European Review for Medical and Pharmacological Sciences 2020; 24: 1877-1886 Identification of lung adenocarcinoma-specific exosome RNAs in peripheral blood by RNA-Seq analysis X.-Q. LIU1, A. TUFMAN1, R. KIEFL1, G.-F. LI2, Q.-L. MA2, R.M. HUBER1 1Department of Internal Medicine V, Division of Respiratory Medicine and Thoracic Oncology, Ludwig-Maximilians University (LMU), Thoracic Oncology Centre Munich; Comprehensive Pneumology Center Munich, Member of the German Center for Lung Research (DZL), Munich, Bavaria, Germany 2Third Affiliated Hospital of Kunming Medical University, Yunnan Cancer Center. Kunming, Yunnan, China Abstract. – OBJECTIVE: Several plasma-de- carcinoma patients, who were more abundant/ rived exosome RNAs have been identified as key detectable than healthy volunteers. regulators in cancer development. They have CONCLUSIONS: Our data indicate that been considered as potential biomarkers for a plasma-derived exosome RNAs, UGT1A1, non-invasive “liquid biopsy” to diagnose and and BAIAP2L1, as well as the eight isolated assess the progression of cancer. This study pseudogenes could serve as diagnostic and aimed to identify human lung adenocarcino- prognostic biomarkers for an effective non-inva- ma-specific exosome RNAs in peripheral blood, sive “liquid biopsy” of lung adenocarcinomas. while assessing the feasibility and efficiency of this recently developed deep-sequencing tech- Key Words: nology for transcriptome profiling. Lung adenocarcinoma, Pseudogenes, Exosome, PATIENTS AND METHODS: Plasma-derived Biomarkers. exosome RNAs were isolated from 13 lung ad- enocarcinoma patients, 3 patients with benign lung diseases, and 15 healthy volunteers. RNA- seq analysis of ribosomal RNA-depleted total RNA was performed. RNAs differentially ex- Introduction pressed between lung adenocarcinoma and be- nign lung diseases or healthy volunteers were Lung cancer is the predominant cause of identified, followed by GO and KEGG pathway cancer-related death worldwide1. Non-small cell enrichment analyses for the identification of key lung cancer (NSCLC) accounts for approxi- exosome RNAs associated with lung adenocar- mately 80% of all lung cancer cases, main- cinomas. RESULTS: Significant differentially ex ly including adenocarcinomas, squamous cell - 2-4 pressed RNAs, such as UDP glucuronosyl- carcinomas, and large cell carcinomas . In transferase family 1 member A1 (UGT1A1) and spite of great advances in NSCLC therapy, the BAI1-associated protein 2 like 1 (BAIAP2L1), prognosis for NSCLC patients remains poor5. A were identified as differentially expressed be- previous research6 has indicated that the use of tween lung adenocarcinoma patients and pa- biomarkers to guide lung cancer therapies will tients with benign lung diseases. Eight pseudogenes, including Tropomyosin 1 (Al- be beneficial for the treatment of this malignan- pha) Pseudogene (LOC100129096), Prothymo- cy. However, there are no reliable diagnostic and sin, Alpha Pseudogene 2 (PTMAP2), Cell Di- prognostic tools for the detection and identifica- vision Cycle 14C, Pseudogene (CDC14C), Tro- tion of changes in the disease status. Therefore, pomyosin 1 (Alpha) Pseudogene (LOC643634), seeking biomarkers is an important component Ferritin Heavy Chain 1 Pseudogene 2 (FTH1P2), of the lung cancer research. Actin Related Protein 2/3 Complex Subunit 3 Exosomes are 30-100 nm diameter mem- Pseudogene 3 (ARPC3P3), Ferritin Heavy Chain 1 Pseudogene 11 (FTH1P11), and Prothymosin brane-enclosed extracellular vesicles originating 7 Alpha Pseudogene 5 (PTMAP5) were identified from endosomes . Under normal and stressful from plasma-derived exosomes in lung adeno- conditions, almost all cell types can secrete exo- Corresponding Author: Rudolf M. Huber, MD; e-mail: [email protected] 1877 X.-Q. Liu, A. Tufman, R. Kiefl, G.-F. Li, Q.-L. Ma, R.M. Huber somes, and cancer cells are confirmed to pro- amine Tetraacetic Acid (EDTA)-anticoagulated duce more exosomes than normal cells from the blood collection tubes (BD Vacutainer, Plymouth, same organ8. Moreover, exosomes are found to UK). After centrifugation, plasma samples were carry various biological molecules, such as lip- then collected and saved at –80° C. Plasma-de- ids, proteins, and nucleic acid species, including rived exosome RNAs were isolated using the mRNAs, miRNA, lncRNAs, rRNAs, genomic exoRNeasy Serum Plasma Kit (cat. no. 77064; DNA, and pseudogenes8,9. Increasing evidence Qiagen GmbH, Hilden, Germany), which was has shown that those exosomal-derived mole- designed for the rapid purification of total vesic- cules play a crucial role in intercellular signal ular RNAs, including non-coding RNA, mRNA, transduction, immunoregulation, and tumorigen- miRNA, and other small RNAs from serum or esis. Also, these