Mechanism Of TRF-59:75-Gly-TCC-1-M3-mediated Proliferation And Drug Resistance Regulation In Multiple Myeloma

Background: Multiple myeloma,the second most common hematologic malignancy,still remains an incurable disease.Understanding the mechanism of progression and drug resistance of multiple myeloma will contribute to early identification of risk factors,development of new treatment strategies,and improvement of prognosis of patients with refractory relapse disease.In previous studies,noncoding RNA and many other factors have been demonstrated to be associated with the progression or drug-resistance of multiple myeloma.tRNA derived small RNAs are non-coding RNAs that originally thought to be nonfunctional.In recent years,more and more evidences have indicated that these small fragments play significant roles in cancer,neurodegenerative disease,virus infection,inflammation,metabolic disorder,immunity,aging and inheritance by participating in different molecular and physiological processes.To date,the significance of tRNA-derived small RNA fragments in the progression and drug resistance of multiple myeloma remain largely unknown.Methods: In part Ⅰ,we collected marrow plasma cells from 5relapsed and refractory multiple myeloma patients and 5 newly diagnosed multiple myeloma patients.We compared the subtypes of tRNA-derived small RNA between the two groups and identified significantly dysregulated molecules through RNA sequencing.Differential expression molecules in RNA sequencing data were screened by R language edge R package,and data analysis and visualization were performed by R language or Perl environment.In part Ⅱ,firstly we verified the expression levels of three tRNA-derived small RNAs that were most significantly up-regulated in relapsed and refractory multiple myeloma in RNA sequence by q PCR,and selected the fragment with most significant fold change for subsequent functional studies.Specifically,RNA interference technology was used to inhibit the function of this fragment in drug-resistant myeloma cell lines.Cell proliferation was detected by CCK-8,apoptosis was detected by Annexin V/PI double staining flow cytometry,and drug resistance was measured by bortetomib.The effect of inhibition of this fragment on tumor proliferation was also observed in BALB/c mice.Finally,the potential downstream pathway of this fragment was predicted by bioinformatics and public database information,and the expression of key proteins and angiogenic factors were detected by westernblot.The interaction between ts RNA and Ago protein was preliminarily explored by Ag O-RIP assay.SPSS 23.0software was used for statistical analysis.For normally distributed data,two-tailed unpaired T test was used,otherwise,non-parametric test was selected.P <0.05 was considered statistically significant.Graph Pad 8.4.3,Cytoscape 3.8.0,and Adobe Illustrator software were used for data visualization.Results: tRNA-derived small RNAs fragments were dysregulated in relapsed and refractory multiple myeloma.Further analysis showed that10 of them were significantly up-regulated(t RF-1:16-chr M.Lys-TTT,t RF-59:75-Gly-TCC-1-M3,t RF-+1:T26-Ser-GCT-4-3,etc.)and 16 of them were significantly down-regulated(t RF-1:23-His-GTG-1,t RF-1:29-Thr-TGT-2,t RF-1:32-Ala-CGC-3,etc.)in relapsed and refractory multiple myeloma.q PCR in clinical samples showed that t RF-59:75-Gly-TCC-1-M3 was the most significantly up-regulated.In ARP1 and RPMI-8226 drug-resistant cells we found that inhibition of t RF-59:75-GLy-TCC-1-M3 led to decreased cell proliferation,increased apoptosis and increased drug sensitivity.We also observed inhibited tumor growth rate and increased sensitivity to bortezomib in the subcutaneous tumorigenesis model in BALB/c mice by inhibiting t RF-59:75-GLy-TCC-1-M3.Additionally,we found the downregulation of PI3K/Akt/m TOR pathway and VEGF by westernblot.Ago-RIP assay suggested that t RF-59:75-Gly-TCC-1-M3 interacted with Ago protein.Conclusions: tRNA-derived small RNAs were disregulated in relapsed and refractory multiple myeloma,among which t RF-59:75-GLy-TCC-1-M3 was the most significantly up-regulated.Functional loss of t RF-59:75-GLy-TCC-1-M3 inhibited the proliferation and drug resistance of myeloma cells and promoted apoptosis.t RF-59:75-GLyTCC-1-M3 may be involved in the regulation of angiogenesis or the PI3K/Akt/ m TOR pathway.