Study Of Differential Expression Profile Of CircularRNA In Tongue Squamous Cell Carcinoma

Tongue squamous cell carcinoma?TSCC?is one of the most common malignant tumors in the oral and maxillofacial head and neck cancer[1].The invasion of the cancer is strong,and it grows fast and easily metastases to the neck lymph nodes.Traditional methods of treatment for tongue squamous cell carcinoma include surgical treatment,radiotherapy and chemotherapy.But the prognosis of patients,especially in advanced patients,is poor.The death rate and recurrence rate are high[2]Therefore,it is of great significance to strengthen the study on the mechanism of tongue squamous cell carcinoma,to explore new clinical diagnostic markers for early detection and early treatment.Enhancing the research on the mechanism of invasion and metastasis of tongue squamous cell carcinoma and seeking new therapeutic targets are of scientific significance for improving the cure rate and reducing the recurrence rate of tongue squamous cell carcinoma.Previous studies have shown that the formation of malignant tumors is often not caused by genetic mutations themselves,but closely related to the process of regulating gene expression levels[3-5].The study of the regulation mechanism of genes in tongue squamous cell carcinoma is of great significance to elucidate the mechanism of tongue squamous cell carcinoma.CircRNA is a kind of newly recognized special RNA,most of which are formed by more than one exons through reverse splicing.They are abundant in eukaryotic cells,with tissue,time and disease specificity[6].CircRNA can act as a sponge for miRNA,and can inhibit its activity by binding to miRNA,thus regulating the target of miRNA[7].In addition,circRNA can regulate gene expression by regulating gene transcription,regulating RNA binding protein and participating in protein translation[8-10].Studies have shown that circRNA plays an important role in the development of a variety of cancers,such as gastric cancer,glioma,and lung cancer[11-13].Up to now,the study of circRNA is in its infancy,and its relationship with the tongue squamous cell carcinoma has not yet been reported.This is the first study to use circRNA microarray technology,screening the differentially expressed circRNA in tongue squamous cell carcinoma,to explore the circRNA which is related to tongue squamous cell carcinoma,and provide experimental basis for further study on the regulation mechanism of circRNA in tongue squamous cell carcinoma.Considering the structural stability of circRNA,it has great potential to be a clinical diagnostic marker or new therapeutic targets in the future.This study is divided into three parts:Part I Screening of differentially expressed circRNA between the tongue squamous cell carcinoma tissue and the para cancerous tissueObjectivesTo detect the different expression of circRNA in tongue squamous cell carcinoma tissue and para cancerous tissue using circRNA microarray,screening of differentially expressed circRNA between the tongue squamous cell carcinoma tissue and the para cancerous tissue,explore the relationship between circRNA and squamous cell carcinoma of tongue.Materials and methods1.Randomly selected 4 cases of tongue squamous cell carcinoma in Southern Medical University stomatological hospital during November 2016 to May 2017.Get the tongue squamous cell carcinoma tissues and the para cancerous tissues.2.Use the Invitrogen's Trizol kit to extract totol RNA of all samples.Determine the optical density of samples at 260nm and 280nm wavelengths by DU70 spectrophotometer to obtain A260/A280.Use the formaldehyde denatured gel to detect total RNA in all samples by electrophoresis.The synthesis of cDNA with fluorescent group by qualified RNA can be used for chip hybridization.3.Feature Extraction software is used for preprocessing and analysis of the chip after cross scanning.GeneSpring GX software is used to calculate the difference of gene expression and statistical significance p value.The standard of differential gene was the expression in the tumor tissue is 2 times more or less than in the para cancerous tissue,and the p value was less than 0.05.The differentially expressed circRNA between the tissues of tongue squamous cell carcinoma and the para cancerous tissues is screened according to this standard.Results1.Every total RNA which was extracted from the 8 samples was more than 1?g.The value of A260/A280 was between 1.8 and 2.1.The electrophoresis strips were clear.The ratio of strip 28S to strip 18S was greater than or close to 2:1.It is suggested that the quality of RNA in all samples conform to the requirements of the chip experiment.2.CircRNA microarray experiments were carried out in the 4 cases of cancer tissues and 4 para cancerous tissues.After pretreatment and normalization of the results,the differential expression circRNA is selected according to the standard.The results showed that 17171 differentially expressed circRNA were screened,accounting for 10.8%of the detectable circRNA of the chip.More than 2 times the high expression