The Mechanism Of TRNA-derived Fragments In Regulation Of NSCLC

Objective:Non-small cell lung cancer(NSCLC)is one of the most common tumors with the highest incidence and mortality in the world.Over the past decades,although significant progress has been made in the diagnosis and treatment,the early diagnosis rate of NSCLC is still less than 30% and the 5-year overall survival rate is only about 20%.Thus,it is extremely necessary to investigate the molecular mechanisms of NSCLC in order to find novel biomarker for early detection and new therapeutic targets for improving the prognosis of NSCLC.tRNA derived-fragments(tRFs)is a new class of non-coding small-molecule RNA.Recent studies suggest that tRFs are involved in the development and progress of several cancers.However,whether tRFs may also play regulatory roles in NSCLC remains largely unknown.The research objectives of this study include:(1)To identify NSCLC-associated tRFs;(2)To determine the regulatory effects of tRFs on NSCLC;and(3)To explore the mechanism of tRFs in NSCLC cells.Methods: RNA sequencing was used to screen and identify NSCLC-related tRFs.qRT-PCR was used to analyze and confirm the expression levels of tRFs in tumor tissues,in pre-operation plasma,in NSCLC cell lines(PC9,HCC827,NCI-H226,A549),and in normal cells(BEAS-2B).Candidate tRF and corresponding inhibitor were synthesized and were separately transfected into NSCLC cells.Cell proliferation,migration,and apoptosis were determined using CCK-8 kit,transwell plate,and Fluor 488 Annexin V and PI kit,respectively.RNA-Pull down and MS techniques were used to find the binding proteins of tRFs.RIP assay and qPCR method were applied to verify the existence of binding proteins.Lastly,si RNA-based assays were used to assess the impacts of silencing tRF-binding protein on cell phenotypes.Results: RNA-seq found that tRFs profiles in pre-operation plasma were different from that in post-operation plasmas.7 differentially expressed tRFs were identified.Among them the tRF-007333 was the most significant differentially expressed ones.Further qPCR analysis showed that tRF-007333 was also up-regulated in NSCLC tumor tissues and in several NSCLC cell lines(PC9,HCC827,NCI-H226,A549).Overexpression of tRF-007333 in NSCLC cells(PC9,HCC827,A549)promoted the cell proliferation,while knockdown of tRF-007333 inhibited cell proliferation.No significant effects of tRF-007333 on cell migration and apoptosis were observed in NSCLC cells.The RNA pull-down assay and MS analysis showed that tRF-007333 can bine directly with the heat shock protein beta-1(HSPB-1).RIP and qPCR analyses confirmed the interactions between tRF-007333 and HSPB-1.Silencing of HSPB-1 was found to significantly inhibit cell proliferation,suggesting that HSPB-1was a major target of tRF-007333.Conclusion:(1)RNA sequencing of plasma samples from NSCLC patients identified NSCLCassociated tRF-007333;(2)tRF-007333 was up-regulated in NSCLC tumor tissues,preoperation plasma and in NSCLC cells,but down-regulated in adjacent normal tissues and in normal human bronchial epithelium cells;(3)Up-regulation of tRF-007333 could promote proliferation of NSCLC cells;(4)tRF-007333 increased NSCLC cell proliferation by targeting its binding protein HSPB-1.