Mediation of multiple pathways regulating cell proliferation, migration, and apoptosis in the human malignant glioma cell line U87MG via unphosphorylated STAT1

Laboratory investigation

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Object

Signal transducer and activator of transcription 1 (STAT1) is thought to be a tumor suppressor protein. The authors investigated the expression and role of STAT1 in glioblastoma.

Methods

Immunohistochemistry was used to detect the expression of STAT1 in glioblastoma and normal brain tissues. Reverse transcription–polymerase chain reaction and Western blot analysis were used to detect mRNA and protein expression levels of STAT1. Cell growth, proliferation, migration, apoptosis, and the expression of related genes and proteins (Bcl-2, Bax, cleaved caspase-3, caspase-9, p21, and proliferating cell nuclear antigen) were examined in vitro via cell counting kit-8, wound-healing, flow cytometry, Rhodamine B, TUNEL, and Western blot assays.

Results

Human glioblastoma had decreased expression of STAT1 proteins. Transfection of the U87MG cells with STAT1 plasmid in vitro demonstrated significant inhibition of cell growth and an increase in apoptotic cell death compared with cells transfected with vector or mock plasmids. These effects were associated with the upregulation of cleaved caspase-3, Bax, and p21 and the downregulation of Bcl-2 expression.

Conclusions

The results of this study suggest that increased expression of STAT1 by transfection with STAT1 plasmid synergistically inhibits human U87MG glioblastoma cell growth in vitro.

Abbreviations used in this paper:CCK-8 = cell counting kit-8; GBM = glioblastoma multiforme; OD = optical density; PCNA = proliferating cell nuclear antigen; RT-PCR = reverse transcription–polymerase chain reaction; rTdT = terminal deoxynucleotidyl transferase, recombinant; STAT = signal transducer and activator of transcription; TMZ = temozolomide.

Article Information

* Mr. Dou and Dr. Zhao contributed equally to this work.

Address correspondence to: Gang Zhao, M.D., Department of Neurosurgery, First Bethune Hospital of Jilin University, Changchun 130021, Jilin Province, People's Republic of China. email: Baotoujuhaitao@yahoo.com.cn.

Please include this information when citing this paper: published online April 19, 2013; DOI: 10.3171/2013.3.JNS122051.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Expression of STAT1 in glioblastoma and normal brain tissues and the transfection rate of STAT1 vector in U87MG cells. Representative photomicrographs (A–D) of the tissues demonstrating different STAT1 expression levels. Typical glioblastoma area (A) samples with < 5% positive cells were designated as negative (−). Samples stained slightly (B), with between 5% and 25% positive cells, were designated as (+). Samples stained moderately (C), with between 25% and 50% positive cells, were designated as (++). Normal brain tissue (D). Samples stained deeply, with > 50% strongly positive cells, were designated as (+++). Arrows point to positive normal cells. Graphs (E and F) showing transfection rates of STAT1. Control group (E) and Lipofectamine 2000/STAT1 group (F). Bars = 20 μm. The U87MG cell count is measured on the y axis, FL1-H on the x axis refers to FITC-Annexin V, and the line marked M1 signifies a positive cell count.

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    STAT1 plasmids induce U87MG cell apoptosis. A: Analysis of apoptosis after transfection with various plasmids in each group. Bright field photo was observed with an optical microscope (bar = 50 μm). Rhodamine B: Red fluorescence represents living cells (bar = 100 μm). TUNEL assay: Propidium iodide stains both apoptotic and nonapoptotic cells red, and TUNEL-positive cells had green fluorescence (bar = 100 μm). B: Flow cytometry analysis of apoptotic cells with Annexin VFITC and propidium iodide staining at 48 hours after transfection. *p < 0.05, compared with mock and vector groups. The x axis refers to FL1-H (FITC-Annexin V), and the y axis refers to propidium iodide. C: Cleaved caspase-3, caspase-9, Bax, and Bcl-2 protein expression was determined using Western blot analyses. **p < 0.01, compared with mock and vector groups. Data were presented as the means ± standard errors (SEs) of 3 separate experiments.

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    STAT1 plasmids inhibit U87MG cell migration. Upper: Migration abilities were determined using wound-healing assays. Lower: The wound gaps were measured at 6 reference points along the wound, and the results were expressed as the average wound gap. Data represent the means ± SEs of triplicate experiments. **p < 0.01, STAT1 compared with mock and vector groups.

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    Analysis of cell growth and proliferation after transfection with various plasmids. Upper: Growth inhibitory effects were evaluated with CCK-8 assays for the U87MG cells transfected with different plasmids, and the data were presented as the ratio to control (mock). Data represent the means ± SEs of 3 separate experiments. **p < 0.01, compared with mock and vector groups. Lower: Expression of p21 and PCNA was determined by Western blotting. Data represent the means ± SEs of 3 separate experiments. **p < 0.01, compared with mock and vector groups.

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