Please use this identifier to cite or link to this item:
標題: 利用IP-SELEX篩選HLJ1特異性適體及其功能探討
Functional characterization of specific HLJ1 aptamer by IP-SELEX
作者: 陳之容
Chih-Jung Chen
關鍵字: 非小細胞肺癌;HLJ1;介白素-8;適體;免疫沉澱法結合配體指數增強系統進化技術;NSCLC;HLJ1;Interleukin 8;aptamer;IP-SELEX
引用: 蔡銘珈: 腫瘤抑制基因HLJ1經由類鐸受體二之訊息傳遞路徑調控介白素八之表現。國立中興大學生物醫學研究所。碩士學位論文 (2006)。 Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomised clinical trials. Non-small Cell Lung Cancer Collaborative Group. BMJ 1995;311:899-909. Amini A, Yeh N, Gaspar LE, Kavanagh B, Karam SD: Stereotactic body radiation therapy (SBRT) for lung cancer patients previously treated with conventional radiotherapy: a review. Radiat Oncol 2014;9:210. Andrews S: FastQC: a quality control tool for high throughput sequence data. . Available online at: http://wwwbioinformaticsbabrahamacuk/projects/fastqc 2010. Baghirova S, Hughes BG, Hendzel MJ, Schulz R: Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells. MethodsX 2015;2:440-445. Bernhart SH, Hofacker IL, Will S, Gruber AR, Stadler PF: RNAalifold: improved consensus structure prediction for RNA alignments. BMC Bioinformatics 2008;9:474. Blind M, Blank M: Aptamer Selection Technology and Recent Advances. Mol Ther Nucleic Acids 2015;4:e223. Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ: Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 1992;355:564-566. Brahmer JR: Harnessing the immune system for the treatment of non-small-cell lung cancer. J Clin Oncol 2013;31:1021-1028. Butt AQ, Mills KH: Immunosuppressive networks and checkpoints controlling antitumor immunity and their blockade in the development of cancer immunotherapeutics and vaccines. Oncogene 2014;33:4623-4631. Cerchia L, de Franciscis V: Targeting cancer cells with nucleic acid aptamers. Trends Biotechnol 2010;28:517-525. Chang YC, Kao WC, Wang WY, Wang WY, Yang RB, Peck K: Identification and characterization of oligonucleotides that inhibit Toll-like receptor 2-associated immune responses. FASEB J 2009;23:3078-3088. Chen C, Zhou S, Cai Y, Tang F: Nucleic acid aptamer application in diagnosis and therapy of colorectal cancer based on cell-SELEX technology. NPJ Precis Oncol 2017;1:37. Dela Cruz CS, Tanoue LT, Matthay RA: Lung cancer: epidemiology, etiology, and prevention. Clin Chest Med 2011;32:605-644. Detterbeck FC, Boffa DJ, Kim AW, Tanoue LT: The Eighth Edition Lung Cancer Stage Classification. Chest 2017;151:193-203. Edgar RC: Search and clustering orders of magnitude faster than BLAST. Bioinformatics (Oxford, England) 2010;26:2460-2461. El-Kebir M, Satas G, Raphael BJ: Inferring parsimonious migration histories for metastatic cancers. Nat Genet 2018. El-Zein RA, Abdel-Rahman S, Santee KJ, Yu R, Shete S: Identification of Small and Non-Small Cell Lung Cancer Markers in Peripheral Blood Using Cytokinesis-Blocked Micronucleus and Spectral Karyotyping Assays. Cytogenet Genome Res 2017;152:122-131. Elle IC, Karlsen KK, Terp MG, Larsen N, Nielsen R, Derbyshire N, Mandrup S, Ditzel HJ, Wengel J: Selection of LNA-containing DNA aptamers against recombinant human CD73. Mol Biosyst 2015;11:1260-1270. Ettinger DS, Wood DE, Aisner DL, Akerley W, Bauman J, Chirieac LR, D'Amico TA, DeCamp MM, Dilling TJ, Dobelbower M, Doebele RC, Govindan R, Gubens MA, Hennon M, Horn L, Komaki R, Lackner RP, Lanuti M, Leal TA, Leisch LJ, Lilenbaum R, Lin J, Loo BW, Jr., Martins R, Otterson GA, Reckamp K, Riely GJ, Schild SE, Shapiro TA, Stevenson J, Swanson SJ, Tauer K, Yang SC, Gregory K, Hughes M: Non-Small Cell Lung Cancer, Version 5.2017, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2017;15:504-535. Fidler IJ: The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nat Rev Cancer 2003;3:453-458. Francisco LM, Salinas VH, Brown KE, Vanguri VK, Freeman GJ, Kuchroo VK, Sharpe AH: PD-L1 regulates the development, maintenance, and function of induced regulatory T cells. J Exp Med 2009;206:3015-3029. Georganas C, Liu H, Perlman H, Hoffmann A, Thimmapaya B, Pope RM: Regulation of IL-6 and IL-8 expression in rheumatoid arthritis synovial fibroblasts: