Serum mitochondrial tsRNA serves as a novel biomarker for hepatocarcinoma diagnosis

Abstract

Hepatocellular carcinoma (HCC), which makes up the majority of liver cancer, is induced by the infection of hepatitis B/C virus. Biomarkers are needed to facilitate the early detection of HCC, which is often diagnosed too late for effective therapy. The tRNA-derived small RNAs (tsRNAs) play vital roles in tumorigenesis and are stable in circulation. However, the diagnostic values and biological functions of circulating tsRNAs, especially for HCC, are still unknown. In this study, we first utilized RNA sequencing followed by quantitative reverse-transcription PCR to analyze tsRNA signatures in HCC serum. We identified tRF-Gln-TTG-006, which was remarkably upregulated in HCC serum (training cohort: 24 HCC patients vs. 24 healthy controls). In the validation stage, we found that tRF-Gln-TTG-006 signature could distinguish HCC cases from healthy subjects with high sensitivity (80.4%) and specificity (79.4%) even in the early stage (Stage I: sensitivity, 79.0%; specificity, 74.8%; 155 healthy controls vs. 153 HCC patients from two cohorts). Moreover, in vitro studies indicated that circulating tRF-Gln-TTG-006 was released from tumor cells, and its biological function was predicted by bioinformatics assay and validated by colony formation and apoptosis assays. In summary, our study demonstrated that serum tsRNA signature may serve as a novel biomarker of HCC.

Keywords

tsRNA / biomarker / hepatocarcinoma

References

[1] SungH, FerlayJ, SiegelRL, LaversanneM, SoerjomataramI, JemalA, BrayF. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021; 71( 3): 209– 249

[2] KimTH, KimSY, TangA, LeeJM. Comparison of international guidelines for noninvasive diagnosis of hepatocellular carcinoma: 2018 update. Clin Mol Hepatol 2019; 25( 3): 245– 263

[3] LlovetJM, KelleyRK, VillanuevaA, SingalAG, PikarskyE, RoayaieS, LencioniR, KoikeK, Zucman-RossiJ, FinnRS. Hepatocellular carcinoma. Nat Rev Dis Primers 2021; 7( 1): 6

[4] ZhangY, LiuD, ChenX, LiJ, LiL, BianZ, SunF, LuJ, YinY, CaiX, SunQ, WangK, BaY, WangQ, WangD, YangJ, LiuP, XuT, YanQ, ZhangJ, ZenK, ZhangCY. Secreted monocytic miR-150 enhances targeted endothelial cell migration. Mol Cell 2010; 39( 1): 133– 144

[5] ChenX, BaY, MaL, CaiX, YinY, WangK, GuoJ, ZhangY, ChenJ, GuoX, LiQ, LiX, WangW, ZhangY, WangJ, JiangX, XiangY, XuC, ZhengP, ZhangJ, LiR, ZhangH, ShangX, GongT, NingG, WangJ, ZenK, ZhangJ, ZhangCY. Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res 2008; 18( 10): 997– 1006

[6] WangY, LiangH, JinF, YanX, XuG, HuH, LiangG, ZhanS, HuX, ZhaoQ, LiuY, JiangZY, ZhangCY, ChenX, ZenK. Injured liver-released miRNA-122 elicits acute pulmonary inflammation via activating alveolar macrophage TLR7 signaling pathway. Proc Natl Acad Sci USA 2019; 116( 13): 6162– 6171

[7] DawsonSJ, TsuiDWY, MurtazaM, BiggsH, RuedaOM, ChinSF, DunningMJ, GaleD, ForshewT, Mahler-AraujoB, RajanS, HumphrayS, BecqJ, HalsallD, WallisM, BentleyD, CaldasC, RosenfeldN. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 2013; 368( 13): 1199– 1209

[8] ForshewT, MurtazaM, ParkinsonC, GaleD, TsuiDW, KaperF, DawsonSJ, PiskorzAM, Jimenez-LinanM, BentleyD, HadfieldJ, MayAP, CaldasC, BrentonJD, RosenfeldN. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med 2012; 4( 136): 136ra68

[9] ColeC, SobalaA, LuC, ThatcherSR, BowmanA, BrownJW, GreenPJ, BartonGJ, HutvagnerG. Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs. RNA 2009; 15( 12): 2147– 2160

[10] PengH, ShiJ, ZhangY, ZhangH, LiaoS, LiW, LeiL, HanC, NingL, CaoY, ZhouQ, ChenQ, DuanE. A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm. Cell Res 2012; 22( 11): 1609– 1612

