A novel tsRNA, tRF-33-6978WPRLXN4V0O inhibits breast cancer development via regulating PTEN/AKT pathway in BHLHA15-mediated manner.

Transfer RNA-derived fragments (tRFs) are a novel class of small non-coding RNAs. Recent studies have identified diverse tRNA-derived fragments (tRFs) in various diseases, confirming their distinct roles in transcriptional and post-transcriptional regulation. However, the biological functions and clinical significance of tRFs in breast cancer (BC) remain largely unexplored. In the present study, plasma samples were collected from patients with breast cancer (BC) and healthy donors for tRFs sequencing analysis to identify BC-related tRFs. tRF-33-6978WPRLXN4V0O (tRF-33) was then screened. A detection method for tRF-33 was developed to determine its abundance in tissue samples and analyze its clinical value in BC. In vitro, ethynyl-2'-deoxyuridine experiments, cell cloning, and flow cytometry were performed to determine the effects of tRF-33 regulation on BC. In vivo, xenograft tumor formation using MDA-MB-231 cells was performed to investigate the molecular function of tRF-33. To investigate the mechanism, RNA immunoprecipitation, dual luciferase assay, and western blotting were used to identify tRF-33's target gene and the possible pathway regulated by tRF-33. The results showed that tRF-33 was significantly downregulated in BC tissues and exhibited diagnostic value for BC. Lower tRF-33 expression correlated with more lymphatic node metastasis and higher Ki-67 expression levels. tRF-33 reduced the growth of BC cells and slowed tumor growth in nude mice. Mechanistically, tRF-33 directly silenced BHLHA15 by binding to Argonaute 2 (Ago2) and regulated the PTEN/AKT pathway. We identified tRF-33 as a promising diagnostic marker for BC, with suppressive effects on BC progression in vitro and in vivo. Mechanistically, tRF-33 cleaves BHLHA15 mRNA and regulates the PTEN/AKT pathway by interacting with Ago2. KEY MESSAGES: • In our study, plasma samples were collected from breast cancer (BC) patients and healthy donors for tsRNA sequencing analysis. • tRF-33-6978WPRLXN4V0O (tRF-33) was screened as BC-associated tRF. • The detection method for BC-related tRF was constructed. • Ethynyl-2'-deoxyuridine experiment, cell cloning, flow cytometry, and xenograft tumor formation using MDA-MB0231 cells were carried out to investigate the molecular function of tRF-33. • Various techniques including RNA immunoprecipitation, dual luciferase assay, and western blotting were performed in the investigation of mechanisms. • tRF-33 was significantly downregulated in BC tissues and exhibited diagnostic value for BC. • Lower tRF-33 expression correlated with more lymphatic node metastasis and higher Ki-67 expression levels. • tRF-33 reduced cell growth of BC cells and slowed tumor growth in nude mice. • Mechanistically, tRF-33 directly silenced BHLHA15 via binding to Argonaute 2 (Ago2) and regulated PTEN/AKT pathway. • We recognized tRF-33 as a promising diagnostic marker for BC and have suppressive effects on BC progression in vitro and in vivo. • Mechanically, tRF-33 cleaves BHLHA15 mRNA and regulates the PTEN/AKT pathway via interacting with Ago2.