STAT3 promotes tumour progression in glioma by inducing FOXP1 transcription

Abstract Objective This paper investigated the effects of STAT3 through promoting FOXP1 transcription on proliferation, apoptosis and invasion in glioma cells. Methods Quantitative real‐time PCR (qRT‐PCR) and Western blot assay were administered to assess the mRNA and protein expression levels of STAT3 and FOXP1 in glioma tissues and cells, respectively. Luciferase reporter and Chromatin Immunoprecipitation (ChIP) assays were implemented to determine the correlation between STAT3 and FOXP1. MTT and colony formation assays were conducted to identify cell growth. Flow cytometry was run to detect the cell apoptosis rate of glioma cells. Transwell assays were conducted to reveal cell invasion ability. Results The mRNA and protein expression levels of STAT3 were highly expressed in glioma tissues and cells. After cells transfected with siRNA of STAT3, both STAT3 and FOXP1 were simultaneously downregulated. STAT3 directly regulated FOXP1 transcription. STAT3 promoted cell proliferation, inhibited cell apoptosis and enhanced cell invasion through promoting FOXP1 transcription in glioma cells. Conclusion In summary, STAT3 gene was a transcriptional regulator of FOXP1. Depleted STAT3 restrained cell proliferation and invasion, promoted cell apoptosis in glioma cells. This molecular mechanism between STAT3 and FOXP1 can serve as a therapeutic target for glioma treatment.

transmitting signals form cytokines and growth factors. 10,11 The activation of the STAT3 protein is composed of a chain of events including dimerization of the N-terminal domain, then Src Homology 2 (SH2) domain, the subsequent DNA-binding domain and the final transcription activation of the C-terminal domain. [12][13][14] and the transcription activation are of great significance for STAT3 activation. 13 However, STAT3 is constantly activated in a wide range of human malignant tumours including glioma. [15][16][17][18] And once activated, STAT3 is associated with oncogenesis and cancer progression through promoting the transcription of a few genes which control tumour cell viability, and inhibit cell apoptosis, vascularization and cell cycle. [19][20][21][22] To be more specific, the constitutively activated STAT3 elevates the expression of the downstream gene including Bcl-2, Bcl-xl, c-myc, survivin and vascular endothelial growth factor (VEGF). [23][24][25] Forkhead box protein P1 (FOXP1), a member of FOXP subfamily, is a transcriptional factor that has extensive functions. 26 The FOXP subfamily is part of FOX proteins superfamily which has a highly preserved "fork-head" DNA-binding domain. 27 FOX proteins mediate cell-division cycle, cell viability, differentiation and apoptosis. 28 FOXP1 serves as both potential oncogene and tumour suppressor due to its distinguishing expression levels in diverse tumour types. 29 On the one hand, FOXP1 was reported to be an oncogene with overexpressed mRNA levels and protein levels of various human Bcell lymphomas. [30][31][32][33][34] On the other hand, silencing of FOXP1 has been associated with prostate cancer, renal cell cancer and breast cancer. [30][31][32]35,36 Findings of the molecular mechanism of oncogenesis and progression of glioma are vital to ameliorate present treatments and develop new therapy. Up to now, however, the associations between STAT3 and FOXP1 expression in human glioma have not been examined. Our findings facilitate the perception of molecular mechanism and effects of STAT3 on glioma progression via inducing FOXP1 and usher in a novel therapy for gliomagenesis.

| Tissue samples and clinical characteristics
All glioma tissue samples were gained from a total of 71 patients undergoing operative treatment at the Zhujiang Hospital, Southern Medical University, after they signed the written informed consent.
In total, 71 glioma tissues and 71 adjacent glioma tissues were stored frozen at −80°C until for the use of qPCR or western blot measurement. The current study was confirmed by the Ethics Committee of Zhujiang Hospital, Southern Medical University. Clinical characteristics were seen in Table 1. Thermo Fisher Scientific). Media were placed in incubators at 37°C with 5% CO 2 .

| Western blot
Total proteins were isolated from the cell lines.

