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Abstract

Background

Glioma is one of the most malignant tumors, which leads to high mortality in cancer patients. At present, there is no effective therapy for glioma. Therefore, it is urgent and necessary to find new molecular targets for anti-glioma therapy.

Objective

The present study aimed to investigate the role of signal transducer and activator of transcription 1 (STAT1) in the development and progression of human glioma and related mechanisms.

Methods

According to the instructions of Lipofectamine TM²⁰⁰⁰ transfection reagent, we transiently transfected the plasmid pcDNA3.1-STAT1 into glioma U251 cells. Then STAT1 expression in glioma U251 and LN382 cells was detected by Western blot. MTT was performed to assay the proliferative activity of U251 cells after STAT1 transduction, flow cytometry was used to detect cell cycle and apoptosis indicators, cell migration indicator was determined by Wound healing, and Western blot was used for detecting the expression level and change trend of p53, p21, bcl-2, Caspase-8, Cyclin A and Cyclin E in transfected cells.

Results

Overexpressed STAT1 significantly inhibited U251 cell proliferation and promoted U251 cell apoptosis. Meanwhile, high expression of STAT1 can increase the expression of p53, p21, and Caspase-8 while inhibiting the expression of bcl-2, Cyclin A, and Cyclin E.

Conclusion

Highly expressed STAT1 inhibits the proliferative activity of human glioma U251 cells and can promote tumor cell apoptosis and block cell cycle progression while regulating the expression of various signal transduction molecules. Thus, STAT1 has a critical function in the development and progression of glioma and is a novel target for glioma therapy.

Keywords

glioma U251 and LN382 cells signal transducer and activator of transcription-1 proliferation cell cycle apoptosis

Introduction

Glioma is the most common malignant and invasive tumor of the central nervous system, with an average morbidity of 4.67-5.73/10000.¹ It originates from the neuroepithelial cell, accounting for 40%∼50% of intracranial tumors.² Glioma is widely invasive, with high malignancy and unclear pathogenesis. It is challenging to be entirely removed by surgery, with a fast growth rate, easy recurrence, and poor prognosis. In recent years, age is strongly associated with higher incidence and mortality rates in GBM.³ According to statistics, the mean survival time (MST) of patients with brain glioma after surgery is about 12-18 months, and the 5 years survival rate is less than 10%.⁴ In the latest years, with the introduction of the concept of molecular diagnosis and the promotion of precision medicine, the survival rate of glioma patients after surgery has improved. However, the average survival period is still less than 15 months.

Signal transducer and activator of transcription-1 (STAT1), which is a very crucial upstream regulator of IFN signal transduction,⁵ can promote apoptosis and inhibit cell proliferation in tumor cells.⁶ Studies show mice that defect STAT1 are more likely to develop tumors compared with wild mice.⁷ STAT1 has a critical effect on the process of tumor formation, including cell cycle progression, cell apoptosis, tumor angiogenesis, and tumor immune surveillance. However, the relationship between the STAT1 gene and glioma development has not been systematically reported, and how the STAT1 regulates and acts on neuroepithelial tissue has not been clarified.

In this study, we examined the expression level of STAT1 in U251 and LN382 cells. Furthermore, we tested the influence of STAT1 overexpression on the proliferation and apoptosis of glioma U251 cells in vitro and also studied the effects of STAT1 overexpression on p53, p21, bcl-2, Caspase-8, Cyclin A and Cyclin E. Then we further explored the effects of STAT1 on the proliferation, cycle, and apoptosis of glioma U251 cells and its possible mechanism, providing a theoretical basis for a new strategy of glioma treatment.

Materials and methods

Cell culture

Human glioma U251 and LN382 cells were provided by the Molecular Pathology Laboratory of Inner Mongolia Medical University. Cells were cultured in DMEM medium, which contained 10% fetal bovine serum, and cells were incubated at 5% CO2 atmosphere. Cells in the logarithmic growth phase were selected during the experiment.

Transient cell transfection

According to the instructions of the Lipofectamine^TM 2000 liposome (Invitrogen, USA) transfection kit, Lipofectamine 2000 complex was prepared with 240 μL per well serum-free and double antibody-free medium +10 μL Lipofectamine^TM 2000 (total volume 250 μL), incubated at indoor temperature for 5 min; The DNA complex consisted in 246 μL per well serum-free and double antibody-free medium +4 μg plasmid (plasmid concentration is 1.0 μg/μL), total volume 250 μL; Mix the two compounds to make DNA-Lipofectamine^TM 2000 composite, and let it stand at room temperature for 20 min. The EV and pcDNA3.1-STAT1 plasmids were obtained from the Molecular Pathology Laboratory of Inner Mongolia Medical University. Using Lipofectamine^TM 2000 transfection reagent, EV and pcDNA3.1-STAT1 were repeatedly transfected into U251 cell (1 × 10⁶/well) in 6 wells. After 48 h of transient transfection, log-phase cells were collected and used for subsequent experiments.