[11] JinF, GuoZ. Emerging role of a novel small non-coding regulatory RNA: tRNA-derived small RNA. ExRNA 2019; 1 : 39

[12] KumarP, KuscuC, DuttaA. Biogenesis and function of transfer RNA-related fragments (tRFs). Trends Biochem Sci 2016; 41( 8): 679– 689

[13] IvanovP, EmaraMM, VillenJ, GygiSP, AndersonP. Angiogenin-induced tRNA fragments inhibit translation initiation. Mol Cell 2011; 43( 4): 613– 623

[14] ThompsonDM, ParkerR. The RNase Rny1p cleaves tRNAs and promotes cell death during oxidative stress in Saccharomyces cerevisiae. J Cell Biol 2009; 185( 1): 43– 50

[15] HondaS, LoherP, ShigematsuM, PalazzoJP, SuzukiR, ImotoI, RigoutsosI, KirinoY. Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers. Proc Natl Acad Sci USA 2015; 112( 29): E3816– E3825

[16] MauteRL, SchneiderC, SumazinP, HolmesA, CalifanoA, BassoK, Dalla-FaveraR. tRNA-derived microRNA modulates proliferation and the DNA damage response and is down-regulated in B cell lymphoma. Proc Natl Acad Sci USA 2013; 110( 4): 1404– 1409

[17] BalattiV, PekarskyY, CroceCM. Role of the tRNA-derived small RNAs in cancer: new potential biomarkers and target for therapy. Adv Cancer Res 2017; 135 : 173– 187

[18] PekarskyY, BalattiV, PalamarchukA, RizzottoL, VenezianoD, NigitaG, RassentiLZ, PassHI, KippsTJ, LiuCG, CroceCM. Dysregulation of a family of short noncoding RNAs, tsRNAs, in human cancer. Proc Natl Acad Sci USA 2016; 113( 18): 5071– 5076

[19] BalattiV, RizzottoL, MillerC, PalamarchukA, FaddaP, PandolfoR, RassentiLZ, HertleinE, RuppertAS, LozanskiA, LozanskiG, KippsTJ, ByrdJC, CroceCM, PekarskyY. TCL1 targeting miR-3676 is codeleted with tumor protein p53 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2015; 112( 7): 2169– 2174

[20] JinF, YangL, WangW, YuanN, ZhanS, YangP, ChenX, MaT, WangY. A novel class of tsRNA signatures as biomarkers for diagnosis and prognosis of pancreatic cancer. Mol Cancer 2021; 20 : 95

[21] GoodarziH, LiuX, NguyenHCB, ZhangS, FishL, TavazoieSF. Endogenous tRNA-derived fragments suppress breast cancer progression via YBX1 displacement. Cell 2015; 161( 4): 790– 802

[22] ZhangY, ZhangY, ShiJ, ZhangH, CaoZ, GaoX, RenW, NingY, NingL, CaoY, ChenY, JiW, ChenZJ, ChenQ, DuanE. Identification and characterization of an ancient class of small RNAs enriched in serum associating with active infection. J Mol Cell Biol 2014; 6( 2): 172– 174

[23] WangJ, MaG, LiM, HanX, XuJ, LiangM, MaoX, ChenX, XiaT, LiuX, WangS. Plasma tRNA fragments derived from 5′ ends as novel diagnostic biomarkers for early-stage breast cancer. Mol Ther Nucleic Acids 2020; 21 : 954– 964

[24] GuX, WangL, CoatesPJ, BoldrupL, FåhraeusR, WilmsT, SgaramellaN, NylanderK. Transfer-RNA-derived fragments are potential prognostic factors in patients with squamous cell carcinoma of the head and neck. Genes (Basel) 2020; 11( 11): 1344

[25] YuM, LiuZ, LiuY, ZhouX, SunF, LiuY, LiL, HuaS, ZhaoY, GaoH, ZhuZ, NaM, ZhangQ, YangR, ZhangJ, YaoY, ChenX. PTP1B markedly promotes breast cancer progression and is regulated by miR-193a-3p. FEBS J 2019; 286( 6): 1136– 1153

[26] KrügerJ, RehmsmeierM. RNAhybrid: microRNA target prediction easy, fast and flexible. Nucleic Acids Res 2006; 34(Web Server issue): W451− 454 doi: 10.1093/nar/gkl243

[27] XieC, Mao X, HuangJ, DingY, WuJ, Dong S, KongL, GaoG, Li CY, WeiL. KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res 2011; 39(Web Server issue): W316– 322 doi: 10.1093/nar/gkr483

