Abstract
The role of transfer RNA (tRNA)-derived fragment (tRF) in various diseases has been established. However, the effect of tRF-3023b on inflammation remains unclear. Inflammation was imitated in RAW264.7 cells by adding Lipopolysaccharide (LPS). Cells were first divided into control, LPS, and LPS + Bulleyaconitine A (BLA) groups. The contents of TNF-α, IL-6, and MCP-1 were quantified using ELISA. The levels of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), and the phosphorylation of nuclear factor-kappa B (NF-κB)-P65 (p-P65) were detected by Western blotting. RNA sequencing was utilized to find differentially expressed tRFs (DE-tRFs) among three groups. The levels of various tRFs were checked by quantitative real-time PCR (qRT-PCR). Cell cycle and apoptosis were checked by flow cytometry. Dluciferase reporter assay was applied to predict and confirm the interaction between tRF-3023b and Cullin 4A (Cul4a), subsequently RNA pull-down followed by mass spectrometry analysis were conducted. BLA treatment decreased the contents of TNF-α, IL-6, MCP-1, and the expression levels of COX2, iNOS, p-P65. We found 6 DE-tRFs in LPS + BLA group compared to LPS group, tRF-3023b was high expression in control and BLA groups, and the lowest in LPS group. Cul4a was a direct target of tRF-3023b. tRF-3023b mimic affected the cell cycle distribution, promoted cells apoptosis, and suppressed the TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. The suppression of Cul4a affected the cell cycle distribution, resulted in an increase of cell apoptosis while a decrease of TNF-α, IL-6, MCP-1, COX2, iNOS and p-P65. Furthermore, Cul4a overexpression reversed the effect of tRF-3023b mimic. Cul4a knockdown reversed the effect of tRF-3023b inhibitor. Our study positions tRF-3023b as a compelling candidate, through its interaction with Cul4a, the underlying mechanism on inflammation maybe related to NF-κB pathway. The study provides a basis for exploring new therapeutic strategies for inflammation.
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Funding
This research was supported by the National Natural Science Foundation of China (No. 81671586 and 81270091), Research Start-up Fund Project of Jiaxing University for Introducing Talents (No. CD70519066), and the Municipal Public Welfare Research Project (No. 2022AY10001) from Jiaxing, the Scientific Foundation of Jiaxing (No. 2021AD30154), Zhejiang Provincial Natural Science Foundation of China (No. LY20C010004), Zhejiang Provincial Medical Scientific Research Foundation of China (No. 2021KY1108), 2019 Jiaxing Key Discipline of Medicine (No. 2019-zc-02), Key Project of Natural Science Research in Anhui Universities (KJ2020A0860), and Natural Science Foundation-funded Project of Anhui Province (No. 2023AH052599).
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YZ, HZ and YJ wrote the main manuscript text. YZ, HZ, XC, QQ, MD and YJ prepared figures 1-4. YZ, QQ, MD, YJ, NW, YZ and ZH prepared figures 5 and 6. All authors reviewed the manuscript.
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Zhang, Y., Zhou, H., Chen, X. et al. A novel tRNA-derived fragment tRF-3023b suppresses inflammation in RAW264.7 cells by targeting Cul4a through NF-κB signaling. Funct Integr Genomics 24, 9 (2024). https://doi.org/10.1007/s10142-024-01285-3
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DOI: https://doi.org/10.1007/s10142-024-01285-3