Cripto-1 Regulates The Radiosensitivity Of Oral Squamous Carcinoma Cells

Background: Oral squamous cell carcinoma(OSCC)is the most common type of tumor in the oral cavity,with a poor prognosis and a 5-year survival rate of only 50%to 55%.Because of its special growth location,patients mostly have different degrees of eating difficulties and psychological barriers,and their survival quality is poor.Radiation therapy is one of the important tools in the clinical treatment of oral squamous carcinoma,but the resistance of cancer cells to radiation often leads to failure of radiation therapy.Therefore,exploring biological targets that can effectively increase the sensitivity of radiotherapy for oral squamous cell carcinoma is a hot spot and direction of current research.As a member of the epidermal growth factor family,Cripto-1 expression largely influences tumorigenesis and progression.Purpose: This study was conducted to evaluate the key role of Cripto-1 in regulating PI3K/Akt/m TOR signaling pathway,and then to investigate the possible mechanism of its influence on the radiosensitivity of oral squamous cell carcinoma cells,with the aim of using Cripto-1 as a new target for altering radiosensitivity and providing a new idea and theoretical basis for improving radiation therapy in oral squamous cell carcinoma.Methods:1.Two oral squamous carcinoma cell lines,CAL-27 and SCC-4,were used as the subjects of this study,and oral squamous carcinoma cells(CAL-27 and SCC-4)were transfected with Cripto-1 overexpression plasmids and antibiotic screened using genomycin(G418),thereby constructing Cripto-1 stably overexpressed cell lines;2.X-ray irradiation treatment of the overexpressed cell lines followed by CCK-8assay for cell proliferation ability,clone formation assay for cell viability and flow cytometry for apoptosis;3.qRT-PCR assay was used to verify whether Cripto-1 was successfully transfected and to determine the transfection efficiency;then the expression level of Cripto-1 m RNA in the irradiated CAL-27/SCC-4 stable overexpression cells was verified;4.Protein immunoblotting assay(Western Blot)was performed to detect the expression of PI3 K,Akt,m TOR,PFKFB2,PKM1,PKM2,Bax and Bcl-2 proteins in the irradiated overexpression cells and evaluate the effect of Cripto-1 on PI3K/Akt/m TOR signaling pathway.Results:1.Cripto-1 stably overexpressed cell lines(CAL-27-OE,SCC-4-OE)were successfully constructed,and the proliferation ability of the two oral squamous carcinoma cells was significantly enhanced after irradiation,and the irradiated-treated cells showed strong anti-apoptotic ability;2.qRT-PCR assay revealed that the expression levels of Cripto-1 m RNA were significantly higher in CAL-27-OE and SCC-4-OE cells after irradiation treatment than in wild-type CAL-27 and SCC-4 cells after irradiation;3.Protein immunoblotting experiments revealed that overexpressed Cripo-1activated the PI3K/Akt/m TOR signaling pathway,and the activation of PI3 K enhanced the phosphorylation levels of Akt and m TOR,and the elevated oxidative phosphorylation level of Akt increased the energy production capacity of tumor cells,which in turn stimulated the expression levels of the rate limiting enzymes of the aerobic glycolytic pathway,PKM1,PKM2 and The increased expression level of PFKFB2 promoted the aerobic glycolytic process in tumor cells,which resulted in more energy production.Overexpression of Cripto-1 caused changes in the expression levels of apoptosis-related genes Bax and Bcl-2,and the ratio of Bcl-2/Bax was upregulated,which inhibited apoptosis.Conclusion:In this study,we found that overexpression of Cripto-1 increased the proliferation ability of oral squamous carcinoma cells by constructing CAL-27-OE,SCC-4-OE cell lines,while Cripto-1 could affect the aerobic glycolytic process of cells through regulating PI3K/Akt/m TOR signaling pathway,which in turn decreased the radiosensitivity of CAL-27,SCC-4 cells.