Gene Manipulation with Micro RNAs at Single-Human Cancer Cell
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.
Key wordsGene manipulation Cancer Single-cell
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.).
- 4.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 17:985–989Google Scholar
- 5.Lingel A, Simon B, Izaurralde E, Sattler M (2004) Nucleic acid 3’-end recognition by the Argonaute2 PAZ domain. Nat Struct Mol Biol 11:576–577Google Scholar
- 15.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 15:2–10Google Scholar
- 18.Freshney RI (2016) Culture of animal cells: a manual of basic technique and specialized applications. Wiley-Blackwell, Hoboken, NJGoogle Scholar