| Objective: Oral squamous cell carcinoma(OSCC)is the most common oral malignant tumor,accounting for 90%,and the 5-year survival rate of patients has remained at about 50% in recent decades.Cancer stem cells(CSCs)are a subset of tumor cells with self-renew,multi-lineage differentiation,and regenerate homogeneous tumors,so they are called "roots of cancer".As we all know,CSCs not only have the ability of traditional stem cells to divide and proliferate,thereby generating tumor tissue,but also play an important role in tumor invasion,metastasis,recurrence and drug resistance.The regulation of tumor microenvironment on tumor cells,especially tumor stem cells,plays a significant role in the occurrence,development and treatment of tumors.Therefore,it is important to understand the regulation and specific mechanism of tumor microenvironment on tumor cells and tumor stem cells for improving the survival rate and the prognosis of tumor patients.The purpose of this study was to investigate the effects of interleukin-17(IL-17)in the tumor microenvironment on the stemness and biological behavior of oral squamous cell carcinoma tumor cells and tumor stem cells,and investigate whether Epithelial-Mesenchymal Transition(EMT)is the main mechanism of IL-17 regulating oral squamous cell carcinoma.It is expected that this study will provide new strategies for the treatment of oral squamous cell carcinoma,thereby improving the efficacy and quality of life.Methods: 1.Cell classification: Oral squamous cell carcinoma cells SCC9 were divided into two types according to the expression of stem marker CD133.The tumor cells expressing CD133 were cancer stem-like cells(CSLCs),and other tumor cells that do not express CD133 were cancer non-stem-like cells(Non-CSLCs).2.In view of the fact that the number of Non-CSLCs in the solid tumor tissue is greater than 95%,CD133-SCC9 cells were treated with IL-17 at different concentrations,and the concentration gradients were set to 25ng/ml,50ng/ml,75ng/ml,100ng/ml,150ng/ml and 200ng/ml.CCK8 proliferation experiments were used to measure the proliferation of cells in each group after 0h,24 h,48h and 72 h,and selecting the IL-17 concentration with the strongest cell proliferation ability as optimal concentration to construct a tumor microenvironment that stably expresses IL-17.3.Cell grouping: CD133+SCC9 and CD133-SCC9 cells were set in the experimental group and the control group,respectively,and the experimental group was treated with the same amount of optimal concentration of IL-17 for the same time(133+/17+,133-/17+),the control group added the same amount of cell culture medium(133+/17-,133-/17-).4.Stemness detection: The expression of stem cell marker protein CD133 of CD133+SCC9 and CD133-SCC9 cells was detected by immunofluorescence experiments and Western-blot technology.5.Detection of migration and invasion ability: the scratch migration test and transwell invasion test were used to detect the migration and invasion ability of the four groups cells.6.Detection of EMT-related markers: western-blot technology was used to detect the changes of E-cadherin,N-cadherin and Vimentin protein expression levels in the four groups cells.Results: 1.The results of the CCK8 experiment suggest that certain concentrations of IL-17(25ng/ml,50ng/ml,75ng/ml,100ng/ml,150ng/ml)can promote the proliferation of CD133-SCC9,and the ability to promote proliferation is the strongest at 75ng/ml,and the high concentration(200ng/ml)will inhibit the cell proliferation(all P <0.05).2.The results of IF experiments showed that under the same conditions,the number and expression intensity of CD133 positive cells in CD133+SCC9 cells were significantly higher than that of CD133-SCC9 cells,and the expression of CD133 increased after IL-17 intervention in the two groups of SCC9 cells,and the increased trend is more obvious in CD133+SCC9 cells(P <0.05).3.The results of WB experiments showed that under the same conditions,the expression of CD133 protein in CSLCs is significantly more than that of Non-CSLCs.Compared with the control group,the expression of stem cell marker protein CD133 in CD133+SCC9,CD133-SCC9 cell was up-regulated,and the increased trend of CD133+SCC9 is more obvious(P <0.05).4.The scratch test results suggest that under the same conditions,CD133+SCC9 cells have more migration distances than CD133-SCC9 cells.Compared with the respective control groups,the migration distance of CD133+SCC9 and CD133-SCC9 cells in the experimental group was longer and their migration capacity was enhanced,and this enhanced trend is more obvious in CD133+ SCC9 cells(P <0.05).5.Transwell experiment results suggest that under the same conditions,CD133+SCC9 cells penetrated Matrigel more than CD133-SCC9 after 48 h.Compared with the respective control groups,a larger number of the CD133+SCC9 and CD133-SCC9 cells in the experimental group passed through the Matrigel,and the increased trend was more prominent in CD133+SCC9 cells(P <0.05).6.WB results suggest that under the same conditions,CD133+SCC9 cells had lower epithelial marker protein expression and higher interstitial marker protein than CD133-SCC9.Compared with the respective control groups,the E-cadherin protein expression was down-regulated in CD133+SCC9 and CD133-SCC9 cells,while N-cadherin and Vimentin protein expression were up-regulated,and the changed trend of the three proteins was more obvious in CD133+SCC9 cells(P <0.05).Conclusion: Under the action of IL-17,EMT occurred in CD133-SCC9 and CD133+SCC9 cells of oral squamous cell carcinoma,which caused the stemness,migration ability and invasion ability of the two cells to be enhanced,and this enhancement trend was more prominent in CD133+SCC9 cells.It is manifesting that IL-17 may regulate the stemness of OSCC through EMT,which in turn affects its biological behavior in vitro. |
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