Kasamatsu Atsushi, Nozaki Ryunosuke, Kawasaki Kohei, Saito Tomoaki, Minemura Chikashi, Seki Naohiko, Moss Joel, Uzawa Katsuhiro
Department of Dentistry and Oral-Maxillofacial Surgery, Chiba University Hospital, 1-8-1 Inohana, Chuo-ku, Chiba-shi 260-8677, Chiba, Japan.
Department of Oral Science, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi 260-8670, Chiba, Japan.
Cancers (Basel). 2024 Mar 21;16(6):1242. doi: 10.3390/cancers16061242.
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.
微小RNA（miR）作为转录后调节因子在癌症进展中发挥作用。我们之前报道过，内源性环状RNA（circRNA）作为有效的miR海绵发挥作用，并可作为口腔鳞状细胞癌（OSCC）中的新型基因调节因子。在本研究中，我们进行了细胞和荧光素酶报告基因检测，以研究包含miR - 1269a结合序列的合成circRNA - 1269a对在多种癌症中表达上调的miR - 1269a的竞争性抑制作用。我们还使用了数据非依赖采集（DIA）蛋白质组学和计算机分析来确定circRNA - 1269a处理如何影响miR - 1269a下游的分子。首先，我们使用发散/收敛引物的逆转录聚合酶链反应（RT - PCR）和头对头circRNA连接点的直接测序，证实了线性miR - 1269a结合位点序列的环化。在荧光素酶报告基因和细胞功能检测中，circRNA - 1269a显著降低了miR - 1269a的功能，导致细胞增殖和迁移显著减少。对用circRNA - 1269a处理的OSCC细胞进行的DIA蛋白质组学和基因集富集分析表明，284种蛋白质有高度差异表达，这些蛋白质主要富集在凋亡途径中。特别是，与OSCC临床分期和总生存期相关的磷脂酶Cγ2（PLCG2）受circRNA - 1269a/miR - 1269a轴的影响。综上所述，合成circRNA - 1269a通过miR - 1269a及其下游靶点抑制肿瘤进展，表明人工circRNA可能是一种有效的OSCC治疗方法。
