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当代肿瘤药物靶点

Editor-in-Chief

ISSN (Print): 1568-0096
ISSN (Online): 1873-5576

Research Article

全基因组CRISPR-Cas9筛选和鉴定促进前列腺癌生长和转移的潜在基因

卷 23, 期 1, 2023

发表于: 20 August, 2022

页: [71 - 86] 页: 16

弟呕挨: 10.2174/1568009622666220615154137

价格: $65

摘要

目的:鉴定和验证与前列腺癌生长和转移有关的功能基因。 方法:用全基因组敲除文库包装的慢病毒转染DU145细胞构建DU145- ko细胞系。将DU145-KO细胞移植到免疫功能低下的Nu/Nu小鼠腋窝中,然后分别在接种后第3周(早期肺组织)或第7周(有微转移的晚期肺组织)的肺组织和第7周(晚期原发性肿瘤)的原发肿瘤部位收集DU145-KO细胞。在不同的时间点提取肺转移灶进行DNA测序分析,以确定富集的sgRNAs,从而获得候选基因/miRNAs。进一步的生物信息学分析和有限的功能验证研究被进行。 结果:与对照组(DU145-NC细胞)相比,DU145-KO细胞促进小鼠移植瘤的形成,并促进原发肿瘤的生长和转移。序列数据分析表明,sgRNAs的丰度在原发肿瘤和微转移部位发生了显著变化。我们选取了15个靶基因——c1qtnf9b、FAM229A、hsa-mir-3929、KRT23、TARS2、CRADD、GRIK4、PLA2G15、LOXL1、SLITRK6、CDC42EP5、SLC2A4、PTGDS、MYL9和ACOX2(富集sgRNAs的靶基因)进行实验验证,结果表明,敲除这些基因中的任何一个都会增强DU145细胞的侵袭转移潜能。 结论:全基因组CRISPR-Cas9敲除筛选技术结合高通量测序分析发现了可能与前列腺肿瘤侵袭转移相关的基因。对这些基因的分析提供了与疾病相关的生物途径的见解,并揭示了诊断或预后的创新标志物以及潜在的治疗靶点。