| Chromatin immunoprecipitation assays
The chromatin immunoprecipitation (ChIP) assay kit was purchased through Upstate Biotechnology (Lake Placid, NY, USA). U87 or SHG-44 cells were treated with formaldehyde to cross-link protein and DNA to the FOXP1 promoter, and sonication was used to shear DNA. Chromatin was immunoprecipitated using antibodies against STAT3 or IgG isotype control (all from Abcam). IgG was applied as the negative control for the ChIP assay. The quantification of immunoprecipitated DNA was measured by qRT-PCR. All data were normalized to the percentage of input.

| Colony formation assays
In total, 1 × 10 3 U87 or SHG-44 cells were placed in six-well plates and incubated at 37°C. After transfection, living cells were detected via trypan blue staining and calculated. A lower number of viable cells were then planted in 6-well dishes and went on incubation for 2-3 weeks. Subsequently, cells were rinsed with phosphate-buffered saline (PBS) buffer and set with methanol. Crystal violet with methanol was used to stain transfected U87 cells for 10 minutes at room temperature. Finally, distilled water was used to wash and dry the plates. Each measurement was run in triplicate.

| Flow cytometry
Cell apoptosis rate was caculated by flow cytometry assay. Experiments were randomly divided into NC group and transfected groups including siRNA1-STAT3 group, pcDNA3.

| Statistical analysis
The significance of the data between experimental groups was mea-  Then qRT-PCR analysis revealed that mRNA levels of STAT3 and FOXP1 were simultaneously down-regulated in groups of siRNA1-STAT3 and siRNA2-STAT3 compared with NC group (Figure 2A and B). Western blot analysis revealed that the protein levels of STAT3 F I G U R E 1 STAT3 was highly expressed in glioma tissues and cells. A, The STAT3 expression levels in adjacent and tumour glioma tissues were analysed by qRT-PCR. ***P < 0.001, compared with adjacent tissues. B-C, The STAT3 protein levels in adjacent and tumour glioma tissues were analysed by western blot assay. GAPDH was regarded as an internal control. n = 10, ***P < 0.001, compared with adjacent tissues. D, Relative STAT3 expression levels was detected in astrocyte cells (SVG P12 and HA) and glioma cell lines (SHG-44, U87, GOS-3 and TJ905) by qRT-PCR. **P < 0.01, compared with SVG P12; # P < 0.05 and ## P < 0.01, compared with HA. (E-F) Relative STAT3 protein levels were observed in SVG P12, HA, SHG-44, U87 and GOS-3 and TJ905 cell lines by western blot assay. **P < 0.01, compared with SVG P12; # P < 0.05 and ## P < 0.01, compared with HA and FOXP1 were sharply decreased in siRNA transfected U87 or SHG-44 cells compared with NC cells (Figure 2C and D  Figure 3A and B). Next ChIP assays were conducted to analyse whether STAT3 directly bonded the FOXP1 promoter. As expected, STAT3 protein bound the FOXP1 promoter was dramatically increased in U87 and SHG-44 cells. In other words, binding of STAT3 to the FOXP1 promoter enhanced transcription of FOXP1 ( Figure 3C and D). Besides, the inhibition effects of siRNA1-STAT3 were stronger than siRNA2-STAT3. Therefore, siRNA1-STAT3 was selected to continue the next experiments. In short, STAT3 was the transcriptional regulator of FOXP1.

| DISCUSSION
Related reports had revealed that STAT3 expressions were associated with prognosis of diverse types of tumours such as breast cancer, acute myelogenous leukaemia and glioma, among others. [38][39][40] Previously, a few studies had reported the overexpression of STAT3 in certain cancer types including intrahepatic cholangiocarcinoma, breast cancer and high-grade glioma. 18,41,42 Additionally, Carro et al 43 reported a relationship between STAT3 expression and the mesenchymal transformation of glioma, suggesting that STAT3 was a "master regulator" of glioma transformation. In view of this, we hypothesized that STAT3 overexpression is associated with glioma.
As expected, our findings demonstrated that STAT3 expression levels were elevated at both mRNA and protein levels in human glioma tissues and cells.

| 5635
The molecular mechanisms of STAT3 regulation in glioma remain elusive. To determine the mechanism by which STAT3 regulates glioma progression, we predicted its target genes according to a series of related reports. Subsequently, FOXP1 was revealed to be closely connected with glioma oncogenesis and progression with proliferation, migration and invasion suppression of glioma cells. 44  and FOXP1 may be underlying therapeutics for glioma.

CONFLI CT OF INTEREST
The authors confirm that there are no conflicts of interest.