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RESEARCH ARTICLEDOI: 10.4274/tjh.galenos.2019.2019.0312Turk J Hematol 2020;37:104-110Expression Profile Screening and Bioinformatics Analysis ofcircRNA, LncRNA, and mRNA in Acute Myeloid Leukemia Drug-Resistant CellsAkut Myeloid Lösemi İlaç-Dirençli Hücrelerde circRNA, LncRNA, ve mRNA EkspresyonProfili Tarama ve Biyoinformatik AnaliziMeiling Li 1,2 , Fuxue Meng 2 , Quanyi Lu 11Zhongshan Hospital Affiliated to Xiamen University, Department of Hematology, Xiamen, China2The Third Affiliated Hospital of Guizhou Medical University, Department of Hematology and Rheumatology, Duyun, ChinaAbstractObjective: Acute myeloid leukemia (AML) is a highly heterogeneoushematological malignancy, and drug resistance and relapse arekey factors in the failure of leukemia treatment. Studies haveincreasingly shown that circRNA and LncRNA play important rolesin the development of tumors, but their roles remain unclear in themechanism of AML resistance.Materials and Methods: Resistant AML cell line HL-60/ADM(adriamycin, ADM) was constructed and circRNA, LncRNA, and mRNAexpression profiles were screened followed by high-throughputsequencing. Bioinformatics analysis was then carried out, and thecircRNA-miRNA ceRNA network was constructed and confirmed usingqRT-PCR analysis.Results: A total of 1824 circRNAs, 2414 LncRNAs, and 5346 mRNAswere screened for differentially expressed genes. Enrichment analysiswas performed utilizing Gene Ontology and the Kyoto Encyclopedia ofGenes and Genomes, which mainly involved protein domain specificbinding, transforming growth factor-β (TGF-β) receptor, and cellularmetabolism. The mTOR signaling pathway, MAPK signaling pathway,RAP1 signaling pathway, and Akt signaling pathway were closelyrelated to drug resistance.Conclusion: Our study provides a systematic outlook on the potentialfunction of ncRNA in the molecular mechanisms of resistant AML cells.Hsa-circ-0000978 and hsa-circ-0000483 might serve as potentialprognostic biomarkers and therapeutic targets of AML resistance.Keywords: Acute myeloid leukemia, Drug resistance, CircRNA, LncRNA,Bioinformatics analysisÖzAmaç: Akut myeloid lösemi (AML) oldukça heterojen bir hematolojikmalignitedir, ve tedavi başarısızlığında ilaç direnci ve nüks anahtar roloynamaktadır. Çalışmalar, circRNA ve lncRNA’nın tümör gelişimindeönemli rol oynadığını artarak göstermektedir, ancak AML dirençmekanizmasında rolleri belirsizliğini korumaktadır.Gereç ve Yöntemler: Dirençli AML hücre hattı HL-60/ADM (adriamisin,ADM) oluşturuldu ve circRNA, LncRNA, ve mRNA ekspresyon profilleriyüksek-kapasitede dizileme sonrası tarandı. Sonra biyoinformatikanaliz gerçekleştirildi ve circRNA-miRNA ceRNA ağı oluşturuldu veqRT-PCR analizi kullanılarak doğrulandı.Bulgular: Farklı ifade edilen genler için toplam 1824 circRNA, 2414LncRNA, ve 5346 mRNA tarandı. Başlıca protein domain spesifikbağlama, transforme edici büyüme faktörü-β (TGF-β) reseptörü, vehücresel metabolizma ile ilgili ‘Gene Ontology’ ve ‘Kyoto Encyclopediaof Genes and Genome’ kullanılarak zenginleştirme analizigerçekleştirildi. mTOR sinyal yolağı, MAPK sinyal yolağı, RAP1 sinyalyolağı ve Akt sinyal yolağı ilaç direnci ile yakından ilişkili idi.Sonuç: Çalışmamız, dirençli AML hücrelerinin molekülermekanizmasında ncRNA’ın potansiyel fonksiyonuna sistematik birbakış açısı sağlamıştır. Hsa-circ-0000978 ve hsa-circ-0000483potansiyel prognostik biyogöstergeler ve AML direncinin terapötikhedefleri olarak işlev görebilirler.Anahtar Sözcükler: Akut myeloid lösemi, İlaç direnci, CircRNA,LncRNA, Biyoinformatik analiz©Copyright 2020 by Turkish Society of HematologyTurkish Journal of Hematology, Published by Galenos Publishing HouseAddress for Correspondence/Yazışma Adresi: Quanyi Lu, M.D., Zhongshan Hospital Affiliated to XiamenUniversity, Department of Hematology, Xiamen, ChinaPhone : 86-18375129409E-mail : wzst666@126.com ORCID: orcid.org/0000-0001-7173-6080Received/Geliş tarihi: August 22, 2019Accepted/Kabul tarihi: December 3, 2019104
Turk J Hematol 2020;37:104-110Li M, et al: Expression Profile of Resistant Leukemia CellsIntroductionAcute myeloid leukemia (AML) is a highly heterogeneoushematological malignancy. Although its treatment has madesignificant progress, the prognosis is still unsatisfactory.Recurrence and drug resistance are the main factors [1]. Atpresent, there are many studies on the molecular mechanismsof AML resistance [2,3]. However, with the development ofbioinformatics, the epigenetic mechanism in the pathogenesisof AML still remains unclear.Among human transcripts, about 10%-20% are proteinencodingRNA, and the remaining 80%-90% are noncodingRNAs (ncRNAs) [4,5]. Long noncoding RNAs (LncRNAs) are aclass of noncoding RNAs