molecules, present in exosomes plasma. Sterility was maintained and all proce- in bodily fluids, are considered promising bio- dures were performed according to the protocols markers for the diagnosis and prognosis of many recommended by the manufacturers. diseases9,10, including a variety of cancers11-13. Authors14,15 have shown that several plasma-de- Construction of the cDNA Library and rived exosome cargoes, such as exosome RNAs High-Throughput Sequencing and proteins, are overexpressed in NSCLC and The rRNA removal reagents in the Ribo-Zero have been suggested as potential biomarkers Gold Kit (Epicentre Biotechnologies, Madison, for a non-invasive “liquid biopsy”. Noteworthy, WI, USA) were used for the rRNA removal. Us- exosomal LRG1 has been identified as a poten- ing the ScriptSeq™ mRNA-Seq Library Prepa- tial urinary biomarker for detecting NSCLC16. ration Kit (Epicentre, Biotechnologies, Madison, Meanwhile, a number of techniques have been WI, USA), the rRNA-depleted RNA was used for used and are under development for exosome the construction of cDNA libraries. The cDNA li- genetic cargo analysis, such as quantitative Real braries were then sequenced on Illumina® GAIIx Time Polymerase Chain Reaction (qRT-PCR), and HiSeq 2000 platforms. microarray analyses, and next-generation se- quencing. Due to the power of RNA sequencing, Preprocessing and Quality Control of which involves a combination of identification Sequencing Data and quantification in a single high-throughput For the retrieval of the clean reads, prepro- sequencing assay, the adoption of this technolo- cessing was performed to remove the adapt- gy has spread quickly. er sequences and the reads with a low quality In the present study, our findings provide a of < 16 were removed. The quality control of theoretical basis for the development of novel clean reads was then conducted using FastQC diagnostic and predictive biomarkers for lung (http://www.bioinformatics.bbsrc.ac.uk/projects/ adenocarcinomas. fastqc/). Moreover, RNA-seq intrinsic biases, such as nucleotide composition bias and GC bias, were assessed. Patients and Methods Sequence Alignment and Patient Samples and Exosome Transcriptome Assembly RNAs Isolation Genomic sequences of various organisms and A total of 13 lung adenocarcinoma cancer pa- their annotation information were deposited in tients with newly diagnosed, untreated, histolog- the UCSC Genome Browser database (http:// ically confirmed lung adenocarcinoma according genome.ucsc.edu)17. Thus, the clean reads were to the World Health Organization classification mapped to the human reference genome (hg19) were enrolled. Three patients with benign lung in the UCSC database using Tophat2.1.0 (http:// diseases and 15 healthy volunteers were recruited tophat.cbcb.umd.edu)18 with default parameters. as controls. All the patients and healthy volunteer According to the results of the sequence align- blood samples came from The Third Affiliated ment, transcript assembly was then performed Hospital of Kunming Medical University, Yun- using StringTie19. The transcript assemblies were nan Cancer Center, China. This investigation further merged using the Cuffmerge tool20. Cuff- was approved by the local Ethics Committee and merge results were compared with the Ensem- all participants gave informed consent. About 10 bl database (http://ensemblgenomes.org/) using ml blood samples were collected in Ethylene Di- Cuffcompare20 to find known and novel RNAs. 1878 Identification of lung adenocarcinoma-specific exosome RNAs Bioinformatics Analysis Notably, LOC100129096, PTMAP2, CDC14C, Differentially expressed RNAs (including LOC643634, FTH1P2, ARPC3P3, FTH1P11, and mRNAs and non-coding RNAs) were identified PTMAP5 were pseudogenes. in lung adenocarcinoma patient plasma-derived Differentially expressed RNAs between lung exosomes using the DESeq2 package21. The cut- adenocarcinoma and patients with benign lung off values were an absolute fold change ≥ 2, with diseases were significantly enriched in the GO p ≤ 0.05. BP functions, associated with steroid metabolic To understand the biological functions of processes, protein activation cascades, and the differentially expressed RNAs, functional en- acute inflammatory response. While, GO CC richment analyses were performed. Gene On- functions related to blood microparticles, the tology (GO, http://www.geneontology.org/)22 cytoplasmic membrane-bounded vesicle lumen, contains a set of terms for the unification and vesicle lumen, and GO MF functions were of the biology of large-scale gene lists. GO associated with monooxygenase activity, steroid terms mainly include cellular component (CC), hydroxylase activity, and iron ion binding (Table molecular function (MF), and biological pro- I, Figure 1). cess (BP).