MTT assay

Cells were seeded in 1 × 10⁵ wells in 96-well plates; each well was incubated in a serum-free DMEM in a 37°C, 5% CO₂ incubator for 24 h; Add 1 μg/μL plasmid 0.2 μL and LipofectamineTM2000 transfection reagent 0.5 μL per well; 24 h and 48 h after transfection, 5 mg/mL MTT 20 μl were added to per well, the culture was terminated after 4 hours, and the medium was removed. DMSO 150 μL was inserted per well, and the MTT reagent was fully dissolved by shaking at low speed for 10 min. The absorbance values of the immunoassay were measured on the microplate reader at a length of 490 nm, and the average of 10 composite wells was calculated.

Flow cytometric analysis

After 48 h of transient transfection of cells, logarithmic phase cells were collected. Flow cytometry analysis (BD, FACSCalibur, USA) were performed to detect the percentage of apoptotic cells according to Annexin V-FITC/PI double staining.

Wound-healing and transwell assays

Draw a horizontal line on the back of the 6-hole plate for marking, with the line to line spacing of about 1 cm, and added U251 cells (5 × 10⁵/well) in each hole; After the cells adhered to the wall, the gun head scratched, PBS cleaned the cells for three times, and added the serum-free and double antibody-free medium; After 4∼6 h of plasmid transfection, replaced the serum-free and double antibody free medium, and put it into the cell incubator for culture. Took photos after 0 and 24 h, respectively. Image J software was used to measure the scratch distance of cells at 0 h and 24 h and calculate the scratch healing rate of cells. Transwell assays were conducted using a transwell chamber coated with Matrigel. The 10% FBS medium was added in the lower chamber, and U251 cells (4 × 10⁴/well) in serum-free medium were added to the upper chamber. After incubation at 37°C with 5% CO₂ for 24 h, the cells on the lower surface were fixed with methanol and stained with 0.1% crystal violet.

Western blotting

Total protein was extracted 48 hours after the transient transfection of cells according to the protein extraction kit instructions. After the determination of protein concentration by BCA method, cell lysates (20 µg/l) were separated by 10% SDS-PAGE and transferred to PVDF membranes. The PVDF was blocked with 5% non-fat dry milk in Tris-buffered saline plus Tween-20 and incubated at 4°C overnight with anti-STAT1 (1:2,000 dilution) (Santa Cruz, USA), anti-p53 (1:500 dilution), anti-p21 (1:500 dilution) (Santa Cruz, USA), anti-bcl-2 (1:500 dilution), anti-Caspase-8 (1:500 dilution), anti-Cyclin A (1:500 dilution) and anti-Cyclin E (1:500 dilution) (Abcam, UK), and anti-β-actin (1:2000 dilution) (Boster, China). The membranes were washed twice with TBST for 10 min/time, hatched with a fluorescent secondary antibody for 1 hour at indoor temp, and the PVDF membranes were washed three times with TBST for 5 mins each time. The PVDF membranes were scanned, photographed and recorded. Immunoblots were quantified with optical methods using the ImageJ software. The results were normalized using β-actin as an internal control.

Statistical analysis

SPSS17.0 software was used to analyze the data. The continuous variable conforming to the normal distribution is expressed as mean ± standard deviation ( $\bar{x} \pm s$ ). The difference between groups was analyzed by one-way ANOVA. p < .05 was considered statistically significant.

Results

Overexpression of STAT1 Suppresses glioma proliferation and Promotes Apoptosis In Vitro