关键词: 全基因组,CRISPR-Cas9, DU145,前列腺癌,转移,筛选。

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[1]
Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics. CA Cancer J. Clin., 2019, 69(1), 7-34.
[http://dx.doi.org/10.3322/caac.21551] [PMID: 30620402]
[2]
Bray, F.; Ferlay, J.; Soerjomataram, I.; Siegel, R.L.; Torre, L.A.; Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin., 2018, 68(6), 394-424.
[http://dx.doi.org/10.3322/caac.21492] [PMID: 30207593]
[3]
Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell, 2011, 144(5), 646-674.
[http://dx.doi.org/10.1016/j.cell.2011.02.013] [PMID: 21376230]
[4]
Zugazagoitia, J.; Guedes, C.; Ponce, S.; Ferrer, I.; Molina-Pinelo, S.; Paz-Ares, L. Current challenges in cancer treatment. Clin. Ther., 2016, 38(7), 1551-1566.
[http://dx.doi.org/10.1016/j.clinthera.2016.03.026] [PMID: 27158009]
[5]
Gebler, C.; Lohoff, T.; Paszkowski-Rogacz, M.; Mircetic, J.; Chakraborty, D.; Camgoz, A.; Hamann, M.V.; Theis, M.; Thiede, C.; Buchholz, F. Inactivation of cancer mutations utilizing CRISPR/Cas9. J. Natl. Cancer Inst., 2016, 109(1)
[PMID: 27576906]
[6]
Barrangou, R.; Fremaux, C.; Deveau, H.; Richards, M.; Boyaval, P.; Moineau, S.; Romero, D.A.; Horvath, P. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 2007, 315(5819), 1709-1712.
[http://dx.doi.org/10.1126/science.1138140] [PMID: 17379808]
[7]
González, F.; Zhu, Z.; Shi, Z.D.; Lelli, K.; Verma, N.; Li, Q.V.; Huangfu, D. An iCRISPR platform for rapid, multiplexable, and inducible genome editing in human pluripotent stem cells. Cell Stem Cell, 2014, 15(2), 215-226.
[http://dx.doi.org/10.1016/j.stem.2014.05.018] [PMID: 24931489]
[8]
Chira, S.; Gulei, D.; Hajitou, A.; Zimta, A.A.; Cordelier, P.; Berindan-Neagoe, I. CRISPR/Cas9: Transcending the reality of genome editing. Mol. Ther. Nucleic Acids, 2017, 7, 211-222.
[http://dx.doi.org/10.1016/j.omtn.2017.04.001] [PMID: 28624197]
[9]
Wiedenheft, B.; Sternberg, S.H.; Doudna, J.A. RNA-guided genetic silencing systems in bacteria and archaea. Nature, 2012, 482(7385), 331-338.
[http://dx.doi.org/10.1038/nature10886] [PMID: 22337052]
[10]
Ishino, Y.; Shinagawa, H.; Makino, K.; Amemura, M.; Nakata, A. Nucleotide sequence of the iap gene, responsible for alkaline phospha-tase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol., 1987, 169(12), 5429-5433.
[http://dx.doi.org/10.1128/jb.169.12.5429-5433.1987] [PMID: 3316184]
[11]
Mojica, F.J.; Juez, G.; Rodríguez-Valera, F. Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites. Mol. Microbiol., 1993, 9(3), 613-621.
[http://dx.doi.org/10.1111/j.1365-2958.1993.tb01721.x] [PMID: 8412707]
[12]
Mojica, F.J.; Díez-Villaseñor, C.; García-Martínez, J.; Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol., 2005, 60(2), 174-182.
[http://dx.doi.org/10.1007/s00239-004-0046-3] [PMID: 15791728]
[13]
Cong, L.; Ran, F.A.; Cox, D.; Lin, S.; Barretto, R.; Habib, N.; Hsu, P.D.; Wu, X.; Jiang, W.; Marraffini, L.A.; Zhang, F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121), 819-823.
[http://dx.doi.org/10.1126/science.1231143] [PMID: 23287718]
[14]