that regulate gene expression at thetranscriptional or posttranscriptional level [6]. LncRNA playsan important regulatory role in the drug resistance process. Liet al. [7] reported that the LncRNA HOTTIP can promote thedevelopment of pancreatic cancer and regulate gemcitabineresistance by regulating HOXA13, while HOTTIP regulatescisplatin resistance in osteosarcoma cells by activating theWnt/β-catenin pathway [8]. In addition, Qu et al. [9] found thatin sunitinib-resistant renal cell carcinoma, when FOX0 and AKTexpression decreased, LncRNA increased, knocking out LncRNAand then reversing drug resistance. Endogenous competitionbetween mir-34 and mir-449 promotes the expression of AXLand c-MET in sunitinib-resistant renal cell carcinoma to regulatethe drug resistance process, confirming that LncRNA can be usedas a target to repair drug resistance. Circular RNAs (circRNAs)are novel noncoding RNAs characterized by a covalently closedstructure with nonrandom spiking and RNase degradationresistance [10,11]. CircRNA is present in the cytoplasm and isextremely abundant and highly conserved and stable in theblood [12]. CircRNAs are increasingly found in various diseasesand show cell or tissue specificity [13,14,15].At present, the molecular mechanism of LncRNA and circRNAin resistant AML cells remains unclear. In this study, highthroughputsequencing of HL-60 and HL-60/ADM (adriamycin,ADM) was performed utilizing Gene Ontology (GO) and theKyoto Encyclopedia of Genes and Genomes (KEGG). A circRNAmiRNAceRNA network was constructed to provide newtherapeutic targets and the theoretical basis for treatment ofdrug resistance in AML.Materials and MethodsMaterialsHL-60 cells were donated by Professor Lu Quanyi of the KeyLaboratory of Hematology, Xiamen University, Xiamen, China.Basic RPMI 1640 Medium (GIBCO, Carlsbad, CA, USA), fetalbovine serum (FBS; ScienCell, Carlsbad, CA, USA), adriamycin(Haizhenghuirui Pharmaceutical Co. Ltd., Fuyang, Zhejiang,China), the Cell Counting Kit-8 (Dojindo, Tokyo, Japan), RIPAbuffer (Beijing Solarbio Science & Technology Co. Ltd., Beijing,China), the BCA Protein Assay Kit and One-Step Western Kit HRP(Beijing Kangwei Century Biotechnology Co. Ltd., Beijing, China),GAPDH monoclonal antibody (ImmunoWay Biotechnology Co,Plano, TX, USA); P-gp monoclonal antibody (Abcam, Cambridge,MA, USA), and Immobilon Western Chemiluminescent HRPSubstrate (Millipore Corp., Billerica, MA, USA) were also obtained.MethodsCell Culture and Drug Resistance InductionHL-60 cells were incubated in basic RPMI 1640 Medium containing10% FBS, 100 µg/mL streptomycin, and 100 U/mL penicillin at37 °C and 5% CO 2under saturated humidity conditions afterrecovery. The liquid was changed once every 2 days, and 10 6cells/mL were amplified at a 1:3 ratio. ADM induction wasperformed in HL-60 cells by combining the concentrationgradient increasing method and the impact method (high-doseintermittent induction) as referenced in the literature [16]. Theinitial induction concentration was 0.1 µg/mL, shock inductionwas performed for 1 h, and culturing was continued until HL-60cells grew and proliferated normally at 1 µg/mL ADM. It took 8months to successfully induce ADM resistance in HL-60 cells.CCK-8 Assay and Cell IC 50ValuesHL-60 cells were collected and centrifuged at 1000 rpm/min for5 min, and then they were resuspended in RPMI 1640 Mediumand counted. Furthermore, 10 4 cells of cell suspensions of 100µL were placed in 96-well plates at 37 °C in a 5% CO 2incubatorfor culturing for 24 h. ADM was added with differences inconcentrations of 10 µL and cells were incubated for 24 h, andthen 10 µL of CCK-8 solution was added to each well. Cultureplates were further incubated in the incubator for 4 h. OD valueswere measured and data were collected to calculate IC 50values,or ADM concentrations required for 50% inhibition in vitro.Western Blot Detection of the Expression of Drug-ResistantProteinRIPA buffer was added with phenylmethanesulfonyl fluorideto collect the cells showing logarithmic growth, and proteinswere extracted from the cells. Protein concentration wasdetermined using the BCA Protein Assay Kit. Protein samplescontaining sample buffer were denatured for 5 min in boilingwater. SDS-PAGE electrophoresis was performed with 25 µg ofsample in each hole with the addition of 5 µL of prestainedprotein marker. When the bromophenol blue dye ran off thegel layer, the electrophoresis was terminated, and furtherexperiments were performed on a 35 mA transmembraneovernight. The One-Step Western Kit HRP was used according tothe manufacturer’s instructions. Rabbit P-gp antibody, antibodypretreatment solution, and dilution buffer solution were added,105
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