Snail homolog 1 is involved in epithelial-mesenchymal transition-like processes in human glioblastoma cells

Abstract

Despite advancements in neurosurgery, chemotherapy and radiation therapy, the outcome of patients with glioblastoma remains poor. The migration of tumor cells from the primary tumor site with subsequent invasion of these cells into the surrounding normal brain tissue is frequently responsible for relapse and treatment failure. The present study hypothesized that snail homolog 1 (SNAI1), a factor critically involved in the epithelial-mesenchymal transition (EMT) of human carcinoma cells, may also contribute to an invasive EMT-like phenotype of glioblastoma cells. The majority of glioblastoma cell lines investigated in the present study expressed SNAI1 at basal levels. The present study overexpressed SNAI1 in glioblastoma cell lines by lentiviral transfer of human SNAI1 complementary DNA. In addition, the inhibition of SNAI1 expression was achieved by lentiviral transfer of a short hairpin RNA specific for SNAI1. SNAI1 overexpression increased proliferation of one of the cell lines, U251MG, but exhibited only a weak effect on the migration and invasion of glioblastoma cells. However, downregulation of SNAI1 significantly decreased the invasive capacity of all investigated cell lines. In parallel, regained expression of E-cadherin, a marker that is usually lost during EMT, was observed subsequent to SNAI1 knockdown in the glioblastoma cell lines U87MG and U251MG. The data of the present study suggest that certain key genes of the EMT in carcinoma are also involved in the migration and invasion of human glioblastoma cells.

Keywords: E-cadherin; epithelial-mesenchymal transition; glioblastoma; snail homolog 1; tumor cell invasion.

Figure 1.

Expression of SNAI1 in glioblastoma cell lines. Results are presented from a single reverse transcription-quantitative polymerase chain reaction analysis, subsequent to overexpression of SNAI1 by a lentiviral vector. Control vector-transfected cells served as controls. For semi-quantitative analysis, β-actin was used as a housekeeping control, and expression in mock-transfected U87MG cells was set as 1. SNAI1, snail homolog 1.

Figure 2.

Effect of SNAI1 on the proliferation of glioblastoma cells. Proliferation of the indicated cell lines was assessed by MTT assay. Control vector-transfected cells and cells with SNAI1 overexpression were analyzed. Means and standard deviations from triplicate experiments are presented. *P<0.05. SNAI1, snail homolog 1.

Figure 3.

Effect of SNAI1 on the migration and invasion of glioblastoma cells. (A) The migration of the indicated cell lines was assessed. Control vector-transfected cells and cells with SNAI1 overexpression were analyzed. Means and standard deviations from three independent experiments are presented. (B) The invasion ability of the indicated cell lines was assessed by Matrigel assay. Control vector-transfected cells and cells with SNAI1 overexpression were analyzed. Means and standard deviations of three independent experiments are presented. SNAI1, snail homolog 1.

Figure 4.

Expression of epithelial-mesenchymal transition target genes in SNAI1-transgenic cells. Representative results from reverse transcription-polymerase chain reaction experiments with specific primers for the indicated genes are presented. ACTB was used as a housekeeping control. Control vector-transfected cells and cells overexpressing SNAI1 were compared. One out of two experiments with identical results is presented. ACTB, β-actin; SNAI1, snail homolog 1; TWIST, twist homolog; LEF1, lymphoid enhancer-binding factor 1; NF-κB, nuclear factor κ-light-chain-enhancer of activated B cells.

Figure 5.

Knockdown of SNAI1 in glioblastoma cells and re-expression of CDH1. (A) The expression of SNAI1 in the indicated cell lines was assessed by reverse transcription-PCR. Representative results from control vector-transfected cells and cell lines with knockdown of SNAI1 (shSNAI1) are presented. PCR with primers with specificity for enhanced green fluorescent protein were used to prove that transduction of the different cell lines had occurred. ACTB was used as a housekeeping control. (B) The proliferation of the indicated cell lines was assessed by MTT assay. Control vector-transfected and SNAI1 knockdown cells were analyzed. Means and standard deviations from triplicate experiments are presented. *P<0.05. SNAI1, snail homolog 1; PCR, polymerase chain reaction; ACTB, β-actin; CDH1, E-cadherin; ntc, no template control; sh, short hairpin.

Figure 6.

Effect of knockdown of SNAI1 on the migration and invasion of glioblastoma cells. (A) The migration of the indicated cell lines was assessed. Control vector-transfected and SNAI1 knockdown cells were analyzed. Means and standard deviations from triplicates are presented. (B) The invasion capacity of the indicated cell lines was assessed by Matrigel assay. Results from control vector-transfected cells and the cell lines subsequent to knockdown of SNAI1 of three independent experiments are presented as the mean + standard deviation. *P<0.05. SNAI1, snail homolog 1; sh, short hairpin.