Pourcel, C.; Salvignol, G.; Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology, 2005, 151(Pt 3), 653-663.
[http://dx.doi.org/10.1099/mic.0.27437-0] [PMID: 15758212]
[15]
Bolotin, A.; Quinquis, B.; Sorokin, A.; Ehrlich, S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology, 2005, 151(Pt 8), 2551-2561.
[http://dx.doi.org/10.1099/mic.0.28048-0] [PMID: 16079334]
[16]
Makarova, K.; Slesarev, A.; Wolf, Y.; Sorokin, A.; Mirkin, B.; Koonin, E.; Pavlov, A.; Pavlova, N.; Karamychev, V.; Polouchine, N.; Shakhova, V.; Grigoriev, I.; Lou, Y.; Rohksar, D.; Lucas, S.; Huang, K.; Goodstein, D.M.; Hawkins, T.; Plengvidhya, V.; Welker, D.; Hughes, J.; Goh, Y.; Benson, A.; Baldwin, K.; Lee, J.H.; Díaz-Muñiz, I.; Dosti, B.; Smeianov, V.; Wechter, W.; Barabote, R.; Lorca, G.; Altermann, E.; Barrangou, R.; Ganesan, B.; Xie, Y.; Rawsthorne, H.; Tamir, D.; Parker, C.; Breidt, F.; Broadbent, J.; Hutkins, R.; O’Sullivan, D.; Steele, J.; Unlu, G.; Saier, M.; Klaenhammer, T.; Richardson, P.; Kozyavkin, S.; Weimer, B.; Mills, D. Comparative genomics of the lactic acid bacteria. Proc. Natl. Acad. Sci. USA, 2006, 103(42), 15611-15616.
[http://dx.doi.org/10.1073/pnas.0607117103] [PMID: 17030793]
[17]
Makarova, K.S.; Grishin, N.V.; Shabalina, S.A.; Wolf, Y.I.; Koonin, E.V. A putative RNA-interference-based immune system in prokaryotes: Computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol. Direct, 2006, 1(1), 7.
[http://dx.doi.org/10.1186/1745-6150-1-7] [PMID: 16545108]
[18]
Brouns, S.J.; Jore, M.M.; Lundgren, M.; Westra, E.R.; Slijkhuis, R.J.; Snijders, A.P.; Dickman, M.J.; Makarova, K.S.; Koonin, E.V.; van der Oost, J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science, 2008, 321(5891), 960-964.
[http://dx.doi.org/10.1126/science.1159689] [PMID: 18703739]
[19]
Marraffini, L.A.; Sontheimer, E.J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science, 2008, 322(5909), 1843-1845.
[http://dx.doi.org/10.1126/science.1165771] [PMID: 19095942]
[20]
Wu, W.; Yang, Y.; Lei, H. Progress in the application of CRISPR: From gene to base editing. Med. Res. Rev., 2019, 39(2), 665-683.
[http://dx.doi.org/10.1002/med.21537] [PMID: 30171624]
[21]
Zhang, C.; Quan, R.; Wang, J. Development and application of CRISPR/Cas9 technologies in genomic editing. Hum. Mol. Genet., 2018, 27(R2), R79-R88.
[http://dx.doi.org/10.1093/hmg/ddy120] [PMID: 29659822]
[22]
Ma, H.; Dang, Y.; Wu, Y.; Jia, G.; Anaya, E.; Zhang, J.; Abraham, S.; Choi, J.G.; Shi, G.; Qi, L.; Manjunath, N.; Wu, H. A CRISPR-Based screen identifies genes essential for West-Nile-Virus-Induced cell death. Cell Rep., 2015, 12(4), 673-683.
[http://dx.doi.org/10.1016/j.celrep.2015.06.049] [PMID: 26190106]
[23]
Henser-Brownhill, T.; Monserrat, J.; Scaffidi, P. Generation of an arrayed CRISPR-Cas9 library targeting epigenetic regulators: From high-content screens to in vivo assays. Epigenetics, 2017, 12(12), 1065-1075.
[24]
Wang, T.; Wei, J.J.; Sabatini, D.M.; Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science, 2014, 343(6166), 80-84.
[http://dx.doi.org/10.1126/science.1246981] [PMID: 24336569]
[25]
Zhou, Y.; Zhu, S.; Cai, C.; Yuan, P.; Li, C.; Huang, Y.; Wei, W. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature, 2014, 509(7501), 487-491.
