CXCR4 Silencing Inhibits The Proliferation And Metastasis Of Human Salivary Mucoepidermoid Carcinoma Mc3 Cells

Mucoepidermoid carcinoma (MEC) is the most common malignacy in salivary glands and accounts for about 30% of malignant tumors in salivary glands in Chinese population. Although it sometimes shows a slow growth resembling a benign lesion, this neoplasm can be highly aggressive with a dismal prognosis. A 5-year disease-free survival rate between 0 and 43% has been demonstrated in the patients with high-grade tumors.Chemokines belong to the small molecule chemoattractive cytokine family that can induce cytoskeletal rearrangement, firm adhesion to endothelial cells and directional migration, through their interaction with G protein-coupled receptors. CXCR4 is a seven-transmembrane G protein-coupled receptor and it can usually bind more than one chemokine. Stromal cell derived factor-1 (SDF-1),anα-chemokine, binds to CXCR4 and CXCR4 is its one of the important receptor. Recent studies suggested that CXCR4/SDF-1 axis may play an important role in the metastasis of many tumors such as breast cancer, prostate cancer, malignant glioma, nasopharyngeal carcinoma, non-small cell lung cancer and pancreatic cancer.RNA interference (RNAi) is a cellular mechanism in which double-stranded RNA triggers the sequence-specific"silencing"or"knockdown"of gene expression. In mammalian cells this double-stranded RNA may be either a small hairpin RNA (shRNA) or a simple RNA duplex with two unpaired nucleotides on the 3'-ends (siRNA). The transfection of synthetic siRNAs causes only transient suppression of target genes, which is often limited to the cell lines that are easy to be transfected. However, compared with siRNA, shRNA is more efficient to achieve stable long-term RNAi effects. Recently, silencing of CXCR4 of breast cancer by transfection of synthetic siRNAs has been reported.Up to date, the role of SDF-1/CXCR4 interaction in the proliferationing and metastatic potential of salivary gland mucoepidermoid carcinoma remains unknown. We assume that CXCR4 may also be involved in the growth and metastasis of the neoplasm. Therefore, in this study, we examined the expression of CXCR4 and SDF-1αin mucoepidermoid carcinoma MEC-1 cell line and in a highly metastatic mucoepidermoid carcinoma cell line Mc3. Furthermore, we constructed CXCR4-shRNA expression vector and silenced CXCR4 in Mc3 cells to study the function of CXCR4 in salivary gland mucoepidermoid carcinoma in vitro and in vivo.The study includs five parts as follows:1. Expression of CXCR4 in salivary gland mucoepidermoid carcinoma MEC-1 and Mc3 cells.Expression of CXCR4 and SDF-1αwas examinated in Mc3 and MEC-1 cells by BioStarH-I gene chip and confirmed by RT-PCR, immunocy -tochemistry and flow cytometry analysis. Overexpression of CXCR4 was detected in Mc3 cells and SDF-1αexpressed in both cell lines at nearly equal levels.2. Construction of CXCR4-shRNA eukaryotic expression vector.The plasmid expressing shRNA homologous to CXCR4 mRNA was constructed with shRNA expressing vector pWH1. The combinant plasmid was transfected stably into Mc3 Cells using Lipofectamine 2000. Finally, the shRNA efficiency was detected by RT-PCR, immunocytochemistry and flow cytometry analysis. We found that CXCR4-shRNA could effectively block the expression of CXCR4 in Mc3 cells and had no influence on the expression of SDF-1α.3. CXCR4 inhibited shRNA on the proliferation of Mc3 CellsCell growth curve, colonic formation assay in soft agar and tumor growth assay in nude mice were used to evaluate the effects of CXCR4-shRNA on cell proliferation. The population doubling times (hr) of CXCR4-shRNA shRNA transfected cells, empty vector transfected cells and the control cells were 26.5,20.6 and 20.4, respectively. CXCR4-shRNA inhibited the clonogenecity of the cells by 33.9% in soft agar; and the CXCR4-shRNA shRNA transfected cells showed an accumulation in G1 phase and a decrease in S and G2 phase compared with Mc3 groups. In vivo, CXCR4-shRNA inhibited the growth of cell-transplantation-induced tumor by 17.1% in nude mice.4. CXCR4-shRNA inhibited the metastasis and invasion of Mc3 cells.CXCR4-shRNA inhibited cell-to-Matrigel adhesion by 21.4% in a 30 min adhesion assay, The chemotaxis and the invasion of CXCR4-shRNA transfected Mc3 cells decreased to 43.8% and 52.7% of the control cells respectively. The lung metastastasis was inhibited in nude mice by 67.8%.5. The effect of CXCR4-shRNA on the expression of VEGF in Mc3 CellsImmunocytochemistry assay showed that VEGF expression was decreased in CXCR4-shRNA transfected cells.Conclusion:1. CXCR4 is overexpressed in Mc3 cells.2. CXCR4 silencing may inhibit the cell proliferation, metastasis and invasion. And the results demonstrate the effectiveness of RNAi in suppressing CXCR4, implicating an important role of CXCR4 in Mc3 cells proliferation and metastasis.3. CXCR4 regulates the expression of VEGF in Mc3 Cells4.The results suggest a potential application of RNAi-mediated therapeutic strategy for salivary gland mucoepidmoid carcinoma.