Silencing of CXCR4 inhibits the proliferation, adhesion, chemotaxis and invasion of salivary gland mucoepidermoid carcinoma Mc3 cells in vitro.

Mucoepidermoid carcinoma is the most common malignant tumor in salivary glands and high-grade mucoepidermoid carcinoma is often accompanied with poor prognosis. Many recent research works demonstrated that stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor-4 (CXCR4) interaction was critical for metastasis of various cancers. In this study, the immunoexpression of CXCR4 in human salivary gland mucoepidermoid carcinoma in different grades was detected by immunohistochemical analysis and the expression of CXCR4 and its ligand SDF-1 in mucoepidermoid carcinoma MEC-1 cell line and its highly metastatic clone Mc3 was examined by RT-PCR, flow cytometry and immunocytochemical analysis. It was found that CXCR4 was over-expressed in Mc3 cell line and SDF-1 was expressed in both cell lines at a nearly equal level. We further constructed CXCR4-shRNA expression vector to stably transfect Mc3 cells. We found that silencing of endogenous CXCR4 gene expression in Mc3 cells resulted in inhibition of the proliferation, adhesion, chemotaxis and invasion of Mc3 cells in vitro. This study implies that CXCR4 molecule is a potential factor controlling the proliferation and metastasis of Mc3 cells.

[Construction of eukaryotic expression vector of short hairpin RNA for transforming growth factor-beta1].

OBJECTIVE: To construct the plasmid containing short hairpin RNA (shRNA) of TGF-beta1 expression vector. METHODS: Short chain oligonucleotide was designed according to the TGF-beta1 mRNA sequence provided by Genebank, then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pWH1 vector. The recombinant TGF-beta1 shRNA expression vector was evaluated by using enzyme cutting. At last, the constructed TGF-beta1 expression vector was transfected into salivary gland mucoepidermoid carcinoma (Ms) cells by Lipofectomine TM 2000, and its effect on TGF-beta1 expression was observed by RT-PCR and immunohistochemistry. RESULTS: Successful construction was identified by enzyme cutting and the constructed plasmid was called pWH1-TGF-beta1. The shRNA and it inhibited the TGF-beta1 mRNA and protein expression effectively. CONCLUSION: The constructed TGF-beta1 shRNA expression vector can block the TGF-beta1 expression in salivary gland mucoepidermoid carcinoma cells.

[Screening and identification of metastasis-related gene expression in tongue carcinoma with cDNA microarray assay].

OBJECTIVE: To identify metastasis-associated genes in tongue carcinoma and to better understand the mechanism underlying tongue carcinoma metastasis. To compared mRNA expression profiles of two tongue carcinoma cell strains with high and low metastatic potentials using microarray technology. METHODS: Tca8113 and Tb cells were used as model systems to study the molecular mechanism of tumor metastasis. Two fluorescent cDNA probes labeled with Cy3 and Cy5 dyes were prepared from the mRNA samples of Tca8113 and Tb cells by reverse transcription method. The two color probes were then mixed and hybridized to the cDNA chip constructed by double dots of 1 152 human genes, and scanned at two wave lengths. Differential expression genes from the above two cell lines were analyzed using computer. Then six of the different expression genes were further validated by RT-PCR technique. RESULTS: In the 1 152 clones of known genes and expressed sequence tags that were analyzed, 37 showed significantly different (minimum 2 folds) expression levels in two cell lines. Among the 37 genes, 15 were up regulated (with ratio more than 2) and 22 down regulated (with ratio less than 1/2). The results of RT-PCR analysis were coincident with those of microarray assay. CONCLUSION: Some of these genes are known to be involved in human tumor antigen, immune surveillance, adhesion, cell signaling pathway and growth control. It is suggested that the microarray in combination with a relevant analysis facilitates rapid and simultaneous identification of multiple genes of interests and in this study it provided a profound clue to screen candidate targets for early diagnosis and intervention.

[Effects of 17 beta-estradiol on the adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells].

OBJECTIVE: To study the effects of 17 beta-estradiol on the adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells. METHODS: The effects of 17 beta-estradiol on adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells were investigated with cell attachment assay on fibronectin (FN), wound assay, chemotaxis assay, and gelatin-incorporated SDS-PAGE electrophoresis. The expression of estrogen receptor in Mc3 cells was determined by immunohistochemistry assay. RESULTS: Attachment rates of Mc3 cells treated with E2 at 10(-9), 10(-8), 10(-7), 10(-6) mol/L were 38.3%, 50.4%, 69.2% and 91.1% respectively, and the rate in control was 25.0%. When exposed to 17 beta-estradiol at 10(-9), 10(-8), 10(-7) and 10(-6) mol/L for 48 h, motility of Mc3 cells on FN increased by 16.9%, 40.9%, 36.4% and 38.8% respectively. When at 10(-6) mol/l, 17 beta-estradiol increased chemotaxis potential of Mc3 cells to FN by 60.3%. The activity of 68 000 matrix metalloproteinase (MMP-2) of Mc3 cells was enhanced at different levels by 10(-9), 10(-8), 10(-7), 10(-6) mol/L of 17 beta-estradiol, and estrogen receptor was also detected in nucleus of Mc3 cells by immunohistochemistry assay. CONCLUSIONS: 17 beta-estradiol at physiological concentration may enhance the adhesion, invasion and motility potential of salivary mucoepidermoid carcinoma Mc3 cells.