Screening And Validation Of Core CircRNAs In Laryngeal Squamous Cell Carcinoma

Objective:High-throughput sequencing was performed on laryngeal squamous cell carcinoma cell lines and normal mucosal epithelial cell lines to screen differentially expressed circ RNAs in laryngeal squamous cell carcinoma cell lines.Combined with literature,target circ RNAs was screened out.RT-q PCR was used to verify the expression trend of target circ RNAs in laryngeal squamous cell carcinoma tissues and adjacent normal tissues,and its clinical significance in laryngeal squamous cell carcinoma was further analyzed to screen circ RNAs related to the occurrence and development of laryngeal squamous cell carcinoma.To search for potential biomarkers associated with laryngeal squamous cell carcinoma.Methods:Laryngeal squamous cell carcinoma cell lines(Hep-2,TU686,TU212,M2e)and normal mucosal epithelial cell lines(Hacat)were sequenced by high-throughput sequencing.The circ RNAs expression profile of laryngeal squamous cell carcinoma cell lines was constructed by comparing the existing circ RNAs databases.The differential circ RNAs were screened by DEGseq algorithm,and the genes from the differential circ RNAs were annotated in GO and KEGG databases,and the GO function analysis,KEGG pathway enrichment and Passway function enrichment analysis were performed.Combined with the literature,four closely related circ RNAs were selected as target circ RNAs.Laryngeal carcinoma tissue samples and adjacent normal tissues samples were collected from patients diagnosed with laryngeal squamous cell carcinoma during November 2020 to November 2021 who underwent laryngeal carcinoma resection in the Department of Otorhinolaryngology-Head and Neck Surgery of the Affiliated Hospital of Guizhou Medical University.RT-q PCR was used to preliminarily verify the expression trend of target circ RNAs in 5laryngeal squamous cell carcinoma tissues and adjacent normal tissues.According to the results of RT-q PCR,the one circ RNA with the most significant differential expression trend was selected for RT-q PCR verification in 20 laryngeal squamous cell carcinoma tissues and adjacent normal tissues,and its clinical significance in laryngeal squamous cell carcinoma was further analyzed.Results:By high-throughput sequencing,4621 circ RNAs were up-regulated and 5477 were down-regulated in laryngeal squamous cell carcinoma cell lines compared with normal mucosal epithelial cell lines.Combined with the literature,four circ RNAs with significant expression differences were selected as the target circ RNAs for subsequent verification.They were hsa＿circ＿0007203(circ-DGL1),hsa＿circ＿0001869(circ-ZCCHC6),hsa＿circ＿0000471(circ-N4BP2L2),and hsa＿circ＿0007444(circRHOBTB3.RT-q PCR was used to preliminarily verify the expression trend of target circ RNAs in 5 laryngeal squamous cell carcinoma tissues and adjacent normal tissues.The results showed that hsa＿circ＿0007203(circ-DGL1)、hsa＿circ＿0001869(circ-ZCCHC6)and hsa＿circ＿0007444(circ-RHOBTB3)had obvious differential expression trend in laryngeal squamous cell carcinoma tissues and adjacent normal tissues.There was no significant difference in the expression of hsa＿circ＿0000471(circ-N4BP2L2),and the expression trend of hsa＿circ＿0007203(circ-DGL1)and hsa＿circ＿0001869(circ-ZCCHC6)was consistent with the sequencing results.However,the expression trend of hsa＿circ＿0007444(circRHOBTB3)was contrary to the results of sequencing.According to the preliminary validation results of RT-q PCR,hsa＿circ＿0007203(circ-DGL1)with the most significant expression trend was selected for RT-q PCR validation in 20 cases of laryngeal squamous cell carcinoma tissues and adjacent normal tissues,and statistical analysis was conducted.The results showed that hsa＿circ＿0007203(circ-DGL1)was homogeneity with high-throughput sequencing and down-regulated in laryngeal squamous cell carcinoma.A ROC curve was constructed to test the diagnostic sensitivity of hsa＿circ＿0007203(circ-DGL1).The cut-off value was 0.7586,the area under the curve was 0.9550,the sensitivity was 0.7189,and the specificity was0.7635.Further analysis of the relationship between hsa＿circ＿0007203(circ-DGL1)expression and clinical case parameters of laryngeal squamous cell carcinoma showed that the down-regulation of hsa＿circ＿0007203(circ-DGL1)expression was related to the differentiation and lymphatic metastasis of laryngeal squamous cell carcinoma.But there was no correlation with age,sex or T stage.Conclusions:Laryngeal squamous cell carcinoma cell lines and normal mucosal epithelial cell lines were sequenced by high-throughput sequencing technology.Compared with normal mucosal epithelial cell lines,laryngeal squamous cell carcinoma cell lines had a large number of different circ RNAs.Hsa＿circ＿0007203(circ-DGL1)is differentially expressed in laryngeal squamous cell carcinoma and para-carcinoma tissues,and down-regulated in laryngeal squamous cell carcinoma tissues.Moreover,it is related to the differentiation and lymphatic metastasis of laryngeal squamous cell carcinoma,and may be a potential tumor suppressor gene,and as a potential biomarker for early diagnosis of laryngeal squamous cell carcinoma.