[28] HurK, ToiyamaY, OkugawaY, IdeS, ImaokaH, BolandCR, GoelA. Circulating microRNA-203 predicts prognosis and metastasis in human colorectal cancer. Gut 2017; 66( 4): 654– 665

[29] YamadaA, HorimatsuT, OkugawaY, NishidaN, HonjoH, IdaH, KouT, KusakaT, SasakiY, YagiM, HigurashiT, YukawaN, AmanumaY, KikuchiO, MutoM, UenoY, NakajimaA, ChibaT, BolandCR, GoelA. Serum miR-21, miR-29a, and miR-125b are promising biomarkers for the early detection of colorectal neoplasia. Clin Cancer Res 2015; 21( 18): 4234– 4242

[30] KuscuC, KumarP, KiranM, SuZ, MalikA, DuttaA. tRNA fragments (tRFs) guide Ago to regulate gene expression post-transcriptionally in a Dicer-independent manner. RNA 2018; 24( 8): 1093– 1105

[31] PliatsikaV, LoherP, MageeR, TelonisAG, LondinE, ShigematsuM, KirinoY, RigoutsosI. MINTbase v2.0: a comprehensive database for tRNA-derived fragments that includes nuclear and mitochondrial fragments from all The Cancer Genome Atlas projects. Nucleic Acids Res 2018; 46( D1): D152– D159

[32] RehmsmeierM, SteffenP, HochsmannM, GiegerichR. Fast and effective prediction of microRNA/target duplexes. RNA 2004; 10( 10): 1507– 1517

[33] HynesRO. The extracellular matrix: not just pretty fibrils. Science 2009; 326( 5957): 1216– 1219

[34] SeikiM. Membrane-type 1 matrix metalloproteinase: a key enzyme for tumor invasion. Cancer Lett 2003; 194( 1): 1– 11

[35] Brandão-CostaRM, Helal-NetoE, VieiraAM, Barcellos-de-SouzaP, Morgado-DiazJ, Barja-FidalgoC. Extracellular matrix derived from high metastatic human breast cancer triggers epithelial-mesenchymal transition in epithelial breast cancer cells through αvβ3 integrin. Int J Mol Sci 2020; 21( 8): 2995

[36] MaL, YoungJ, PrabhalaH, PanE, MestdaghP, MuthD, Teruya-FeldsteinJ, ReinhardtF, OnderTT, ValastyanS, WestermannF, SpelemanF, VandesompeleJ, WeinbergRA. miR-9, a MYC/MYCN-activated microRNA, regulates E-cadherin and cancer metastasis. Nat Cell Biol 2010; 12( 3): 247– 256

[37] RubinsteinMR, WangX, LiuW, HaoY, CaiG, HanYW. Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA adhesin. Cell Host Microbe 2013; 14( 2): 195– 206

[38] SchmalhoferO, BrabletzS, BrabletzT. E-cadherin, β-catenin, and ZEB1 in malignant progression of cancer. Cancer Metastasis Rev 2009; 28(1−2): 151− 166 doi:10.1007/s10555-008-9179-y

[39] RenD, LinB, ZhangX, PengY, YeZ, MaY, LiangY, CaoL, LiX, LiR, SunL, LiuQ, WuJ, ZhouK, ZengJ. Maintenance of cancer stemness by miR-196b-5p contributes to chemoresistance of colorectal cancer cells via activating STAT3 signaling pathway. Oncotarget 2017; 8( 30): 49807– 49823

[40] CalvisiDF, LaduS, GordenA, FarinaM, ConnerEA, LeeJS, FactorVM, ThorgeirssonSS. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006; 130( 4): 1117– 1128

[41] ShuaiK, LiuB. Regulation of JAK-STAT signalling in the immune system. Nat Rev Immunol 2003; 3( 11): 900– 911

[42] YangJD, HainautP, GoresGJ, AmadouA, PlymothA, RobertsLR. A global view of hepatocellular carcinoma: trends, risk, prevention and management. Nat Rev Gastroenterol Hepatol 2019; 16( 10): 589– 604

[43] TrevisaniF, D’IntinoPE, Morselli-LabateAM, MazzellaG, AccogliE, CaraceniP, DomenicaliM, DeNotariis S, RodaE, BernardiM. Serum alpha-fetoprotein for diagnosis of hepatocellular carcinoma in patients with chronic liver disease: influence of HBsAg and anti-HCV status. J Hepatol 2001; 34( 4): 570– 575

