Development and Validation of a Comprehensive Analysis of the Competing Endogenous CircRNA/miRNA/mRNA Network for Identification of Immune-Related Therapeutic Targets in Esophageal Squamous Cell Carcinoma

Esophageal squamous cell carcinoma (ESCC) immunotherapy exhibits limited efficacy, prompting the investigation of its molecular mechanisms. Circular RNAs (circRNAs) are believed to play a significant role in the development of the tumor microenvironment (TME) in ESCC, but their role in ceRNA regulation remains indeterminate. In this study, we identified the expression of mRNA, circRNA in four tumor samples and their pair normal samples noting their paired samples by high-throughput sequencing to validate the correlation analysis. Overall design: Eight samples (including four tumor samples and paracancerous samples) were used to detect the differential gene expression. Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer's protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) = 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded Total RNA with Ribo-Zero Gold according to the manufacturer's instructions. Then these libraries were sequenced on the Illumina sequencing platform (HiSeqTM 2500 or other platform) and 150 bp/125bp paired-end reads were generated.