The Effects Of Notch Signaling On EMT And Its Mechanism In Oral Squamous Cell Carcinoma Metastasis

Background and objectives:Oral squamous cell carcinoma(OSCC)is one of the most common malignant tumors in oral and maxillofacial region.In recent years,new medical technologies and treatment methods are emerging constantly,effectively raising the local control rate of tumors.However,the 5-year survival rate of oral cancer patients has not decreased and remains as high as 50-55%.The disease mortality is still high,the main reason is that oral squamous cell carcinoma with local invasion and distant metastasis.Therefore,the study of metastasis mechanism in patients with oral squamous cell carcinoma has become the most important research.To elucidate the specific mechanism of tumor metastasis and take effective measures to curb metastasis for the improvement of prognosis and treatment of patients with oral squamous cell carcinoma is of great significance.Epithelial to mesenchymal transition(EMT)is a complex biological process and plays an important role in embryogenesis,wound healing and malignant tumor metastasis.Notch signaling pathway is composed of a series of proteins that are highly conserved during evolution and involve in a variety of biological processes including the regulation of cell proliferation,differentiation and apoptosis.With the deepening of research on Notch signaling pathway,we find that this pathway is related to EMT,which not only involved in the development of biological individuals,but also participates in the occurrence and development of tumors.The activation of Notch signaling pathway can induce EMT,but the relationship between this signal transduction and EMT activity in oral squamous cell carcinoma remains unclear.Therefore,the aim of this study was to research the mechanism of Notch signaling pathway and EMT in OSCC metastasis using two oral squamous cell carcinoma cell lines,Tca8113 and CAL27.Methods:1)RNA interference was used to inhibit the expression of the Snail in Tca8113 and CAL27,the m RNA expression levels of Snail,E-cadherin and Vimentin in Tca8113 and CAL27 cells were detected by q RT-PCR assay.The protein expression levels of Snail,E-cadherin and Vimentin in two cell lines were detected by Western-blot.Transwell assay and scratch test were performed to detect the effect of Snail on cell migration in Tca8113 and CAL27 cells.It is a way to discuss the influence of Snail and EMT related genes in oral squamous cell carcinoma.2)DAPT was used to block the Notch signaling pathway.The effects of DAPT at different dose(1 ~ 40umol/L)on the growth and viability of Tca8113 and CAL27 cells were detected by MTT assay.According to the MTT results,the appropriate concentration and time were selected for subsequent experiments.Cells were treated with DAPT at a concentration of 1 umol/L and 5 umol/L for 24 h or 48 h,The expression of Hes1,Snail,Vimentin and E-cadherin were detected by q RT-PCR after DAPT blocked Notch signaling pathway.Western-blot was used to measure the expression of N1 ICD,Snail,E-cadherin and Vimentin.Transwell experiment and scratch test were used to detect the cell migration ability after DAPT blocked the Notch signaling pathway,so as to explore the effects of the Snail,Notch signaling pathway and EMT related genes on oral squamous cell carcinoma.Results:1)The results of q RT-PCR and Western-blot showed that si RNA could effectively inhibit the m RNA and protein expression of Snail in Tca8113 and CAL27 cells,while reducing the expression of Vimentin and increasing the expression of E-cadherin.Meanwhile,compared with the control group,Transwell assay and scratch assay results showed that silencing Snail gene also significantly inhibited the migration of Tca8113 and CAL27 OSCC cells.2)1umol/L,2umol/L,and 5umol/L concentrations of DAPT in Tca8113 and CAL27 cells had no significant effect on cell proliferation rate after 24 h or 48 h.The concentration of 10umol/L,20umol/L,and 40umol/L concentrations in two cells at 24 h,48h,72 h,96h and 120 h can significantly reduce the proliferation rate of cells,and showed a time-dependent and dose-dependent manner.The results of q RT-PCR and Western-blot showed that after blocking the Notch signaling pathway with 1 umol/L and 5 umol/L DAPT,which could down-regulate Snail and Vimentin and up-regulate the expression of E-cadherin m RNA and protein.At the same time,the results of Transwell experiments and scratch tests showed that the migration ability of Tca8113 and CAL27 cells was significantly decreased after DAPT blocked Notch signaling pathway.Conclusions:1)inhibit the expression of the Snail gene in oral squamous cell carcinoma cells,The ability of the cells to migrate decreased.2)Notch signaling promotes the metastasis of oral squamous cell carcinoma by regulating EMT.