the dominant role for NF-kappa B but not C/EBP beta or c-Jun. J Immunol 2000;165:7199-7206. Greenwald RJ, Freeman GJ, Sharpe AH: The B7 family revisited. Annu Rev Immunol 2005;23:515-548. Guo KT, Ziemer G, Paul A, Wendel HP: CELL-SELEX: Novel perspectives of aptamer-based therapeutics. Int J Mol Sci 2008;9:668-678. Hiller M, Pudimat R, Busch A, Backofen R: Using RNA secondary structures to guide sequence motif finding towards single-stranded regions. Nucleic Acids Res 2006;34:e117. Hoinka J, Zotenko E, Friedman A, Sauna ZE, Przytycka TM: Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers. Bioinformatics 2012;28:i215-223. Howington JA, Blum MG, Chang AC, Balekian AA, Murthy SC: Treatment of stage I and II non-small cell lung cancer: Diagnosis and management of lung cancer, 3rd ed: American College of Chest Physicians evidence-based clinical practice guidelines. Chest 2013;143:e278S-e313S. Janssen S, Giegerich R: The RNA shapes studio. Bioinformatics 2015;31:423-425. Keir ME, Butte MJ, Freeman GJ, Sharpe AH: PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677-704. Kulbachinskiy AV: Methods for selection of aptamers to protein targets. Biochemistry (Mosc) 2007;72:1505-1518. Kusser W: Chemically modified nucleic acid aptamers for in vitro selections: evolving evolution. J Biotechnol 2000;74:27-38. Kuwahara M, Sugimoto N: Molecular evolution of functional nucleic acids with chemical modifications. Molecules 2010;15:5423-5444. Lakhin AV, Tarantul VZ, Gening LV: Aptamers: problems, solutions and prospects. Acta Naturae 2013;5:34-43. Lao YH, Phua KK, Leong KW: Aptamer nanomedicine for cancer therapeutics: barriers and potential for translation. ACS Nano 2015;9:2235-2254. Lastwika KJ, Wilson W, 3rd, Li QK, Norris J, Xu H, Ghazarian SR, Kitagawa H, Kawabata S, Taube JM, Yao S, Liu LN, Gills JJ, Dennis PA: Control of PD-L1 Expression by Oncogenic Activation of the AKT-mTOR Pathway in Non-Small Cell Lung Cancer. Cancer Res 2016;76:227-238. Liu J, You M, Pu Y, Liu H, Ye M, Tan W: Recent developments in protein and cell-targeted aptamer selection and applications. Curr Med Chem 2011;18:4117-4125. Mallikaratchy PR, Ruggiero A, Gardner JR, Kuryavyi V, Maguire WF, Heaney ML, McDevitt MR, Patel DJ, Scheinberg DA: A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia. Nucleic Acids Res 2011;39:2458-2469. Marzec M, Zhang Q, Goradia A, Raghunath PN, Liu X, Paessler M, Wang HY, Wysocka M, Cheng M, Ruggeri BA, Wasik MA: Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1). Proc Natl Acad Sci U S A 2008;105:20852-20857. McCarthy WJ, Meza R, Jeon J, Moolgavkar SH: Chapter 6: Lung cancer in never smokers: epidemiology and risk prediction models. Risk Anal 2012;32 Suppl 1:S69-84. Milla P, Dosio F, Cattel L: PEGylation of proteins and liposomes: a powerful and flexible strategy to improve the drug delivery. Curr Drug Metab 2012;13:105-119. Miller KD, Siegel RL, Lin CC, Mariotto AB, Kramer JL, Rowland JH, Stein KD, Alteri R, Jemal A: Cancer treatment and survivorship statistics, 2016. CA Cancer J Clin 2016;66:271-289. Pardoll DM: The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012;12:252-264. Pasut G, Veronese FM: State of the art in PEGylation: the great versatility achieved after forty years of research. J Control Release 2012;161:461-472. Pauken KE, Wherry EJ: Overcoming T cell exhaustion in infection and cancer. Trends Immunol 2015;36:265-276. Pestourie C, Cerchia L, Gombert K, Aissouni Y, Boulay J, De Franciscis V, Libri D, Tavitian B, Duconge F: Comparison of different strategies to select aptamers against a transmembrane protein target. Oligonucleotides 2006;16:323-335. Phillips JA, Lopez-Colon D, Zhu Z, Xu Y, Tan W: Applications of aptamers in cancer cell biology. Anal Chim Acta 2008;621:101-108. Prodeus A, Abdul-Wahid A, Fischer NW, Huang EH, Cydzik M, Gariepy J: Targeting the PD-1/PD-L1 Immune Evasion Axis With DNA Aptamers as a Novel Therapeutic Strategy for the Treatment of Disseminated Cancers. Mol Ther Nucleic Acids 2015;4:e237. Ramalingam S, Belani C: Systemic