Naohiko Seki

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.

Analyses of our microRNA (miRNA) expression signature combined with The Cancer Genome Atlas (TCGA) data revealed that both strands of pre-miR-139 (miR-139-5p, the guide strand, and miR-139-3p, the passenger strand) are significantly downregulated in lung adenocarcinoma (LUAD) clinical specimens. Functional analyses of LUAD cells ectopically expressing miR-139-3p showed significant suppression of their aggressiveness (e.g., cancer cell proliferation, migration, and invasion). The involvement of the passenger strand, miR-139-3p, in LUAD pathogenesis, is an interesting finding contributing to the elucidation of unknown molecular networks in LUAD. Of 1108 genes identified as miR-139-3p targets in LUAD cells, 21 were significantly upregulated in LUAD tissues according to TCGA analysis, and their high expression negatively affected the prognosis of LUAD patients. We focused on thyroid hormone receptor interactor 13 (TRIP13) and investigated its cancer-promoting functions in LUAD cells. Luciferase assays showed that miR-139-3p directly regulated TRIP13. siRNA-mediated TRIP13 knockdown and TRIP13 inhibition by a specific inhibitor (DCZ0415) attenuated the malignant transformation of LUAD cells. Interestingly, when used in combination with anticancer drugs (cisplatin and carboplatin), DCZ0415 exerted synergistic effects on cell proliferation suppression. Identifying the molecular pathways regulated by tumor-suppressive miRNAs (including passenger strands) may aid in the discovery of diagnostic markers and therapeutic targets for LUAD.