RNA-Seq revealing the tumor immunity regulation mechanism of circular RNA in human laryngeal squamous cell carcinomas
Authors: Cui Tianyu , Li Menghua , Lu Cheng , Xia Min , Ke Zhaoyang , Qiao Yuehua , Liu Liangfa , Shi Xi 
Published: May 2020

Abstract
Background

Motivation: Laryngeal squamous cell carcinoma (LSCC) is one of the relatively common malignant tumors occurring in the epithelial tissue of the larynx. Recently study showed that tumor immunity mechanisms have highly correlation with the development and progression of LSCC. With the development of research on circular RNA (circRNA) in recent years, a large number of abnormal expressions of circRNA in various tumors have been reported. There is a large number of evidences indicated that circRNA is widely involved in stage development of tumors and it also plays an important role as a biomarker in diagnosis and treatment. Therefore, the study of the pathways involved in the development of tumor by circRNA is a necessary process for humans to further understand cancer at the transcriptional level. The purpose of this study was to identify the circRNAs in tumor tissues of patients with laryngeal squamous cell carcinoma (LSCC) by NGS technique and to find the circRNAs that were significantly different from normal adjacent tissues. Finally, the mechanism of these differentially expressed tumor immunity circRNAs affecting LSCC development was further analyzed.
Methods

Raw data was mapped to Hg19 human genome and circRNA read count matrix was generated by featureCount software. In this study, the screening criteria for differential expression were p-value < 0.01 and | log2(FoldChange) | > 2.0. Filtered circRNAs were then applied for generating circRNA-miRNA-mRNA interaction network with known human micro RNA (miRNAs). After filtering unmeaning interactions, network-involved mRNAs were implement to KEGG analysis. Among those KEGG terms, immunity-related pathways were selected and their related circRNAs were chosen for further analysis. Second KEGG analysis then performed to these circRNAs for detecting their potential to be biomarkers. Finally a QRT-PCR experiment was used to verify the results.
Results

Among the 8 samples, 4 were LSCC tumor tissue samples and 4 were adjacent normal tissue samples. A total of 75,931 circRNAs were identified in total 8 samples. After filtering through the above screening criteria, we obtained 39 significantly differential expressed circRNAs from those identified candidates. Of the 39 circRNAs, 21 were detected to be significantly less expressed in LSCC tissues than in adjacent normal tissues, and other 18 were significantly higher. Through the interaction analysis of circRNA-miRNA-mRNA, we found that these differential expressed circRNAs were related to tumor immunity. After screening, four circRNAs with the strongest correlation with tumor immunity were obtained. We found that they play an important role in the membrane receptor tyrosine protein kinase signaling pathway pathway (Ras-Raf-MAPK pathway) and p53 signaling pathway.
Conclusion

According to the results, the method of detecting expression of four selected circRNAs has a bright future to be a kind of new LSCC diagnose approach.