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. 2023 Feb 8.
doi: 10.1002/jcsm.13187. Online ahead of print.

tRNA-derived small RNA, 5'tiRNA-Gly-CCC, promotes skeletal muscle regeneration through the inflammatory response

Linyuan Shen  1   2 , Tianci Liao  1   2 , Qiuyang Chen  1   2 , Yuhang Lei  1   2 , Linghui Wang  1   2 , Hao Gu  1   2 , Yanhao Qiu  1   2 , Ting Zheng  1   2 , Yiting Yang  1   2 , Chenggang Wei  1   2 , Lei Chen  1   2 , Ye Zhao  1   2 , Lili Niu  1   2 , Shunhua Zhang  1   2 , Yan Zhu  3 , Mingzhou Li  1   2 , Jinyong Wang  4 , Xuewei Li  1   2 , Mailin Gan  1   2 , Li Zhu  1   2
Affiliations

tRNA-derived small RNA, 5'tiRNA-Gly-CCC, promotes skeletal muscle regeneration through the inflammatory response

Linyuan Shen et al. J Cachexia Sarcopenia Muscle. .

Abstract

Background: Increasing evidence shows that tRNA-derived small RNAs (tsRNAs) are not only by-products of transfer RNAs, but they participate in numerous cellular metabolic processes. However, the role of tsRNAs in skeletal muscle regeneration remains unknown.

Methods: Small RNA sequencing revealed the relationship between tsRNAs and skeletal muscle injury. The dynamic expression level of 5'tiRNA-Gly after muscle injury was confirmed by real-time quantitative PCR (q-PCR). In addition, q-PCR, flow cytometry, the 5-ethynyl-2'-deoxyuridine (Edu), cell counting kit-8, western blotting and immunofluorescence were used to explore the biological function of 5'tiRNA-Gly. Bioinformatics analysis and dual-luciferase reporter assay were used to further explore the mechanism of action under the biological function of 5'tiRNA-Gly.

Results: Transcriptome analysis revealed that tsRNAs were significantly enriched during inflammatory response immediately after muscle injury. Interestingly, we found that 5'tiRNA-Gly was significantly up-regulated after muscle injury (P < 0.0001) and had a strong positive correlation with inflammation in vivo. In vitro experiments showed that 5'tiRNA-Gly promoted the mRNA expression of proinflammatory cytokines (IL-1β, P = 0.0468; IL-6, P = 0.0369) and the macrophages of M1 markers (TNF-α, P = 0.0102; CD80, P = 0.0056; MCP-1, P = 0.0002). On the contrary, 5'tiRNA-Gly inhibited the mRNA expression of anti-inflammatory cytokines (IL-4, P = 0.0009; IL-10, P = 0.0007; IL-13, P = 0.0008) and the mRNA expression of M2 markers (TGF-β1, P = 0.0016; ARG1, P = 0.0083). Flow cytometry showed that 5'tiRNA-Gly promoted the percentage of CD86+ macrophages (16%, P = 0.011) but inhibited that of CD206+ macrophages (10.5%, P = 0.012). Immunofluorescence showed that knockdown of 5'tiRNA-Gly increased the infiltration of M2 macrophages to the skeletal muscles (13.9%, P = 0.0023) and inhibited the expression of Pax7 (P = 0.0089) in vivo. 5'tiRNA-Gly promoted myoblast the expression of myogenic differentiation marker genes (MyoD, P = 0.0002; MyoG, P = 0.0037) and myotube formation (21.3%, P = 0.0016) but inhibited the positive rate of Edu (27.7%, P = 0.0001), cell viability (22.6%, P = 0.003) and the number of myoblasts in the G2 phase (26.3%, P = 0.0016) in vitro. Mechanistically, we found that the Tgfbr1 gene is a direct target of 5'tiRNA-Gly mediated by AGO1 and AGO3. 5'tiRNA-Gly dysregulated the expression of downstream genes related to inflammatory response, activation of satellite cells and differentiation of myoblasts through the TGF-β signalling pathway by targeting Tgfbr1.

Conclusions: These results reveal that 5'tiRNA-Gly potentially regulated skeletal muscle regeneration by inducing inflammation via the TGF-β signalling pathway. The findings of this study uncover a new potential target for skeletal muscle regeneration treatment.

Keywords: TGF-β signalling pathway; Tgfbr1; inflammation; skeletal muscle regeneration; tsRNAs.

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