Papers in Press. Published April 17, 2019 as doi:10.1373/clinchem.2018.301150 The latest version is at http://clinchem.aaccjnls.org/cgi/doi/10.1373/clinchem.2018.301150 Clinical Chemistry 65:7 Cancer Diagnostics 000–000 (2019) Noncoding RNAs Serve as Diagnosis and Prognosis Biomarkers for Hepatocellular Carcinoma Chang Tan,1,2† Jingyi Cao,1,2† Lu Chen,3,4,5† Xiaochen Xi,1 Siqi Wang,1 Yumin Zhu,1 Liuqing Yang,1 Longteng Ma,6 Dong Wang,1 Jianhua Yin,6* Ti Zhang,3,4,5* and Zhi John Lu1* BACKGROUND: Reliable noninvasive biomarkers for hep- fragment alone promoted cancer cell proliferation and atocellular carcinoma (HCC)7 diagnosis and prognosis clonogenic growth. are urgently needed. We explored the potential of not only microRNAs (miRNAs) but other types of noncod- CONCLUSIONS: Our results show that various ncRNA ing RNAs (ncRNAs) as HCC biomarkers. species, not only miRNAs, identified in the small RNA sequencing of plasma are also able to serve as noninvasive METHODS: Peripheral blood samples were collected from biomarkers. Particularly, we identified a domain of sr- 77 individuals; among them, 57 plasma cell-free RNA pRNA RN7SL1 with reliable clinical performance for transcriptomes and 20 exosomal RNA transcriptomes HCC diagnosis and prognosis. were profiled. Significantly upregulated ncRNAs and © 2019 American Association for Clinical Chemistry published potential HCC biomarkers were validated with reverse transcription (RT)-qPCR in an independent validation cohort (60–150 samples). We particularly in- vestigated the diagnosis and prognosis performance and Hepatocellular carcinoma (HCC) is the most common biological function for 1 ncRNA biomarker, RN7SL1,8 type of primary liver cancer (1). Viruses, like hepatitis B and its S fragment. virus, are crucial in the transformation into HCC (2). The overall 5-year survival rate for all stages of HCC is RESULTS: We identified certain circulating ncRNAs es- only 15%; however, if diagnosed early, the survival rate caping from RNase degradation, possibly through bind- can be up to 70% (1). Thus, noninvasive biomarkers for ing with RNA-binding proteins: 899 ncRNAs were early diagnosis of HCC are urgently needed. highly upregulated in HCC patients. Among them, 337 Although many plasma microRNAs (miRNAs) are genes were fragmented long noncoding RNAs, 252 genes differentially produced in HCC patients vs healthy indi- were small nucleolar RNAs, and 134 genes were piwi- viduals (3, 4), most are unreliable HCC biomarkers, ow- interacting RNAs. Forty-eight candidates were selected ing to lack of independent validation and their failure to and validated with RT-qPCR, of which, 16 ncRNAs distinguish HCC from hepatitis B virus. Recently, other were verified to be significantly upregulated in HCC, types of noncoding RNAs (ncRNAs) have gained atten- including RN7SL1, SNHG1, ZFAS1, and LINC01359. tion as potential biomarker candidates. For instance, the Particularly, the abundance of RN7SL1 S fragment dis- expression of long noncoding RNA (lncRNA) H19 is criminated HCC samples from negative controls (area increased in breast cancer plasma compared to plasmas of under the curve, 0.87; 95% CI, 0.817–0.920). HCC healthy controls (5), and plasma concentrations of 2 ln- patients with higher concentrations of RN7SL1 S frag- cRNAs, SNHG1 and RMRP, are higher in lung cancer ment had lower survival rates. Furthermore, RN7SL1 S patients vs cancer-free controls (6). 1 MOE Key Laboratory of Bioinformatics, Center for Synthetic and Systems Biology, School 7 Nonstandard abbreviations: HCC, hepatocellular carcinoma; miRNAs, microRNAs; of Life Sciences, Tsinghua University, Beijing, China; 2 Tsinghua-Peking Joint Center for ncRNAs, noncoding RNAs; lncRNAs, long noncoding RNAs; cfRNAs, cell-free RNAs; EVs, Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China; 3 Tianjin Med- extracellular vesicles; tRNAs, transfer RNAs; HD, healthy donors; CHB, chronic hepatitis ical University Cancer Institute and Hospital, Department of Hepatobiliary Cancer, Na- B; exoRNA, exosomal RNA; srpRNAs, signal recognition particle RNAs; SRP, signal recog- tional Clinical Research for Cancer, Tianjin, China; 4 Key Laboratory of Cancer Prevention nition particle; piRNAs, piwi-interacting RNAs; snoRNAs, small nucleolar RNAs; RBPs, and Therapy, Tianjin, China; 5 Tianjin’s Clinical Research for Cancer, Tianjin, China; 6 De- RNA-binding proteins; mRNAs, messenger RNAs; RT, reverse transcription; seq, se- partment of Epidemiology, Second Military Medical University, Shanghai, China. quencing; NC, negative control; AUC, area under the curve. * Address correspondence to: J.Y. at 800 Xiangyin Rd., Shanghai 200433, People’s Re- 8 HumanGenes:RN7SL1,RNA,7SL,cytoplasmic1;SNHG1,smallnucleolarRNAhostgene public of China. E-mail [email protected]. T.Z. at Huan Hu Xi Road, Ti Yuan Bei, He 1; ZFAS1, ZNFX1 antisense RNA 1; LINC01359, long intergenic non-protein coding RNA Xi District, Tianjin 300060, People’s Republic Of China. E-mail [email protected]. 