The Mechanism Of CircSOX9 Promoting The Proliferation And Invasion Of Nasopharyngeal Carcinoma By Regulating MiR-485-3p/SOX9 Sigal Axis

Introduction:Nasopharyngeal carcinoma is one of the multiple tumors in China and the most common head and neck malignancy.Although the diagnosis and treatment have been greatly improved,due to the concealment of early symptoms,most patients have developed to the middle and late stage at the time of diagnosis,and their 5-year survival rate is still unsatisfactory.Therefore,it is necessary to explore the potential molecular mechanism and find accurate biomarkers in order to better diagnose and treat patients with nasopharyngeal carcinoma.Circular RNA is a kind of Non-coding RNA,which has been identified as an important gene expression regulator and participates in the occurrence and development of tumor diseases.More and more evidence showed that some circular RNAs were abnormally expressed in nasopharyngeal carcinoma,and these circular RNAs may play a role in the occurrence and development of nasopharyngeal carcinoma.However,the mechanism of circular RNA in nasopharyngeal carcinoma is rarely reported.The purpose of this study was to explore the role and molecular mechanism of circSOX9 in the biological process of nasopharyngeal carcinoma.Method:1.The research object circSOX9 was determined by searching the database,and the tumor samples and adjacent normal tissues of clinical patients with nasopharyngeal carcinoma were collected.The expression level of circSOX9 in nasopharyngeal carcinoma and adjacent normal tissues was detected by qRT-PCR to further analyze the relationship between the expression level of circSOX9 and the clinical pathology and prognosis of nasopharyngeal carcinoma.The molecular characteristics of circSOX9 were verified by RNase R digestion test,and the expression and localization of circSOX9 were detected by nucleocytoplasmic separation test and RNA fluorescence in situ hybridization.2.Establishment a stable nasopharyngeal carcinoma cell line transfected with knockdown circSOX9.Cell Counting Kit-8(CCK-8),plate clone formation assay and traswell assay were used to detect the changes of proliferation,invasion and migration ability of nasopharyngeal carcinoma cells after knockdown of circSOX9.qRT-PCR and Western blot were used to further detect the change of epithelial mesenchymal transition(EMT)ability of nasopharyngeal carcinoma cells after knockdown of circSOX9.The effect of knockdown of circSOX9 on the proliferation of nasopharyngeal carcinoma cells in vivo was detected by subcutaneous tumorigenesis experiment in nude mice.3.Analyze the target gene miR-485-3p downstream of circSOX9 by bioinformatics prediction.qRT-PCR was used to detect the correlation between the expression of miR-485-3p and circSOX9 in nasopharyngeal carcinoma,and the targeted interaction was verified by Dual-Luciferase Reporter Assay.RNA binding protein immunoprecipitation(RIP)test was used to detect the binding of circSOX9 and miR-485-3p to Ago2.The expression of SOX9 in nasopharyngeal carcinoma was analyzed by bioinformatics.The correlation between the expression of SOX9 and miR-485-3p in nasopharyngeal carcinoma was detected by qRTPCR.The expression of SOX9 in nasopharyngeal carcinoma cells after overexpression or knockdown of miR-485-3p was detected by qRT-PCR and Western blot.Dual-Luciferase Reporter Assay verified its targeting interaction.The ability of circSOX9 to regulate the proliferation,invasion and metastasis of nasopharyngeal carcinoma cells through miR-485-3p / SOX9 axis pathway was tested by response experiment.Result:1.Bioinformatics analysis showed that circSOX9 was significantly up-regulated in nasopharyngeal carcinoma.At the same time,it was confirmed in collected clinical nasopharyngeal carcinoma tissues and adjacent normal tissues.Its expression level was positively correlated with its malignant progression.RNase R digestion test showed the circular structure of circSOX9,and RNA FISH showed that circSOX9 was mainly expressed in the cytoplasm.2.Knocking down the expression of circSOX9 can inhibit the proliferation,invasion,migration and the ability of EMT of nasopharyngeal carcinoma cells.Knocking down the expression of circSOX9 can also inhibit the tumorigenicity of nasopharyngeal carcinoma cells in nude mice.3.MiR-485-3p is the downstream target gene of circSOX9,the expression of circSOX9 and miR-485-3p in nasopharyngeal carcinoma was negatively correlated,and the DualLuciferase Reporter Assay verified their targeting interaction.RIP test showed that circSOX9 and miR-485-3p combined with Ago2 and participated in the ceRNA mechanism.The expression of miR-485-3p and SOX9 was negatively correlated in nasopharyngeal carcinoma.Dual-Luciferase Reporter Assay verified the targeted interaction between mir-485-3p and SOX9.qRT-PCR and Western blot showed that miR-485-3p could target and regulate the expression of SOX9 in nasopharyngeal carcinoma cells.Further,the analysis of response experiment showed that circSOX9 could promote the up regulation of SOX9 expression through spongy miR-485-3p,and then promote the proliferation and invasion of nasopharyngeal carcinoma cells.Conclusion:CircSOX9 promotes the proliferation,invasion and migration of nasopharyngeal carcinoma through the miR-485-3p/SOX9 signaling axis,indicating that circSOX9 can be used as a potential therapeutic target and predictive marker for nasopharyngeal carcinoma.