is 9982;more than 2 times the low expression is 7189.More than 5 times the high expression is 2032;more than 5 times the low expression is 849.More than 10 times the high expression is 392;more than 5 times the low expression is 179.3.Hsa_circ₀034026 is the highest circRNA,with a ratio of 137.22.Hsa_circ₀113500 is the lowest circRNA,with a ratio of 95.44.Part ? qPCR verification of the differentially expressed circRNA between the tongue squamous cell carcinoma tissue and the para cancerous tissueObjectivesCapitalBio Technology Human CircRNA Array v2 is a kind of high throughput circRNA microarray chip.Using this chip,we efficiently screened the differentially expressed circRNA between the tongue squamous cell carcinoma tissue and the para cancerous tissue.In order to further verify the accuracy of the chip results,we randomly selected 4 circRNA from differentially expressed circRNA,and conducted qPCR experiments in 10 cases of the tongue squamous cell carcinoma tissue and the para cancerous tissue.Check whether the results of the two are consistent.Materials and methods1.The randomly selected 4 circRNA are hsa_circ₀000576,hsa_circ₀026371,hsa_circ₀ 111246 and hsa_circ₀114424.Design their primers.2.qPCR experiments were conducted in the 10 cases of the tongue squamous cell carcinoma tissues and the para cancerous tissues to detect the relative expression of the 4 circRNA.ResultsThe primer design of 4 circRNA was successfully performed.The results of qPCR experiment showed that hsa_circ₀000576 was highly expressed in the tongue squamous cell carcinoma tissues relative to the para cancerous tissues?p<0.05?,hsa_circ₀026371,hsa_circ₀111246,hsa_circ₀114424 were lowly expressed in the tongue squamous cell carcinoma tissues relative to the para cancerous tissues?p<0.05?.The results of qPCR experiment were in good agreement with the results of circRNA chip experiment.Part III Bioinformatics analysis of the differentially expressed circRNA between the tongue squamous cell carcinoma tissue and the para cancerous tissueObjectivesThe experimental data obtained through high throughput circRNA chip experiments were very large.To use these huge experimental data to explore the relationship between the differentially expressed circRNA and the tongue squamous cell carcinoma,we have to perform bioinformatics analysis.Bioinformatics analysis include pathway and function enrichment analysis and circRNA-miRNA interaction analysis.Through these analyses,the important role of circRNA in the occurrence and development of tongue squamous cell carcinoma can be explored.Materials and methods1.KEGG Orthology Based Annotation System software contains 1 functional database and 7 pathway databases.The results of circRNA chip experiment are analyzed with this software,the biological function and signal pathway process can be analyzed at the same time.2.Using miRanda software to predict the most likely combinated miRNA for differentially expressed circRNA and to build circRNA-miRNA network.Preliminarily explore the mechanism of circRNA in the development of tongue squamous cell carcinoma.Results1.GO enrichment analysis showed that the differentially expressed circRNA was mainly involved in cell adhesion,biological adhesion and signaling in the aspect of biological processes?BP?;cell periphery,plasma membrane and membrane in the aspect of cellular component?CC?;molecular transducer activity,receptor activity and signal transducer activity in the aspect of molecular function?MF?.2.KEGG_PATHWAY enrichment analysis showed that the differentially expressed circRNA was mainly related to ECM-receptor interaction,Focal adhesion,Amoebiasis,Fc gamma R-mediated phagocytosis and some other signal pathways.3.Building circRNA-miRNA network with the top 10 differentially expressed circRNA.Hsa_circ₀033967,hsa_circ₀099630 and hsa_circ₀000579 had a large number of miRNA binding sites.Conclutions1.CircRNA microarray is an efficient circRNA research technique.In this study,we first applied it to the study of tongue squamous cell carcinoma,and screened out the differentially expressed circRNA between the tongue squamous cell carcinoma tissue and the para cancerous tissue quickly and efficiently.It is suggested that differential circRNA can correctly distinguish between cancer tissue and para cancerous tissue by cluster analysis of the circRNA microarray experement results.2.In this study,the QPCR experiment was used to verify the results of the circRNA microarray experiment.The results of QPCR detection coincided with the results of the circRNA microarray experiment,which improved the reliability of the results of the circRNA microarray experiment.3.Based on bioinformatics study,we analyzed the biological functions and signaling pathways of the differentially expressed circRNA.Preliminary mechanism exploration has been carried out.Through circRNA-miRNA interaction analysis,it is suggested that some differentially expressed circRNA may regulate the occurrence and development of tongue squamous cell carcinoma through competitive binding of micro RNA.