Abstract
The aim of this study was to investigate the effect of a B-cell-specific MLV integration site-1 (Bmi-1) RNA interference (RNAi) expression vector on the proliferation and invasiveness of laryngeal carcinoma. We constructed a lentiviral vector expressing Bmi-1-specific short hairpin RNA (shRNA), and transfected it into HEp-2 cells. Bmi-1 gene expression was detected by real-time RT-PCR and western blot analysis. We used flow cytometry and TUNEL assay to analyze the apoptosis of transfected cells, and examined cellular growth in vitro by MTT assay. We established an animal model and evaluated the therapeutic effects of small interfering RNA (siRNA) against Bmi-1. siRNA against Bmi-1 significantly knocked down Bmi-1 expression in HEp-2 cells, induced cell cycle arrest at the G1 phase, inhibited cell proliferation and promoted cell apoptosis. Lentiviral Bmi-1-shRNA vector transfection also significantly reduced cell migration. The formation and growth rate of xenograft tumors in mice transfected with siRNA against Bmi-1 was significantly reduced. The loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, and the increased activity of caspase-3, -8 and -9 occurred concomitantly with the inhibition of Bmi-1. Our data indicate that siRNA against Bmi-1 significantly suppresses tumor growth and induces apoptosis in vitro and in vivo.
Highlights
Laryngeal carcinoma is a common head and neck malignancy with high incidence, accounting for approximatelyKey words: apoptosis, cancer gene therapy, small interfering RNA, B-cell-specific MLV integration site-12.4% of new malignancies worldwide each year [1,2]
Among the four candidate target sequences screened, the lentiviral vector containing the human B-cell-specific MLV integration site-1 (Bmi-1) short hairpin RNA (shRNA)-expressing cassette achieved the greatest efficacy in silencing Bmi-1 expression. This construct was denoted as Bmi-1-RNA interference (RNAi)-LV; the negative control containing pGCSIL/U6 mock vector only was denoted as NC-green fluorescent protein (GFP)-LV
After the Bmi-1-RNAi-LV construct was transfected into HEp-2 cells, Bmi-1 mRNA expression levels in the transfected cells were compared with those in the untransfected and control-transfected (NC-GFP-LV) HEp-2 cells by quantitative RT-PCR
Summary
2.4% of new malignancies worldwide each year [1,2]. Despite extensive application of different treatment modalities, invasion and metastasis remain the main causes of mortality for laryngeal squamous cell carcinoma (LSCC) patients. Current treatments, including surgical intervention, radiation therapy and chemotherapy, are moderately effective in the early-stage cases, but are less effective in more advanced cases [3]. The mechanisms behind the occurrence and development of LSCC remain unclear. A novel therapeutic strategy for the treatment of LSCC is urgently required. Research over the past years clearly implicates multiple genetic alterations in the development and progression of laryngeal carcinoma [4]. Alterations in the expression profiles of several genes have been reported, including those that have important functions in cell adhesion, signal transduction, cell differentiation, metastasis, DNA repair and glycosylation [5]
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