chemotherapy for advanced non-small cell lung cancer: recent advances and future directions. Oncologist 2008;13 Suppl 1:5-13. Rudd CE, Taylor A, Schneider H: CD28 and CTLA-4 coreceptor expression and signal transduction. Immunol Rev 2009;229:12-26. Saintigny P, Burger JA: Recent advances in non-small cell lung cancer biology and clinical management. Discov Med 2012;13:287-297. Shepherd FA, Douillard JY, Blumenschein GR, Jr.: Immunotherapy for non-small cell lung cancer: novel approaches to improve patient outcome. J Thorac Oncol 2011;6:1763-1773. Siegel RL, Miller KD, Jemal A: Cancer Statistics, 2017. CA Cancer J Clin 2017;67:7-30. Smith C, Heyne S, Richter AS, Will S, Backofen R: Freiburg RNA Tools: a web server integrating INTARNA, EXPARNA and LOCARNA. Nucleic Acids Res 2010;38:W373-377. Soria JC, Marabelle A, Brahmer JR, Gettinger S: Immune checkpoint modulation for non-small cell lung cancer. Clin Cancer Res 2015;21:2256-2262. Srivastava N, McDermott D: Update on benefit of immunotherapy and targeted therapy in melanoma: the changing landscape. Cancer Manag Res 2014;6:279-289. Sundar R, Soong R, Cho BC, Brahmer JR, Soo RA: Immunotherapy in the treatment of non-small cell lung cancer. Lung Cancer 2014;85:101-109. Tabarzad M, Kazemi B, Vahidi H, Aboofazeli R, Shahhosseini S, Nafissi-Varcheh N: Challenges to design and develop of DNA aptamers for protein targets. I. Optimization of asymmetric PCR for generation of a single stranded DNA library. Iran J Pharm Res 2014;13:133-141. Tan L, Neoh KG, Kang ET, Choe WS, Su X: PEGylated anti-MUC1 aptamer-doxorubicin complex for targeted drug delivery to MCF7 breast cancer cells. Macromol Biosci 2011;11:1331-1335. Tan L, Neoh KG, Kang ET, Choe WS, Su X: Affinity analysis of DNA aptamer-peptide interactions using gold nanoparticles. Anal Biochem 2012;421:725-731. Tang Z, Shangguan D, Wang K, Shi H, Sefah K, Mallikratchy P, Chen HW, Li Y, Tan W: Selection of aptamers for molecular recognition and characterization of cancer cells. Anal Chem 2007;79:4900-4907. Topalian SL, Taube JM, Anders RA, Pardoll DM: Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat Rev Cancer 2016;16:275-287. Tuerk C, Gold L: Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 1990;249:505-510. Vesely MD, Kershaw MH, Schreiber RD, Smyth MJ: Natural innate and adaptive immunity to cancer. Annu Rev Immunol 2011;29:235-271. Woo EY, Yeh H, Chu CS, Schlienger K, Carroll RG, Riley JL, Kaiser LR, June CH: Cutting edge: Regulatory T cells from lung cancer patients directly inhibit autologous T cell proliferation. J Immunol 2002;168:4272-4276. Wu Y, Sefah K, Liu H, Wang R, Tan W: DNA aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells. Proc Natl Acad Sci U S A 2010;107:5-10. Xiao Z, Shangguan D, Cao Z, Fang X, Tan W: Cell-specific internalization study of an aptamer from whole cell selection. Chemistry 2008;14:1769-1775. Xing H, Tang L, Yang X, Hwang K, Wang W, Yin Q, Wong NY, Dobrucki LW, Yasui N, Katzenellenbogen JA, Helferich WG, Cheng J, Lu Y: Selective Delivery of an Anticancer Drug with Aptamer-Functionalized Liposomes to Breast Cancer Cells in Vitro and in Vivo. J Mater Chem B 2013;1:5288-5297. Zappa C, Mousa SA: Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res 2016;5:288-300. Zhang Y, Huang S, Gong D, Qin Y, Shen Q: Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer. Cell Mol Immunol 2010;7:389-395. Zhong R, Liu L, Zou L, Zhu Y, Chen W, Zhu B, Shen N, Rui R, Long L, Ke J, Lu X, Zhang T, Zhang Y, Wang Z, Liu L, Sun Y, Cheng L, Miao X: Genetic variations in TERT-CLPTM1L locus are associated with risk of lung cancer in Chinese population. Mol Carcinog 2013;52 Suppl 1:E118-126. Zhou J, Rossi J: Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov 2017;16:440. Zhu Q, Liu G, Kai M: DNA Aptamers in the Diagnosis and Treatment of Human Diseases. Molecules 2015;20:20979-20997. Zou W, Chen L: Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol 2008;8:467-477. Zuker M: On finding all suboptimal foldings of an RNA molecule. Science 1989;244:48-52.