[http://dx.doi.org/10.1038/nature13166] [PMID: 24717434]
[26]
Parnas, O.; Jovanovic, M.; Eisenhaure, T.M.; Herbst, R.H.; Dixit, A.; Ye, C.J.; Przybylski, D.; Platt, R.J.; Tirosh, I.; Sanjana, N.E.; Shalem, O.; Satija, R.; Raychowdhury, R.; Mertins, P.; Carr, S.A.; Zhang, F.; Hacohen, N.; Regev, A. A Genome-wide CRISPR screen in primary immune cells to dissect regulatory networks. Cell, 2015, 162(3), 675-686.
[http://dx.doi.org/10.1016/j.cell.2015.06.059] [PMID: 26189680]
[27]
Alimirah, F.; Chen, J.; Basrawala, Z.; Xin, H.; Choubey, D. DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: Implications for the androgen receptor functions and regulation. FEBS Lett., 2006, 580(9), 2294-2300.
[http://dx.doi.org/10.1016/j.febslet.2006.03.041] [PMID: 16580667]
[28]
Litvinov, I.V.; Antony, L.; Dalrymple, S.L.; Becker, R.; Cheng, L.; Isaacs, J.T. PC3, but not DU145, human prostate cancer cells retain the coregulators required for tumor suppressor ability of androgen receptor. Prostate, 2006, 66(12), 1329-1338.
[http://dx.doi.org/10.1002/pros.20483] [PMID: 16835890]
[29]
Pulukuri, S.M.; Gondi, C.S.; Lakka, S.S.; Jutla, A.; Estes, N.; Gujrati, M.; Rao, J.S. Retraction: RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo. J. Biol. Chem., 2020, 295(37), 13136.
[http://dx.doi.org/10.1074/jbc.RX120.015588] [PMID: 32917829]
[30]
Stone, K.R.; Mickey, D.D.; Wunderli, H.; Mickey, G.H.; Paulson, D.F. Isolation of a human prostate carcinoma cell line (DU 145). Int. J. Cancer, 1978, 21(3), 274-281.
[http://dx.doi.org/10.1002/ijc.2910210305] [PMID: 631930]
[31]
Longo, P.A.; Kavran, J.M.; Kim, M.S.; Leahy, D.J. Transient mammalian cell transfection with polyethylenimine (PEI). Methods Enzymol., 2013, 529, 227-240.
[http://dx.doi.org/10.1016/B978-0-12-418687-3.00018-5] [PMID: 24011049]
[32]
Faustino-Rocha, A.; Oliveira, P.A.; Pinho-Oliveira, J.; Teixeira-Guedes, C.; Soares-Maia, R.; da Costa, R.G.; Colaço, B.; Pires, M.J.; Colaço, J.; Ferreira, R.; Ginja, M. Estimation of rat mammary tumor volume using caliper and ultrasonography measurements. Lab Anim. (NY), 2013, 42(6), 217-224.
[http://dx.doi.org/10.1038/laban.254] [PMID: 23689461]
[33]
Chan, J.K. The wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology. Int. J. Surg. Pathol., 2014, 22(1), 12-32.
[http://dx.doi.org/10.1177/1066896913517939] [PMID: 24406626]
[34]
Chen, G.; Liang, Y.X.; Zhu, J.G.; Fu, X.; Chen, Y.F.; Mo, R.J.; Zhou, L.; Fu, H.; Bi, X.C.; He, H.C.; Yang, S.B.; Wu, Y.D.; Jiang, F.N.; Zhong, W.D. CC chemokine ligand 18 correlates with malignant progression of prostate cancer. BioMed Res. Int., 2014, 2014, 230183.
[http://dx.doi.org/10.1155/2014/230183] [PMID: 25197632]
[35]
Torres-Ruiz, R.; Rodriguez-Perales, S. CRISPR-Cas9: A revolutionary tool for cancer modelling. Int. J. Mol. Sci., 2015, 16(9), 22151-22168.
[http://dx.doi.org/10.3390/ijms160922151] [PMID: 26389881]
[36]
Mou, H.; Smith, J.L.; Peng, L.; Yin, H.; Moore, J.; Zhang, X.O.; Song, C.Q.; Sheel, A.; Wu, Q.; Ozata, D.M.; Li, Y.; Anderson, D.G.; Emer-son, C.P.; Sontheimer, E.J.; Moore, M.J.; Weng, Z.; Xue, W. CRISPR/Cas9-mediated genome editing induces exon skipping by alternative splicing or exon deletion. Genome Biol., 2017, 18(1), 108.
