The Effect Of Hsa＿circ＿0036988 On The Migration And Invasion Of Oral Squamous Cell Carcinoma

Background:Oral squamous cell carcinoma（OSCC）is one of the most common malignancy in head and neck region.Despite advances in oncology research and surgical treatments,the overall 5-year survival rate was about 60%,with a poor prognosis.Its pathogenesis about OSCC is still unclear.With the rapid development of sequencing technology,circ RNAs were identified as a new type of functional noncoding RNAs（nc RNAs）,characterized by cell-type specific and stability.Currently,increasing evidence have indicated that circ RNAs may be new biomarkers and potential therapeutic targets for the diagnosis of diseases.Purpose:In this study,we aimed to investigate the role of hsa＿circ＿0036988 in the development of OSCC at the cellular level,providing a new theoretical basis for the diagnosis and treatment of OSCC.Methods:Eight pairs of OSCC tissues and adjacent normal tissues of OSCC patients were selected to screen for differentially expressed circ RNAs by high-throughput sequencing.Hsa＿circ＿0036988 expression level in 42 OSCC tissue samples was detected by qRT-PCR,as well as in OSCC cell lines（SCC15,SCC25,CAL27）and Human oral epithelial keratinocyte（HOK）cells.The overexpressed plasmids and small interfering RNA（si RNA）of hsa＿circ＿0036988 were constructed,and then transferred them into the OSCC cell line,to highly expressed or knocked down the level of hsa＿circ＿0036988.Effects of overexpression or knockdown of hsa＿circ＿0036988 on proliferation,migration and invasion of OSCC cell lines were detected by CCK-8 assay,Ed U proliferation assay and Transwell assay.Then,Western blot assay was used to detect changes in proliferation,invasion and epithelial-mesenchymal transition（EMT）-related protein levels.Circ Interactome（http://circinteractome.nia.nih.gov/）was used to predict the mi RNA that may potentially combine with hsa＿circ＿0036988.The expression level of corresponding mi RNA in OSCC tissues was detected by qRT-PCR.The changes of mi RNA expression were observed after overexpression or knockdown of hsa＿circ＿0036988 in cytological experiments.Results:High-throughput sequencing revealed that hsa＿circ＿0036988 expression level was significantly decreased in OSCC samples.qRT-PCR result shows that the expression of hsa＿circ＿0036988 was significantly down-regulated in 42 pairs of cancer tissues compared to adjacent normal tissues（P<0.001）.Compared with HOK cells,the expression level of hsa＿circ＿0036988 in SCC15,SCC25 and CAL27 cell lines was also significantly decreased（P<0.001）.The clinicopathological data analysis demonstrated that the expression of hsa＿circ＿00036988 in OSCC tissues was negatively correlated with lymph nodes metastasis.The qRT-PCR results showed that after transfected with overexpressed plasmid,the expression of hsa＿circ＿0036988 in SCC15 was increased by about 80 times.After si RNA（small interfering RNA）transfection,the expression of hsa＿circ＿0036988 in CAL27 was decreased by about75%(P_SCC15<0.001,P_CAL27<0.001).CCK-8 proliferation assay showed that in the SCC15 cell line which transfected with overexpressed plasmid,the proliferation rate of circ＿0036988 group was lower than that of the Control group.In the CAL27 cell line transfected with si RNA,the proliferation rate of si-circ＿0036988 group was higher than that of si-NC group,the difference was statistically significant(P_SCC15<0.05,P_CAL27<0.05).Similarly,the Ed U proliferation assay results shows that the proliferation rate of circ＿0036988 group was lower than that of Control group,the proliferation rate of si-circ＿0036988 group was higher than that of si-NC group,and the difference was statistically significant(P_SCC15<0.05,P_CAL27<0.05).Transwell migration and invasion assay results showed that the amount of migrated cells in circ＿0036988 group was less than that in the Control group.And migrated cells in si-circ＿0036988 group was higher than that in si-NC group(P_SCC15<0.05,P_CAL27<0.05).Western blot showed that circ＿0036988 group compared with Control group,and si-circ＿0036988 group compared with si-NC group,showing significant changes in proliferation,invasion and EMT-related protein expression levels.The predicted results showed that hsa＿circ＿0036988 might potentially bind to hsa-mi R-1200,hsa-mi R-665 and hsa-mi R-492.qRT-PCR results showed that hsa-mi R-665 was significantly up-regulated in OSCC tissues.After overexpression of hsa＿circ＿0036988 in SCC15,the expression level of hsa-mi R-665 was significantly decreased,while that of hsa-mi R-665 was increased after knockdown of hsa＿circ＿0036988 in CAL27.Conclusion:Hsa＿circ＿0036988 was found to be a downregulated circ RNA in OSCC,and its expression level is closely related to lymph node metastasis of OSCC patients.Hsa＿circ＿0036988 may contribute to disease progression by regulating OSCC proliferation,migration,invasion and EMT process.It may play a significant role in the development of OSCC.