We labeled the transfected cells as a recombinant plasmid (pcDNA3.1-STAT1 group) and control empty vector pcDNA3.1 (EV group), respectively, and set up a blank control group (Mock group). After 48 h of transfection, under the inverted phase contrast microscope, many dead cell floating and cell fragments were seen in the culture medium of the pcDNA3.1-STAT1 group; the number of adherent cells were significantly reduced, and the growth is slow, with an irregular shape, elongation or shrinkage, and poor refraction. The transfection efficiency was observed under the fluorescence microscope. In the pcDNA3.1-STAT1 group, more green fluorescent proteins were expressed (Figure 1(a)). STAT1 expression was measured by western blot. STAT1 protein levels were obviously larger in the pcDNA3.1-STAT1 group than in the EV and Mock group, but there was no marked change in STAT1 expression levels between the EV and Mock groups (Figure 1(b)). In a further, the proliferation of the pcDNA3.1-STAT1 group was considerably reduced in comparison to the EV group (Figure 1(c)). Still, the percentage of apoptotic death in the pcDNA3.1-STAT1 group was remarkably higher than that in the EV group (Figures 1(d) and (e)). The ratio of G0/G1 cells was significantly increased in the pcDNA3.1-STAT1 group compared to the Mock and EV groups, while the proportion of S cells decreased significantly (Figures 1(f) and (g)). The results showed that STAT1 could inhibit DNA synthesis and mitosis of glioma U251 cells, leading to cell arrest in G0/G1 phase. The Wound healing experiments showed that the number of cells entering the scratch was significantly reduced in the pcDNA3.1-STAT1 group, and the ability of cell migration was inhibited. Therefore, the high expression of STAT1 inhibits the migration of glioma cells (Figure 1(h)). In conclusion, high expression of STAT1 inhibited the proliferation of U251 cells and also promoted apoptosis in vitro.

Figure 1.

The overexpression of STAT1 inhibits U251 cell proliferation and promotes U251 cell apoptosis in vitro. 48 h after transfection, MTT was used to detect the proliferation activity of U251 cells after STAT1 transfection, flow cytometry assays were performed to determine the cell cycle and apoptosis indicators, while Wound healing test was used to detect cell migration indicators. Data are representative images or expressed as $\bar{x} \pm s$ of a single group from three separate experiments. (a) Fluorescence microscope observation of pcDNA3.1-STAT1 transfection (×200). (b) The levels of STAT1 proteins in the different groups of cells were determined by western blot assays. (c) U251 cell proliferation. (d)–(g) U251 cell apoptosis and cell cycle. (h) Wound-Healing and Transwell assays (×100). *p < .05, compared with Mock group and EV group, respectively.

Effect of high expression of Stat1 on the expression of P53, P21, Bcl-2, Caspase-8, cyclin A and cyclin E in U251 cells

The very important regulators of cell proliferation and apoptosis include p53, p21, bcl-2, Caspase-8, Cyclin A, and Cyclin E. For the mechanism of STAT1 action, we investigated the levels of this protein expression in several groups of U251 cells. The expression of p53, p21, and Caspase-8 in the pcDNA3.1-STAT1 group was markedly larger than that in Mock or EV groups, while the expression of bcl-2, Cyclin A and Cyclin E was significantly lower than that in Mock group and EV group (p < .05) (Figures 2(a)–(f)). Thus, high expression of STAT1 decreased the expression of bcl-2, Cyclin A, and Cyclin E, but raised the expression of p53, p21, and Caspase-8 in U251 cells.

Figure 2.

Effect of high expression of STAT1 on the expression of p53, p21, bcl-2, Caspase-8, Cyclin A and Cyclin E in U251 cells. U251 cells were transfected with plasmids for 48 h. The relative levels of p53, p21, bcl-2, Caspase-8, Cyclin A and Cyclin E proteins in the different groups of cells were determined by western blot assays (A-F). Data are representative images or expressed as $\bar{x} \pm s$ of a single group from three separate experiments. *p < .05, compared with Mock group and EV group, respectively.

Discussion

Members of the STAT family, which were initially thought to be cytokine-related signaling proteins, are now among the most promising molecular targets for cancer therapy.^8–10 In the STAT family, STAT1 modulates the IFN-γ-dependent signaling pathway and regulates a variety of biological roles, including growth arrest, induction of cell death as well as antiviral defense.^11–13

Thus, our experimental result suggests that STAT1 may be a negative modulator that affects glioma development and progression.^14,15 At the same time, we also proved this by the effect of highly expressed STAT1 on U251 cell proliferation and apoptosis. We found that the overexpression of STAT1 induced by transient transfection inhibited the proliferation of U251 cells and further promoted apoptosis. The experimental results clearly show that STAT1 is an inhibitor of glioma cell proliferation and a catalyst of apoptosis, which is consistent with Ryan N's research.¹⁶

