Dataset Information

A 5'-tRNA halve, tiRNA-Gly promotes cell proliferation and migration via binding to RBM17 and inducing alternative splicing in papillary thyroid cancer.

ABSTRACT:

Background

tRNA-derived small noncoding RNAs (sncRNAs) are mainly categorized into tRNA halves (tiRNAs) and fragments (tRFs). Biological functions of tiRNAs in human solid tumor are attracting more and more attention, but researches concerning the mechanisms in tiRNAs-mediated tumorigenesis are rarely. The direct regulatory relationship between tiRNAs and splicing-related proteins remain elusive.

Methods

Papillary thyroid carcinoma (PTC) associated tRNA fragments were screened by tRNA fragments deep sequencing and validated by qRT-PCR and Northern Blot in PTC tissues. The biological function of tRNA fragments were assessed by cell counting kit, transwells and subcutaneous transplantation tumor of nude mice. For mechanistic study, tRNA fragments pull-down, RNA immunoprecipitation, Western Blot, Immunofluorescence, Immunohistochemical staining were performed.

Results

Herein, we have identified a 33 nt tiRNA-Gly significantly increases in papillary thyroid cancer (PTC) based on tRFs & tiRNAs sequencing. The ectopic expression of tiRNA-Gly promotes cell proliferation and migration, whereas down-regulation of tiRNA-Gly exhibits reverse effects. Mechanistic investigations reveal tiRNA-Gly directly bind the UHM domain of a splicing-related RNA-binding protein RBM17. The interaction with tiRNA-Gly could translocate RBM17 from cytoplasm into nucleus. In addition, tiRNA-Gly increases RBM17 protein expression via inhibiting its degradation in a ubiquitin/proteasome-dependent way. Moreover, RBM17 level in tiRNA-Gly high-expressing human PTC tissues is upregulated. In vivo mouse model shows that suppression of tiRNA-Gly decreases RBM17 expression. Importantly, tiRNA-Gly can induce exon 16 splicing of MAP4K4 mRNA leading to phosphorylation of downstream signaling pathway, which is RBM17 dependent.

Conclusions

Our study firstly illustrates tiRNA-Gly can directly bind to RBM17 and display oncogenic effect via RBM17-mediated alternative splicing. This fully novel model broadens our understanding of molecular mechanism in which tRNA fragment in tumor cells directly bind RNA binding protein and play a role in alternative splicing.

SUBMITTER: Han L

PROVIDER: S-EPMC8256553 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundIncreasing 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.MethodsSmall 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.ResultsTranscriptome 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.ConclusionsThese 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.