Suppression of Survivin Expression by Short Hairpin RNA Induces Apoptosis in Human Laryngeal Carcinoma Cells

Abstract

Purpose: The aim of this study was to evaluate the suppression effect of survivin shRNA on the expression of the survivin gene in the human laryngeal cancer cell line Hep-2. Procedures: 60 cases of laryngeal squamous-cell carcinoma (LSCC) and 10 cases of normal laryngeal mucosa were examined using immunohistochemistry to determine whether the expression of survivin correlated with tumorigenesis. Three plasmid vectors of short hairpin RNA (shRNA) specific for survivin were designed and generated. Western blot and real-time PCR analysis of survivin expression in Hep-2 cells was performed 48 h after transfection. The growth curve was used to determine the cell proliferation. Propidium iodide (PI) single staining was applied to detect the cell cycle. The apoptosis of the cells was analyzed by flow cytometry with the FITC-annexin-V/PI double staining and PI single staining. Results: 68.33% (41 out of 60) of tumors were positive for survivin expression and significantly associated with lymph node metastasis and advanced stage. In contrast, no expression of survivin in normal mucosa was detected. Transfection of Hep-2 cells with survivin shRNA significantly inhibited survivin expression at both the mRNA and the protein level in Hep-2 cells. Downregulation of survivin resulted in increasing the apoptosis index, but the results showed no obvious influence on cell cycle. Conclusions: This study demonstrates that survivin shRNA effectively inhibits survivin gene expression in Hep-2 cells leading to growth suppression and apoptotic induction in Hep-2 cells.