Cyclin A, Cyclin E, p53, p21, bcl-2, and Caspase-8 are closely associated with STAT1-regulated cell survival, proliferation, and apoptosis.⁸ Cyclin A is produced in the G2/S phase of the cell cycle and plays a significant role in the G1/S and G2/M conversion. It has been reported that IFN-βinduces G0/G1 phase arrest in human malignant glioma cells by downregulating cyclin A and cyclin-dependent kinase expression.¹⁷ Cyclin A is a necessary regulatory protein in the G1-S phase. Its overexpression will lead to the increase of cells entering the S phase and promote cell proliferation. If its expression is downregulated, the cell cycle will be blocked, and apoptosis will occur. Cyclin E mainly acts on the late G1 phase. Before the start of the S phase, Cyclin E regulates the cell to enter the S phase from the G1 phase. Luhtala S¹⁸ has shown that Cyclin E overexpression exists in a variety of malignant tumor cells, leading to abnormal proliferation of tumor cells. The high expression is often related to tumor invasiveness and poor prognosis. We revealed that the expression levels of Cyclin A as well as Cyclin E were obviously reduced after induction of STAT1 overexpression in U251 cells. These experimental data strongly suggest that high expression of STAT1 inhibits the expression of Cyclin A and Cyclin E while inducing cell cycle arrest in gliomas. P53 is a suppressor oncogene, and more than 50 percent of malignant tumors have mutations in this gene. Like all other tumor-repressive genes, the p53 gene can slow down the division of the normal cell. Indeed, a previous study demonstrated that p53 is an important negative regulator of the cell cycle in glioma.¹⁹ STAT1 negatively regulates the p53 inhibitor MDM2 gene, thereby increasing the expression of p53.²⁰ In addition, STAT1 can directly interact with p53, acting as a co-activator and regulating the functional activity of p53 responsive genes.²¹ P21, a member of the CIP family, is a cell cycle protein-dependent kinase inhibitor located downstream of the p53 gene. It can inhibit the mitotic activity of some cells through its effect on the cell cycle, thereby inhibiting cell growth.²² In this study, we observed that high expression of STAT1 resulted in elevated expression levels of p53 and p21. Indeed, a previous study demonstrated that the anti-tumor effect of STAT1 is achieved by up-regulating p53.²³ The up-regulation of p53 can promote the expression of p21²⁴ so that the cell cycle is blocked in the G1/S phase, and apoptosis often occurs in the G1 phase, thus leading to apoptosis.²⁵ Hua Wei et al showed that STAT1 totally inhibited P-STAT1 and downregulated p53 and p21, which further indicated the role of the STAT1-p53-p21 axis in the regulation of apoptosis, oxidative stress and senescence in the ADR-induced Chronic nephrosis mouse model.²⁶ Activated STAT1 can specifically bind to the conserved STAT1 response element in the p21 promoter region, and positively regulate the expression of p21, leading to cell cycle arrest or apoptosis.^27,28 Therefore, after activation of STAT1, p21 can be increased through dependent or independent p53 pathways. In summary, the downstream genes p53 and p21 regulated by STAT1 are senescence-related genes. Research has shown that Bmp9 treatment inhibits osteoblast senescence through activating Smad1, which suppresses the transcriptional activity of Stat1, thereby inhibits P21 expression and SASPs production.²⁹ Sun Y et al showed that 82% (82/100) of pancreatic adenocarcinoma cases expressed Stat1 protein, whereas 72% (72/100) of PA cases expressed p21 protein. The expression of these 2 proteins was positively correlated (co-expressed in tumor cells), whereas the loss of expression of Stat1 and p21 proteins was associated with tumor dedifferentiation, advanced clinical stages, and LN metastasis of pancreatic cancer.³⁰ The reports showed the decrease in STAT1 expression significantly leads to a decrease in p21 expression, leading to cell cycle arrest in cancer cells in G0/G1 phase.³¹ The research indicated that the activation of STAT1 plays a major role in the transcriptional activation p21 in a p53-independent manner. The results demonstrated that the increased p21 expression by Treatment with aluminum chloride (AlCl₃) is dependent on STAT1. The results implied that the change of p21 modulated by the STAT1 gene might influence the cell cycle distribution caused by AlCl₃.^28,32 B-cell lymphoma-2 (bcl-2) family proteins are essential modulators of programmed cell death and apoptosis and play a prominent role in human cancer. Bcl-2 protein can regulate the appropriate Ca₂⁺ concentration in the endoplasmic reticulum and mitochondria, keep them in dynamic balance, and avoid cell apoptosis caused by excessive Ca₂⁺ concentration in mitochondria.³³ In addition, it has been reported that bcl-2 inhibits apoptosis by inhibiting cytochrome C release from mitochondria in glioblastoma.³⁴ STAT1 is reported to promote apoptosis of retinal pericytes under high glucose conditions via increasing Bcl-2-like protein 11 (Bim) expression.³⁵ Also, it’s revealed that STAT1 downregulate Bcl-2 expression and to be involved in interferon-γ/tumor necrosis factor-α-induced apoptosis in NIT-1 cells.³⁶ Those results suggested a positive correlation between STAT1 and apoptosis. Caspase-8 is a crucial promoter of death receptor-mediated apoptosis. It can cut and activate itself through oligomerization and activate the downstream cysteine protease to produce an apoptosis effect.³⁷ Research shows that Caspase-8 expression is often lost in glioblastoma. This event may account not only for glioblastoma progression but also for glioblastoma resistance to radiotherapy and chemotherapy.³⁸ Rajendra Karki et al identified that high expression of STAT1 raised the expression of Caspase-8, which was a critical role for STAT1 in promoting inflammatory cell death.³⁹ Jerzy A et al found that the scaffold function of caspase-8 facilitated activation of lethal JAK1/2-STAT1 signaling in intestinal epithelial cells.⁴⁰ Similarly, Iris Stolzer et al reported that cell death, barrier breakdown and systemic infection were abrogated by an additional deletion of STAT1 in Casp8ΔIEC mice. In the absence of epithelial STAT1, loss of epithelial cells was abolished which was accompanied by a reduced Caspase-8 activation. Mechanistically, they demonstrated that epithelial STAT1 acted upstream of caspase-8-dependent as well as -independent cell death.⁴¹ Victoria et al discovered that downregulation of caspase-8 in breast cancer cells completely abolished TRAIL-induced STAT1 phosphorylation and the increase in total STAT1 protein levels.⁴² Thus, STAT1 may promote the expression of p53, p21, and Caspase-8, but inhibit the expression of bcl-2, Cyclin A, and Cyclin E, resulting in cell cycle arrest, apoptosis as well as growth inhibition in glioma cells. The result suggests that STAT1 may inhibit glioma cell proliferation and a catalyst for apoptosis.

