Abstract
Micro RNAs (miRNAs) are small RNAs processed from longer precursor RNA transcripts that can fold back on themselves to form Watson-Crick paired hairpin structures. Once processed from the longer molecule, the small RNA is much too short to code for proteins but can play other very important roles, like gene regulation. The phenomenon of RNA interference was initially observed by Napoli and Jorgensen in transgenic petunia flowers, where gene suppression was observed after introducing a transgene of chalcone synthase (CHS) belonging to the flavonoid biosynthesis pathway. miRNAs were first discovered for their roles in development but it has quickly become evident that they have causal roles in cancer as well. miRNA can also be used to manipulate genes for the investigation of carcinogenesis. Single-cell transcriptome profiling studies in our laboratory suggest that carcinogenesis often is the result of the malfunction of multiple members of a molecular pathway. Here, we describe a protocol to manipulate multiple cancer-related genes in a single human cell to investigate how multiple genes interact during carcinogenesis.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Napoli C, Lemieux C, Jorgensen R (1990) Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-supression of homologous genes in trans. Plant Cell 2:279–289
Romano N, Macino G (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6(22):3343–3353
Kim VN (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat Rev Mol Cell Biol 6(5):376–385
Parmar R, Willoughby JL, Liu J, Foster DJ, Brigham B, Theile CS, Charisse K, Akinc A, Guidry E, Pei Y, Strapps W, Cancilla M, Stanton MG, Rajeev KG, Sepp-Lorenzino L, Manoharan M, Meyers R, Maier MA, Jadhav V (2016) 5’-(E)-Vinylphosphonate: a stable phosphate mimic can improve the RNAi activity of siRNA-GalNAc conjugates. Chembiochem 17:985–989
Lingel A, Simon B, Izaurralde E, Sattler M (2004) Nucleic acid 3’-end recognition by the Argonaute2 PAZ domain. Nat Struct Mol Biol 11:576–577
Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305:1437–1441
Rivas FV, Tolia NH, Song JJ, Aragon JP, Liu J, Hannon GJ, Joshua-Tor L (2005) Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol 12:340–349
MacRae IJ, Ma E, Zhou M, Robinson CV, Doudna JA (2008) In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci U S A 105:512–517
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Macfarlane LA, Murphy PR (2010) MicroRNA: biogenesis, function and role in cancer. Curr Genomics 11:537–561
Cheng CY, Hwang CI, Corney DC, Flesken-Nikitin A, Jiang L, Oner GM, Munroe RJ, Schimenti JC, Hermeking H, Nikitin AY (2014) miR-34 cooperates with p53 in suppression of prostate cancer by joint regulation of stem cell compartment. Cell Rep 6:1000–1007
Kim NH, Kim HS, Kim NG, Lee I, Choi HS, Li XY, Kang SE, Cha SY, Ryu JK, Na JM, Park C, Kim K, Lee S, Gumbiner BM, Yook JI, Weiss SJ (2011) p53 and microRNA-34 are suppressors of canonical Wnt signaling. Sci Signal 4:ra71
Zhang DG, Zheng JN, Pei DS (2014) P53/microRNA-34-induced metabolic regulation: new opportunities in anticancer therapy. Mol Cancer 13:115
Achari C, Winslow S, Ceder Y, Larsson C (2014) Expression of miR-34c induces G2/M cell cycle arrest in breast cancer cells. BMC Cancer 14:538
Zhang YH, Wang QQ, Li H, Ye T, Gao F, Liu YC (2016) miR-124 radiosensitizes human esophageal cancer cell TE-1 by targeting CDK4. Genet Mol Res 15:2–10
Jain CK, Gupta A, Dogra N, Kumar VS, Wadhwa G, Sharma SK (2014) MicroRNA therapeutics: the emerging anticancer strategies. Recent Pat Anticancer Drug Discov 9:286–296
Peek AS, Behlke MA (2007) Design of active small interfering RNAs. Curr Opin Mol Ther 9:110–118
Freshney RI (2016) Culture of animal cells: a manual of basic technique and specialized applications. Wiley-Blackwell, Hoboken, NJ
Landen CN, Chavez-Reyes A, Bucana C, Schmandt R, Deavers MT, Lopez-Berestein G, Sood AK (2005) Therapeutic Eph A2 gene targeting in vivo using neutral liposomal small interfering RNA delivery. Cancer Res 65:6910–6918
Mahmood T, Yang PC (2012) Western blot: technique, theory, and trouble shooting. N Am J Med Sci 4:429–434
Rao DD, Vorhies JS, Senzer N, Nemunaitis J (2009) siRNA vs. shRNA: similarities and differences. Adv Drug Deliv Rev 61:746–759
Acknowledgments
This work was supported by grants R01CA197903 and R01CA1645093 from the National Institutes of Health, USA (J.F.Z.), and CHE1213161 from the National Science Foundation USA (J.F.Z.), and an internal grant from the University of Southern California (J.F.Z. and P.P.S.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Stucky, A., Chen, X., Zhong, J.F. (2018). Gene Manipulation with Micro RNAs at Single-Human Cancer Cell. In: Ying, SY. (eds) MicroRNA Protocols . Methods in Molecular Biology, vol 1733. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7601-0_18
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7601-0_18
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7600-3
Online ISBN: 978-1-4939-7601-0
eBook Packages: Springer Protocols