Synthetic Circular RNA for microRNA-1269a Suppresses Tumor Progression in Oral Squamous Cell Carcinoma

Simple Summary Circular RNAs (circRNAs) are a new class of unique RNAs that have single-stranded, covalently closed, and continuous loop structures, which confer stability and resistance to endonuclease-mediated degradation that in turn affords substantially longer circulatory half-lives relative to linear RNAs. In this study, we established a novel synthetic circRNA that carries miR-1269a binding sequences and investigated its potential application as a therapeutic tool to slow cancer progression. Briefly, phospholipase C gamma 2 (PLCG2), which is related to oral squamous cell carcinoma (OSCC) clinical stage and overall survival, was affected by the circRNA-1269a/miR-1269a axis. Our confirmation that circRNA-1269a/miR-1269a/PLCG2 can inhibit OSCC proliferation and migration by promoting apoptosis in OSCC cells in vitro indicates that PLCG2 overexpression via the circRNA1269a/miR-1269a axis may be a valuable tool for controlling OSCC growth. Therefore, these data suggest that circRNA-1269a/miR-1269a could act as a potential therapeutic axis for OSCCs.Abstract microRNAs (miRs) function in cancer progression as post-transcriptional regulators. We previously reported that endogenous circular RNAs (circRNAs) function as efficient miR sponges and could act as novel gene regulators in oral squamous cell carcinoma (OSCC). In this study, we carried out cellular and luciferase reporter assays to examine competitive inhibition of miR-1269a, which is upregulated expression in several cancers, by circRNA-1269a, a synthetic circRNA that contains miR-1269a binding sequences. We also used data-independent acquisition (DIA) proteomics and in silico analyses to determine how circRNA-1269a treatment affects molecules downstream of miR-1269a. First, we confirmed the circularization of the linear miR-1269a binding site sequence using RT-PCR with divergent/convergent primers and direct sequencing of the head-to-tail circRNA junction point. In luciferase reporter and cellular functional assays, circRNA-1269a significantly reduced miR-1269a function, leading to a significant decrease in cell proliferation and migration. DIA proteomics and gene set enrichment analysis of OSCC cells treated with circRNA-1269a indicated high differential expression for 284 proteins that were mainly enriched in apoptosis pathways. In particular, phospholipase C gamma 2 (PLCG2), which is related to OSCC clinical stage and overall survival, was affected by the circRNA-1269a/miR-1269a axis. Taken together, synthetic circRNA-1269a inhibits tumor progression via miR-1269a and its downstream targets, indicating that artificial circRNAs could represent an effective OSCC therapeutic.