In addition, the empirical results reported herein should be considered in the light of some limitations. First, our studies were performed in low-passage glioma cell lines that may not represent the comprehensive biology of GBM in vivo. Therefore, these results obtained from these low-passage glioma cell lines, ideally, need to be validated in more cell lines or primary cultures of GBM. In addition, it is necessary to understand whether STAT1 can also upregulate other related factors to affect the cycle and apoptosis of glioma cells. At the same time, this topic lacks in vitro experimental studies to further validate the results. These issues are currently being investigated in our laboratory. Nevertheless, understanding the role of STAT1in glioma progression will improve our understanding of the mechanisms underlying glioma survival, as well as help establish STAT1 as a potential therapeutic target for the treatment of glioma.

Conclusion

STAT1 plays a critical role in the development and progression of glioma. The conclusion indicates that STAT1 regulates proliferation and apoptosis through multiple molecular signaling pathways, which may become a novel target for glioma therapy.

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the National Natural Science Foundation of China (grant no. 81660509), Natural Science Foundation of Inner Mongolia Autonomous Region (grant nos. 2014MS0836 and 2015BS0810), Science Research Project of Colleges and Universities in Inner Mongolia Autonomous Region (grant no. NJZY20141), Hohhot Religion High-quality Developmental and Advantageous Key Clinical Project of Neurological System Disease, Inner Mongolia Autonomous Region Clinical Medicine Research Center of Nervous System Diseases and The research project of Inner Mongolia Medical University Affiliated Hospital (grant no. 2023NYFY LHZD002, 2023NYFY LHYB001).

ORCID iDs

Ping Huang

Hongwei Wang

Data Availability Statement

sets used during this study were available from the corresponding author on reasonable request.

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. Induction of interferon-beta and interferon signaling by TRAIL and Smac mimetics via caspase-8 in breast cancer cells. PLoS One 2021; 16(3): e0248175. DOI: 10.1371/journal.pone.0248175.

STAT1 negatively regulates human glioma U251 cell proliferation Huang et al: Role of STAT1 in glioma

Abstract

Background

Objective

Methods

Results

Conclusion

Keywords

Introduction

Materials and methods

Cell culture

Transient cell transfection

MTT assay

Flow cytometric analysis

Wound-healing and transwell assays

Western blotting

Statistical analysis

Results

Overexpression of STAT1 Suppresses glioma proliferation and Promotes Apoptosis In Vitro

Effect of high expression of Stat1 on the expression of P53, P21, Bcl-2, Caspase-8, cyclin A and cyclin E in U251 cells

Discussion

Conclusion

Footnotes

Declaration of conflicting interests

Funding

ORCID iDs

Data Availability Statement

References