Comprehensive Circular RNA Expression Profiles Of Oral Squamous Cell Carcinoma In Chinese Hamsters And The Function And Mechanism Of Hsa_circ₀127523

Objective:An animal model of oral buccal squamous cell carcinoma of Chinese hamster was established and the differential expression profile of circRNA of oral squamous cell carcinoma was established by the second-generation high-throughput sequencing technology.The expression level of the circRNAs with significant difference obtained by the screening was verified.Combined with the database to screen out the hsa_circ₀127523,which plays a key role in oral squamous cell carcinoma and study its function in human oral squamous cell lines.Methods:An animal model of Chinese hamster oral squamous cell carcinoma was successfully established by the DMBA smear method and the model was evaluated by HE pathological staining and ultrastructural observation of transmission electron microscope.The data of the high-throughput sequencing results were integrated and analyzed and the differentially expressed circRNAs were screened and quantitatively verified by qRT-PCR in tissue samples;the target relationship of circRNA-miRNA-mRNA was predicted by miRanda?v3.3a?software and quantitative determination of qRT-PCR was performed;UCSC,circBase and circBank databases were used to screen human differentially expressed hsa_circ₀127523.The effects of hsa_circ₀127523 on the proliferation,migration and invasion of CAL27 and Tca8113 oral squamous cell carcinoma cells were detected by CCK-8,transwell cell migration and invasion methods.Combined with online software CircInteratome,miRanda and Targetscan,the miRNAs and mRNAs targeted by hsa_circ₀127523 were predicted and their expression levels were detected at the gene level.Results:DMBA smear method established four dynamic stages of Chinese hamster oral squamous cell carcinoma animal models: normal group,simple hyperplasia group,abnormal hyperplasia group and squamous cell carcinoma group.Under the normal pathological structure of HE and transmission electron microscope observation,compared with normal,the pathological morphology and internal cell structure of the buccal pouch of the three groups,the simple hyperplasia group,the abnormal hyperplasia group and the squamous cell carcinoma group had different degrees of lesions.The high-throughput sequencing was carried out on 12 samples with 3 replicates in each of the four stages of tissue samples to establish a circRNA differential expression profile of Chinese hamster oral squamous cell carcinoma.The GO function and KEGG pathway analyze of the host genes derived from the differentially expressed circRNA were performed.The total 3485 circRNAs were detected,of which 229 were novel discovered circRNAs and 89 circRNAs were significantly different in statistical analysis.Eight circRNAs significantly different were verified in 12 pairs of tissues,which were consistent with the expression levels of sequencing.The changes of cgr_circ_Man2a1/cgr_miR-1260/Notch2 and cgr_circ_Pdcd4/ cgr-miR-15b-5p/Akt3 were predicted by biological software and the expression levels were verified,which were consistent with the competitive endogenous RNA?ceRNA?mechanism.In addition,combined with UCSC,circBase,circBank database and circRNA differential expression profiles,it was found that the expression level of hsa_circ₀127523 derived from MAN2A1 gene was significantly increased.QRT-PCR was used to detect the expression of hsa_circ₀127523 in oral squamous cell carcinoma cells.SiRNA was designed and transferred into CAL27 and Tca8113 cell lines.The expression level of hsa_circ₀127523 was knocked down.Compared with the transfection-free sequence si-NC group,the silencing efficiency of si-hsa_circ₀127523 in CAL27 cells was about 42.28%;the silencing efficiency in Tca8113 cells was about 38.12%.The results of CCK-8 proliferation activity experiments showed that after hsa_circ₀127523 was silenced,the cell viability of the si-hsa_circ₀127523 group was lower than that of the si-NC group and it was statistically significant.Transwell migration and invasion experiments showed that in the CAL27 and Tca8113 cell lines,the number of cells passing through the bottom of the chamber was significantly reduced in the si-hsa_circ₀127523 group compared to the si-NC group.After hsa_circ₀127523 was silenced,the expressions of the target miRNA?hsa-miR-515-5p?and mRNA?TRIP13?were predicted by the software and measured by qRT-PCR,which found that relative expression level of hsa-miR-515-5p was decreased and the expression level of TRIP13 was increased compared with the si-NC group.Conclusion:In this study,the Chinese hamster oral squamous cell carcinoma animal model and the differential expression profile of circRNA were successfully established,which verified that the expression of the differential circRNAs were consistent with the sequencing results;combined with related databases,the behavioral functions of hsa_circ₀127523 in human oral squamous carcinoma cell lines was explored,which provided a basis for further understanding of hsa_circ₀127523 as a biomarker for the diagnosis of oral squamous cell carcinoma.It's helpful to further understand the mechanism of hsa_circ₀127523 in OSCC.