[44] ColliA, FraquelliM, CasazzaG, MassironiS, ColucciA, ConteD, DucaP. Accuracy of ultrasonography, spiral CT, magnetic resonance, and alpha-fetoprotein in diagnosing hepatocellular carcinoma: a systematic review. Am J Gastroenterol 2006; 101( 3): 513– 523

[45] LokAS, SterlingRK, EverhartJE, WrightEC, HoefsJC, DiBisceglie AM, MorganTR, KimHY, LeeWM, BonkovskyHL, DienstagJL; HALT-C Trial Group. Des-γ-carboxy prothrombin and α-fetoprotein as biomarkers for the early detection of hepatocellular carcinoma. Gastroenterology 2010; 138( 2): 493– 502

[46] HayesJ, PeruzziPP, LawlerS. MicroRNAs in cancer: biomarkers, functions and therapy. Trends Mol Med 2014; 20( 8): 460– 469

[47] SchwarzenbachH, HoonDSB, PantelK. Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 2011; 11( 6): 426– 437

[48] GiordanoS, ColumbanoA. MicroRNAs: new tools for diagnosis, prognosis, and therapy in hepatocellular carcinoma? Hepatology 2013; 57(2): 840− 847 doi:10.1002/hep.26095

[49] FornariF, FerracinM, TrerèD, MilazzoM, MarinelliS, GalassiM, VenerandiL, PollutriD, PatriziC, BorghiA, FoschiFG, StefaniniGF, NegriniM, BolondiL, GramantieriL. Circulating microRNAs, miR-939, miR-595, miR-519d and miR-494, identify cirrhotic patients with HCC. PLoS One 2015; 10( 10): e0141448

[50] BhattacharyaS, SteeleR, ShrivastavaS, ChakrabortyS, Di BisceglieAM, RayRB. Serum miR-30e and miR-223 as novel noninvasive biomarkers for hepatocellular carcinoma. Am J Pathol 2016; 186( 2): 242– 247

[51] CaiJ, ChenL, ZhangZ, ZhangX, LuX, LiuW, ShiG, GeY, GaoP, YangY, KeA, XiaoL, DongR, ZhuY, YangX, WangJ, ZhuT, YangD, HuangX, SuiC, QiuS, ShenF, SunH, ZhouW, ZhouJ, NieJ, ZengC, StroupEK, ZhangX, ChiuBCH, LauWY, HeC, WangH, ZhangW, FanJ. Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma. Gut 2019; 68( 12): 2195– 2205

[52] WenL, LiJ, GuoH, LiuX, ZhengS, ZhangD, ZhuW, QuJ, GuoL, DuD, JinX, ZhangY, GaoY, ShenJ, GeH, TangF, HuangY, PengJ. Genome-scale detection of hypermethylated CpG islands in circulating cell-free DNA of hepatocellular carcinoma patients. Cell Res 2015; 25( 11): 1250– 1264

[53] ShiJ, ZhangY, ZhouT, ChenQ. tsRNAs: the Swiss army knife for translational regulation. Trends Biochem Sci 2019; 44( 3): 185– 189

[54] LeeYS, ShibataY, MalhotraA, DuttaA. A novel class of small RNAs: tRNA-derived RNA fragments (tRFs). Genes Dev 2009; 23( 22): 2639– 2649

[55] ThompsonDM, ParkerR. Stressing out over tRNA cleavage. Cell 2009; 138( 2): 215– 219

[56] FuH, FengJ, LiuQ, SunF, TieY, ZhuJ, XingR, SunZ, ZhengX. Stress induces tRNA cleavage by angiogenin in mammalian cells. FEBS Lett 2009; 583( 2): 437– 442

[57] YinY, CaiX, ChenX, LiangH, ZhangY, LiJ, WangZ, ChenX, ZhangW, YokoyamaS, WangC, LiL, LiL, HouD, DongL, XuT, HiroiT, YangF, JiH, ZhangJ, ZenK, ZhangCY. Tumor-secreted miR-214 induces regulatory T cells: a major link between immune evasion and tumor growth. Cell Res 2014; 24( 10): 1164– 1180

[58] BlackburnSD, ShinH, HainingWN, ZouT, WorkmanCJ, PolleyA, BettsMR, FreemanGJ, VignaliDAA, WherryEJ. Coregulation of CD8⁺ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 2009; 10( 1): 29– 37

[59] KusmartsevS, NefedovaY, YoderD, GabrilovichDI. Antigen-specific inhibition of CD8⁺ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species. J Immunol 2004; 172( 2): 989– 999

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