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.
microRNAs（miRs）作为转录后调控因子在癌症进展中发挥重要作用。我们先前研究发现内源性环状RNA（circRNAs）可作为高效的miR海绵，在口腔鳞状细胞癌（OSCC）中发挥新型基因调控功能。本研究通过细胞实验和荧光素酶报告基因检测，验证了含有miR-1269a结合序列的人工合成环状RNA circRNA-1269a对多种癌症中高表达的miR-1269a的竞争性抑制作用。同时采用数据非依赖性采集（DIA）蛋白质组学与生物信息学分析，探究circRNA-1269a处理对miR-1269a下游分子的调控机制。首先通过发散/收敛引物RT-PCR及头尾连接点直接测序，证实线性miR-1269a结合位点序列的成功环化。荧光素酶报告实验与细胞功能检测显示，circRNA-1269a能显著抑制miR-1269a功能，导致细胞增殖和迁移能力明显下降。对circRNA-1269a处理的OSCC细胞进行DIA蛋白质组学及基因集富集分析发现，284个差异表达蛋白主要富集于凋亡通路，其中与OSCC临床分期及总生存期相关的磷脂酶Cγ2（PLCG2）受到circRNA-1269a/miR-1269a轴调控。综上，人工合成circRNA-1269a通过靶向miR-1269a及其下游靶点抑制肿瘤进展，表明人工环状RNA有望成为OSCC的有效治疗策略。
肿瘤(癌症)患者之家