enhances the cell proliferation via activation of the JAK/STAT pathway in bladder cancer cells.

Figure 6

Regulation of JAK/STAT signaling by IGFBP4-1. A. GSEA plot showing IGFBP4-1 expression was positively associated with JAK/STAT pathway. B. Expression of JAK/STAT pathway key genes were detected by western blotting. C. CCK-8 assay revealed the role of JAK/STAT in the proliferation of IGFBP4-1 -transfected cells. D. Flow cytometry assay revealed the role of JAK/STAT in the G1/S phase transition of IGFBP4-1-transfected cells. E. Flow cytometry assays with Annexin V and PI revealed the role of JAK/STAT in the apoptosis of IGFBP4-1-transfected cells. F. Western blotting revealed the role of JAK/STAT in the downstream cell cycle and cell apoptosis associated genes of IGFBP4-1-transfected cells. ****, p<0.0001.

Discussion

Previous studies indicated that IGFBPs involved in the growth, chemo-resistance and progression in many cancers 17-20. IGF2BPs have been identified as positive regulators of cell proliferation and metastasis. A study by Xu et al revealed that IGF2BPs wee upregulated in colorectal cancer and associated with worse clinical outcome, which implies that IGF2BPs harbor prognostic significance 14. IGF2BPs overexpression has been linked to advanced disease stage and adverse clinical outcome in several cancers 21-23. Furthermore, IGF2BPs was found to act as oncogenic factors promoting proliferation and invasion of glioblastoma via activating PI3K/MAPK pathway 24. However, the role of lncRNA IGFBP4-1 in driving proliferation of bladder cancer has yet be elucidated. In our study, we found IGFBP4-1 could promote tumorigenesis of bladder cancer by JAK/STAT signaling. Thus, we have identified IGFBP4-1 as a bladder-specific pro-proliferation marker that activates JAK/STAT pathway. Through an analysis of the TCGA database, we showed IGFBP4-1 was upregulated in bladder cancer tissues, which was confirmed in experiments, conducted with 5 pairs of bladder cancer tissues and bladder cancer cell lines. In the present study, results of clinically relevant study suggested that bladder cancer patients with higher IGFBP4-1 levels were more prone to poor overall survival, and higher IGFBP4-1 expression was correlated with a significantly increased risk of death in bladder cancer patients. In order to confirm the role of IGFBP4-1 in modulating tumor cell fate, IGFBP4-1 was overexpressed or knocked down in bladder cancer cell lines. The results showed IGFBP4-1 significantly inhibited cell apoptosis and promoted cell growth and cell cycle progression in vitro. The effects of IGFBP4-1 on the tumorigenicity of bladder cancer cells were also confirmed by in vivo assays. IGFBP4-1 silenced cells had a significantly decreased ability to form tumors in nude mice compared with vector transfected cells.

Consistent with previous studies, our study demonstrated that IGFBP4-1 could be considered to play an oncogenic role in the progression of bladder cancer by promoting cell growth. Additionally, we further discovered IGFBP4-1 promote cell growth of bladder cancer cells via JAK/STAT signaling. JAK/STAT signaling plays a crucial role in regulating cell growth, apoptosis and differentiation, and is activated in many tumors 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 signaling in Non-small cell lung cancer 28. Inhibition of JAK/STAT signaling 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 signaling pathway 31. JAK-STAT signaling pathways also play key roles in maintaining the stemness, self-renewal and proliferative potential of bladder cancer stem cells 32. Our results showed that upregulation of IGFBP4-1 could increase the expression of phosphorylation of STAT3 and IGFBP4-1 knockdown significantly reduced the expression of phosphorylation of STAT3. According to the in vitro assays, we concluded that STAT3 affects phenotypes by regulating the cyclin D1, Bcl2 and Bax expression level. Then we treated IGFBP4-1 overexpressed cells with AG490, a JAK/STAT pathway inhibitor, or vehicle (DMSO) and found the inhibition of JAK/STAT rescued the effects of IGFBP4-1 on phosphorylation of STAT3, cyclin D1, Bcl2 and Bax. Besides, the promoting effects of IGFBP4-1 on cell proliferation was impaired by AG490, and the cell apoptosis rate of IGFBP4-1 overexpressed cells cultured in AG490 was at partially increased as compared with IGFBP4-1 overexpressed cells cultured in normal media. Moreover, percentage of cells in the S phase was significantly reduced in IGFBP4-1 overexpressed cells with AG490 treatment compared with DMSO treatment. Therefore, we conclude that IGFBP4-1 functions as a tumor promotor via JAK/STAT signaling pathway in bladder cancer development. In summary, our study found IGFBP4-1 upregulation 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. IGFBP4-1 exhibits an oncogenic role in the development of human bladder cancer.