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Objective To investigate the effect of tRNA fragment differentially expressed between high and low malignant bladder urothelial carcinoma (BUC) cells induced by heat injury on the proliferation and invasion of BUC cells. Methods High-grade BUC cell line T24 and low-grade BUC cell line 5637 were inflicted to heat injury. Then transfer RNA stress-induced fragments (tRFs) in these cells were sequenced before and after injury, and the tRFs whose expression decreased in T24 cells while increased in 5637 cells after heat injury were screened out, and further verified in clinical specimen by RT-PCR. The single-chain mimic and antisense strand (inhibitor) of this target tRF were constructed and transfected into heat injured T24 and 5637 cells respectively, and the cells were assigned into heat injury, vector control, mimic and inhibitor groups. Finally, CCK-8 assay, flow cytometry and Transwell test were conducted respectively to detect the regulatory effect of this tRF on the proliferation, apoptosis, migration and invasion of BUC cells. Results The originally selected tRFs were identified as tRF-chrM.Ser-GCT and tRF-Ile-AAT-1 through sequencing and RT-PCR in cell lines. However, when comparing the specimens from the second surgery of transurethral resection of bladder tumor (TURBT) with those from the first one, the results discovered that only tRF-Ile-AAT-1 levels were increased in low-grade BUC patients (1.61±0.16 times, P < 0.01) while decreased in high-grade BUC patients (0.75±0.14 times, P < 0.05). In the T24 cells experiments, when compared with the T24Heat group, the proliferation of BUC cells in the T24Heat+Mimic group was inhibited (P < 0.05), the apoptotic rate was significantly improved (5.88±1.01 times, P < 0.01), and the number of migrated and invaded cells were both greatly diminished (0.40±0.05 times, P < 0.01; 0.23±0.04 times, P < 0.01). Moreover, the proliferation of BUC cells in the 5637Heat+ Inhibitor group was enhanced (P < 0.05), the apoptotic rate was declined (0.11±0.02 times, P < 0.01), and the number of migrated cells as well as invaded cells were both elevated (1.81±0.29 times, P < 0.01; 2.70±0.15 times, P < 0.01), as compared with the 5637Heat group. Conclusion tRNA fragment tRF-Ile-AAT-1 induced by heat injury can inhibit the proliferation and invasion in BUC cells