Alpha-tubulin was used as the loading control. Knockdown efficiency of siMMP-13 MMPs can degrade all types of ECM proteins; however, no association has been reported between the role of MMP-13 in oral cancer metastasis. of MMP-13 may be utilized to impede the process of metastasis. depends substantially on MMP-13 expression. RESULTS MMP-13 expression in higher in OC3-I5 than in OC3 Metastasis is a phenomenon in invasive cancer cells, and the degradation of the ECM can facilitate the migration of cancer cells. Therefore, the degradation of collagenous ECM by MMPs is essential for the invasion of malignant cells and tumor-associated blood angiogenesis. Considering the role IWR-1-endo of the MMP superfamily in cell invasion, we compared the expression of MMP-13 between the oral cavity squamous cell carcinoma OC3 and the invasive ability-enhanced OC3-I5 cell lines. The primary immunoblotting results revealed a higher expression of MMP-13 in OC3-I5 than in OC3 (Figure ?(Figure1),1), thus supporting our speculation that OC3I5 cells pertain a higher matrix degradation ability. Open in a separate window Figure 1 Expression of MMP-13 in oral cancer OC3 IWR-1-endo and metastasis-enhanced OC3-I5 and the knockdown efficiency of siMMP-13(A) Thirty micrograms of protein samples were diluted in a Laemmli sample buffer and separated using 1D SDS-PAGE following standard procedures. Expression of the target protein MMP-13 was monitored by immunoblotting. (B) OC3 and OC3-I5 cells were transfected with 50 IWR-1-endo nM siMMP-13, and the knockdown efficiency of various siMMP-13 strains and combinations were examined by immunoblotting. None represented for the mock cells. The combination of Strains 1, 9, and 11 revealed the optimal knockdown efficiency; hence, this combination was selected for further investigation. The concentration of 50 nM was selected as the experimental condition. Alpha-tubulin was used as the loading control. Knockdown efficiency of siMMP-13 MMPs can degrade all types of ECM proteins; however, no association has been reported between the role of MMP-13 in oral cancer metastasis. Therefore, we examined the function of MMP-13 in OC3-I5 cell invasion. The OC3-I5 cells with an enhanced invasive ability and higher MMP3 expression were selected from the parental OC3 cells by using a Transwell? invasion assay kit, and thus we inferred that MMP-13 might be responsible at least in part for the higher invasion ability. To inquire about this possibility, we used siRNA knockdown as a tool for downregulating MMP-13, and examined the effect of MMP-13 knockdown on the invasion ability of OC3-I5. Three strains of synthetic siRNA against MMP-13 were obtained from Invitrogen. The sequences 5-CCG AGG AGA AAC AAT IWR-1-endo GAT CTT-3 (Strain 1), 5-GCT CCG AGA AAT GCA GTC TTT-3 (Strain 9), and 5-CTG TCA ATG AGA GCA TAA TTT-3 (Strain 11) were designed against MMP-13. Furthermore, the efficiency of MMP-13 down regulation by various siRNA strains combinations was examined by Western immunoblotting analysis. A combination of Strains 1, 9, and 11 and the working concentration of IL10A 50 nM were found to be effective in down regulating MMP23 and thus selected for further experimentation (Figure ?(Figure11). siMMP-13 downregulated the invasion ability of oral cancer cells in transwell invasion and migration assays The Transwell? invasion assay kit was employed to examine the invasion ability. The assay revealed a significant decrease in the IWR-1-endo invasion ability of both OC3 and OC3-I5 cells transfected with siMMP-13 compared with the scramble siRNA-transfected controls (mock). After siMMP-13 knockdown, the transwell invasion and migration abilities of the OC3 and OC3-I5 cells decreased by 40% and 60%, respectively. These results indicate that MMP-13 plays an important role in the migration and invasion abilities of those oral cancer cells (Figure ?(Figure22). Open in a separate window Number 2 Transwell invasion ability, transwell migration ability, and the number of cell attachments in oral malignancy cells transfected with or without siMMP-13(A) OC3 and OC3-I5 cells were transfected with 50 nM siMMP-13 or scramble siRNA (mock). An equal quantity of cells (50 000 cells/place).