在實驗室先前研究中發現屬於熱休克蛋白40家族成員的Human Liver DnaJ-like (HLJ1) 是腫瘤生長及轉移的抑制基因,在細胞株中HLJ1大量表現能抑制癌細胞增生 (proliferation)、聚落形成 (colony formation) 、侵襲 (invasion) 與移動 (migration) 能力。此外,在過去研究中也發現,非小細胞肺癌 (non-small cell lung cancer,NSCLC) 中HLJ1蛋白大量表現會促使下游途徑的介白素-8 (interleukin 8,IL-8) 表現增加,因此推測HLJ1蛋白可能參與免疫發炎反應。本研究建立指數式擴增配體的系統進化技術與免疫沉澱法做結合 (immunoprecipitation-coupled systematic evolution of ligands by exponential enrichment, IP-SELEX),篩選能夠辨認HLJ1的適體,探討若將HLJ1阻斷後是否會抑制IL-8的表現及對於癌細胞侵襲與移動能力之影響。首先,利用IP-SELEX技術篩選後,透過次世代定序並結合生物資訊分析的方式分析篩選結果獲得HLJ1候選適體序列,進一步藉由免疫沉澱法確認HLJ1適體能夠專一性結合目標蛋白HLJ1。此外,將HLJ1適體轉染至大量表現HLJ1蛋白的肺癌細胞株後,結果顯示HLJ1蛋白受到抑制時會使癌細胞的侵襲及移動能力增加。由即時定量聚合酶連鎖反應分析在IL-8 mRNA層次上的改變,也發現HLJ1蛋白會增加IL-8 mRNA的表現,且同時加入HLJ1適體後,IL-8 mRNA的表現會受到抑制。另外,共轉染 (co-transfection) HLJ1及HLJ1適體至癌細胞中,經由冷光報導基因分析結果證實HLJ1適體會抑制IL-8表現。綜合上述結果證實本研究所篩選出之HLJ1適體具有與HLJ1蛋白的專一性結合能力及拮抗能力。

Our previous studies have shown that Human Liver DnaJ-like protein (HLJ1), which belongs to the Hsp40 family, plays the role of a tumor suppressor gene in non-small cell lung cancer (NSCLC). Overexpression of HLJ1 could inhibit cancer cell proliferation, colony formation, invasion and migration. In addition, HLJ1 could induce Interleukin 8 (IL-8) expression in lung cancer, which implied it may be involved in the immune inflammatory response. This study is aimed to use the immunoprecipitation-coupled systematic evolution of ligands by exponential enrichment (IP-SELEX) to screen HLJ1-specific aptamer, and further to investigate whether cell motility and IL-8 induction would be altered by the HLJ1 aptamer. After IP-SELEX screening, the HLJ1 candidate aptamers were identified through next-generation sequencing and bioinformatics analysis. Immunoprecipitation and pull down assay showed that the aptamer can specifically bind to HLJ1. Furthermore, the invasion and migration ability of lung cancer cell transfected with HLJ1 aptamer were increased. Transfecting HLJ1 aptamer into cells could also down-regulate IL-8 mRNA expression as measured by real-time PCR. In addition, IL-8 promoter reporter construct, HLJ1 expression vector and HLJ1 aptamer were co-transfected into lung cancer cell line and indicated that HLJ1 aptamer decreased IL-8 promoter transcriptional activity. In conclusion, our results indicated that the identified HLJ1 aptamer might be through binding to HLJ1 to reverse the original function of HLJ1 in NSCLC.
Rights: 同意授權瀏覽/列印電子全文服務,2021-08-07起公開。
Appears in Collections:分子生物學研究所

Files in This Item:
File SizeFormat Existing users please Login
nchu-107-7104055016-1.pdf3.64 MBAdobe PDFThis file is only available in the university internal network   
Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.