The Role Of OPN In HNSCC And Its Mechanism Of Mediating Proliferation And Invasion

I.Objective Head Neck Cancer(HNC)is a common malignant tumor,including epithelial malignant tumors originating from the sinus,nasal cavity,mouth,pharynx,and throat,accounting for 6% of malignant tumors.The pathological type is almost squamous cell carcinoma.Approximately 650,000 new cases of Head and Neck Squamous Cell Carcinoma(HNSCC)are reported worldwide each year,of which 35,000 are fatal.Currently,treatment of patients with HNSCC is based on tumor staging and potential location,while treatment based on potential biology is rare,although advances in technology and therapy have improved the quality of life of patients with HNSCC,but due to local recurrence and distantness after surgery Metastasis,the overall prognosis is still very poor.Therefore,it is better to understand the mechanism of action of HNSCC at the molecular level to improve,prevent and treat,and to provide personalized treatment for patients.Osteopontin(OPN)is an N-phosphorylated glycoprotein containing approximately three hundred amino acid residues.In recent years,the study of OPN in tumors has received more and more attention.A large number of experiments have shown that the high expression of OPN is associated with advanced metastasis and poor prognosis.OPN activates a variety of oncogenes and regulates angiogenic molecules to promote tumor growth,metastasis,and angiogenesis.Although the molecular mechanism of tumor promotion in OPN has been studied in other cancers,the expression and specific mechanism of action in HNSCC are still unclear.This paper mainly discusses the role and regulation mechanism of OPN in the occurrence and development of HNSCC.II.Methods 1.Determination of OPN expression in mammalian head and neck squamous cell carcinoma.(1)Clinical specimens of head and neck squamous cell carcinoma and adjacent tissues were collected as a control group;(2)The expression of OPN in the sample was detected by Real-time PCR and Western blot;(3)Immunohistochemistry was used to detect the expression and localization of OPN in squamous cell carcinoma of the head and neck.2.Effect of OPN on proliferation and invasion of head and neck squamous cell carcinoma.(1)Transfection of OPN-si RNA and Over-OPN to UM-SCC6(human oral squamous cell carcinoma cells)and CNE-2(human nasopharyngeal carcinoma cells),respectively,to interfere with and overexpress OPN;(2)The effects of OPN on cell proliferation were detected by CCK8 experiment,colony formation assay and Ki67 assay;(3)Western blot analysis was used to detect the expression of apoptosis-related factors Capase-3 and Bcl-2;(4)Transwell chamber assay was used to detect the effect of OPN on cell invasion;(5)Western blot analysis was used to detect the expression of invasion-related factors MMP2 and MMP9.3.The mechanism of action of OPN in regulating head and neck squamous cell carcinoma.(1)Transfection of OPN-si RNA and Over-OPN into UM-SCC6 cells and CNE-2 cells;(2)The optimal time for the addition of the optimal concentration of p38 MAPK inhibitor(SB032580);(3)The effect of OPN on the p38 MAPK signaling pathway was examined.III.Results 1.Expression of OPN in mammalian head and neck squamous cell carcinoma.(1)Real-time PCR and Westren blot showed that OPN was highly expressed in squamous cell carcinoma of the head and neck compared with adjacent tissues.(2)The results of immunohistochemistry showed that the number of positive cells of OPN in the experimental group was higher than that in the control group;the positive staining of OPN protein was mainly located in the cytoplasm,and there was a small amount of staining in the cell membrane and interstitial space.2.Effect of OPN on proliferation and invasion of head and neck squamous cell carcinoma.(1)OPN can promote cell proliferation.Transfection of OPN-si RNA and Over-OPN down-regulated and up-regulated OPN expression,respectively.CCK8,colony formation and Ki67 results showed that down-regulation of OPN can inhibit cell proliferation,and up-regulation of OPN can promote cell proliferation.The results of Western blot showed that the expression of Capase-3 and Bcl-2 was increased after down-regulation of OPN compared with the control group.(2)OPN can promote cell invasion.Transfection of OPN-si RNA and Over-OPN down-regulated and up-regulated OPN expression,respectively.Transwell chamber results showed that down-regulation of OPN can inhibit cell invasion,and up-regulation of OPN can promote cell invasion.The results of Western blot showed that the expression of MMP2 and MMP9 was decreased after down-regulation of OPN compared with the control group.The expression of MMP2 and MMP9 was increased after up-regulation of OPN compared with the control group.3.The mechanism of action of OPN in regulating head and neck squamous cell carcinoma.OPN is able to activate the p38 MAPK signaling pathway.The results of Western blot showed that the expression of p-p38 was decreased compared with the control group after transfection with OPN-si RNA.The expression of p-p38 was significantly decreased after adding SB032580 inhibitor compared with OPN-si RNA alone.After transfection with Over-OPN the expression of p-p38 was increased,and the expression of p-p38 was decreased after the addition of SB032580 inhibitor and the addition of Over-OPN alone.IV.Conclusions 1.OPN expression in human head and neck squamous cell carcinoma is higher than adjacent tissues.2.OPN can affect the proliferation and invasion of UM-SCC6 cells and CNE-2 cells.3.OPN may regulate the proliferation and invasion of UM-SCC6 cells and CNE-2 cells through the p38 MAPK signaling pathway.