[http://dx.doi.org/10.1186/s13059-017-1237-8] [PMID: 28615073]
[37]
Zuo, E.; Cai, Y.J.; Li, K.; Wei, Y.; Wang, B.A.; Sun, Y.; Liu, Z.; Liu, J.; Hu, X.; Wei, W.; Huo, X.; Shi, L.; Tang, C.; Liang, D.; Wang, Y.; Nie, Y.H.; Zhang, C.C.; Yao, X.; Wang, X.; Zhou, C.; Ying, W.; Wang, Q.; Chen, R.C.; Shen, Q.; Xu, G.L.; Li, J.; Sun, Q.; Xiong, Z.Q.; Yang, H. One-step generation of complete gene knockout mice and monkeys by CRISPR/Cas9-mediated gene editing with multiple sgRNAs. Cell Res., 2017, 27(7), 933-945.
[http://dx.doi.org/10.1038/cr.2017.81] [PMID: 28585534]
[38]
Jiang, F.; Doudna, J.A. CRISPR-Cas9 structures and mechanisms. Annu. Rev. Biophys., 2017, 46(1), 505-529.
[http://dx.doi.org/10.1146/annurev-biophys-062215-010822] [PMID: 28375731]
[39]
Slaymaker, I.M.; Gao, L.; Zetsche, B.; Scott, D.A.; Yan, W.X.; Zhang, F. Rationally engineered Cas9 nucleases with improved specificity. Science, 2016, 351(6268), 84-88.
[http://dx.doi.org/10.1126/science.aad5227] [PMID: 26628643]
[40]
Hartenian, E.; Doench, J.G. Genetic screens and functional genomics using CRISPR/Cas9 technology. FEBS J., 2015, 282(8), 1383-1393.
[http://dx.doi.org/10.1111/febs.13248] [PMID: 25728500]
[41]
Chen, S.; Sanjana, N.E.; Zheng, K.; Shalem, O.; Lee, K.; Shi, X.; Scott, D.A.; Song, J.; Pan, J.Q.; Weissleder, R.; Lee, H.; Zhang, F.; Sharp, P.A. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell, 2015, 160(6), 1246-1260.
[http://dx.doi.org/10.1016/j.cell.2015.02.038] [PMID: 25748654]
[42]
Goodspeed, A.; Jean, A.; Costello, J.C. A whole-genome CRISPR screen identifies a role of MSH2 in cisplatin-mediated cell death in muscle-invasive bladder cancer. Eur. Urol., 2019, 75(2), 242-250.
[http://dx.doi.org/10.1016/j.eururo.2018.10.040] [PMID: 30414698]
[43]
Wang, C.; Jin, H.; Gao, D.; Wang, L.; Evers, B.; Xue, Z.; Jin, G.; Lieftink, C.; Beijersbergen, R.L.; Qin, W.; Bernards, R. A CRISPR screen identifies CDK7 as a therapeutic target in hepatocellular carcinoma. Cell Res., 2018, 28(6), 690-692.
[http://dx.doi.org/10.1038/s41422-018-0020-z] [PMID: 29507396]
[44]
Thiery, J.P.; Acloque, H.; Huang, R.Y.; Nieto, M.A. Epithelial-mesenchymal transitions in development and disease. Cell, 2009, 139(5), 871-890.
[http://dx.doi.org/10.1016/j.cell.2009.11.007] [PMID: 19945376]
[45]
Cho, E.S.; Kang, H.E.; Kim, N.H.; Yook, J.I. Therapeutic implications of cancer epithelial-mesenchymal transition (EMT). Arch. Pharm. Res., 2019, 42(1), 14-24.
[http://dx.doi.org/10.1007/s12272-018-01108-7] [PMID: 30649699]
[46]
Singh, M.; Yelle, N.; Venugopal, C.; Singh, S.K. EMT: Mechanisms and therapeutic implications. Pharmacol. Ther., 2018, 182, 80-94.
[http://dx.doi.org/10.1016/j.pharmthera.2017.08.009] [PMID: 28834698]
[47]
Huang, H. Matrix metalloproteinase-9 (MMP-9) as a cancer biomarker and MMP-9 biosensors: Recent advances. Sensors (Basel), 2018, 18(10), 3249.
[http://dx.doi.org/10.3390/s18103249] [PMID: 30262739]

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