1359; H19, H19, imprinted maternally expressed transcript; RMRP, RNA component of Z.J.L. at School of Life Sciences, Tsinghua University, Beijing, China 100084. Fax +86- mitochondrial RNA processing endoribonuclease; XIST, X inactive specific transcript; 10-62789217; e-mail [email protected]. NEAT1, nuclear paraspeckle assembly transcript 1; MIR122, microRNA 122; HEIH, hepa- † C. Tan, J. Cao, and L. Chen contributed equally. tocellular carcinoma upregulated EZH2-associated long non-coding RNA; LINC01225, Received December 21, 2018; accepted March 12, 2019. LINC01225 pseudogene; CYTOR, cytoskeleton regulator RNA; MIR21, microRNA 21; Previously published online at DOI: 10.1373/clinchem.2018.301150 MIR192, microRNA 192; MIR801, microRNA 801; MIR1228, microRNA 1228. © 2019 American Association for Clinical Chemistry 1 Copyright (C) 2019 by The American Association for Clinical Chemistry Published research on cell-free RNA (cfRNA) in BLOOD SAMPLING AND CELL-FREE RNA PURIFICATION cancer suggests that RNA fragments exist in plasma or Peripheral whole blood samples (5–10 mL) were col- extracellular vesicles (EVs), such as exosomes and mi- lected from participant patients in K2EDTA tubes. crovesicles (7). These RNA fragments may be processed Tubes were inverted 8–10 times and centrifuged (820g, posttranscriptionally and may be developmentally regu- 10 min, 4 °C). Blood cells were removed by use of a lated and tumor-specific. For example, EVs from human centrifuge at high speed (14000g, 1 min, 4 °C). Super- primary fibroblasts contain both full-length human hY5 natant was collected, aliquoted, and stored at Ϫ80 °C, and shorter processed fragments, whereas cancer cell- within 2 h after collection. derived EVs only contain a processed hY5 fragment, A modified protocol was used to isolate cfRNA from which promotes apoptotic cell death (8). In another ex- plasma with ZYMO Quick-cfRNA Serum & Plasma kit ample, the 5Ј fragment of glycine transfer RNA (tRNA) (R1059). DNase I was added, followed by Proteinase K represses the expression of genes associated with the en- treatment. cfRNA was purified with Zymo-Spin IC Col- dogenous retroelement, MERVL, in both embryonic umn according to the manufacturer’s protocol. stem cells and embryos (9). Although biological functions for fragments of Y cfRNA LIBRARY CONSTRUCTION RNA and tRNA in plasma and EVs have been demon- We used NEBNext® Multiplex Small RNA Library Prep strated, little research has focused on other types of Set to construct the small RNA libraries, and Illumina cfRNA fragments, such as lncRNAs (10). XIST has a HiSeq ϫ10 sequencing platform to sequence libraries structural fragment that is necessary for X chromosome– and generate 150-bp paired-end reads. inactivated gene silencing (11). LncRNA NEAT1_2 also has structural fragments (12) that promote NEAT1_2 DATA PROCESSING AND DIFFERENTIAL EXPRESSION stabilization. ANALYSIS Little is known about the biological functions of Library quality was evaluated with FastQC (version extracellular RNA fragments and the utility of extracel- 0.11.7) on raw read1. Adapters were removed by cut- lular RNA fragments as diagnostic biomarkers. To un- adapt (version 1.16) with the parameter setting of: –a ravel the function of extracellular RNA fragments and AGATCGGAAGAGCACACGTCTGAACTCCAGT- find robust noninvasive biomarkers for HCC, we se- CAC. Trimmed reads were mapped to the human quenced extracellular RNA from plasma and exosomes rRNAs, and rRNAs were removed with Bowtie2 (version and comprehensively interrogated their transcriptome. 2.2.9) with the following settings: –norc, –sensitive-local, –no-unal. Unmapped reads from the rRNA alignment Materials and Methods step were sequentially aligned to human miRNAs, piwi- interacting RNAs (piRNAs), Y RNAs, signal recognition STUDY DESIGN In the identification phase of our studies, 45 plasma particle RNAs (srpRNAs), tRNAs, small nucleolar cfRNA libraries containing 31 HCC, 11 healthy donors (snoRNAs), small nuclear RNAs, vault RNAs, lncRNAs, (HDs), and 3 chronic hepatitis B (CHB) samples (15 messenger RNAs (mRNAs), and transcripts of uncertain CHB individuals pooled into 3 libraries) were sequenced. coding potential. The human piRNA genome sequence Four exosomal RNA (exoRNA) libraries containing 2 was downloaded from piwiRNABank, and the other hu- HCC (10 HCC individuals pooled into 2 libraries) and 2 man genome sequences were downloaded from GEN- HD (10 HD individuals pooled into 2 libraries) were also CODE (Release v27). sequenced. In the quantification phase,