Abstract
Wnts are secreted lipid-modified signaling proteins. Activation of Wnt signalling in many tissues has also been associated with cancer. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study we used pAVU6+27 vectors, which have SalI and XbaI clone sites, to construct the siRNA expression vectors for human Wnt9a. Two kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that in the cells, transfected with U6+27 cassettes with anti-Wnt9a hairpin siRNA inserts, dramatically reduced Wnt9a signals were observed as compared to the untransfected cells. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the human Wnt9a in MCF-7 cells by RNAi, but was inhibited after over-expression of human Wnt9a. These results suggests the expression level of human Wnt9a in MCF-7 that breast cancer may play a role in adjusting the rate of cellular proliferation.
Similar content being viewed by others
References
Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR, Nusse R (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423:448–452
Nelson WJ, Nusse R (2004) Convergence of Wnt, catenin, and cadherin pathways. Science 303:1483–1487
Cadigan KM, Nusse R (1997) Wnt signaling: a common theme in animal development. Genes Dev 11:3286–3305
Jamora C, Fuchs E (2002) Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol 4:101–108
Reya1 T, Clevers H (2005) Wnt signalling in stem cells and cancer. Nature 434:843–850
Polakis P (2000) Wnt signaling and cancer. Genes Dev 14:1837–1851
Thiery JP (2002) Epithelial–mesenchymal transitions in tumour progression. Nat Rev Cancer 2:442–454
Pagliarini RA, Xu T (2003) A genetic screen in Drosophila for metastatic behavior. Science 302:1227–1231
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811
Montgomery MK, Xu S, Fire A (1998) RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA 95:15502–15507
Fearon ER, Cadigan KM (2005) Wnt signaling glows with RNAi. Science. 308:801–808
Liu XD, Ma SM, Liu Y, Liu SZ, Sehon A (2004) Short hairpin RNA and retroviral vector-mediated silencing of p53 in mammalian cells. Biochem Biophys Res Commun 324:1173–1178
Zamore PD, Tuschl T, Sharp PA, Bartel DP (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25–33
Elbashir SM, Lendeckel W, Tuschl T (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 15:188–200
Tavernarakis N, Wang SL, Dorovkov M, Ryazanov A, Driscoll M (2000) Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes. Nat Genet 24:180–183
Kennerdell JR, Carthew RW (2000) Heritable gene silencing in Drosophila using double-stranded RNA. Nat Biotechnol 18:896–898
Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363–366
Grishok A, Pasquinelli AE, Conte D, Li N, Parrish S, Ha I, Baillie DL, Fire A, Ruvkun G, Mello CC (2001) Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106:23–34
Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH (2001) Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 15:2654–2659
Knight SW, Bass BL (2001) A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293:2269–2271
Wu MT, Wu RH, Hung CF, Cheng TL, Tsai WH, Chang WT (2005) Simple and efficient DNA vector-based RNAi systems in mammalian cells. Biochem Biophys Res Commun 330:53–59
Samuel CE (2001) Antiviral actions of interferons. Clin Microbiol Rev 14:778–809
Geiss G, Jin G, Guo J, Bumgarner R, Katze MG, Sen GC (2001) A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling. J Biol Chem 276:30178–30182
Gil J, Esteban M (2000) Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of actions. Apoptosis 5:107–114
Player MR, Torrence PF (1998) The 2-5A system: modulation of viral and cellular processes through acceleration of RNA degradation. Pharmacol Ther 78:55–113
Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20:505–508
Novina CD, Sharp PA (2004) The RNAi revolution. Nature 430:161–164
Tuschl T (2002) Expanding small RNA interference. Nat Biotechnol 20:446–448
Agami R (2002) RNAi and related mechanisms and their potential use for therapy. Curr Opin Chem Biol 6:829–834
Mazieres J, You L, He B, Xu Z, Lee AY, Mikami I, McCormick F, Jablons DM (2005) Inhibition of Wnt16 in human acute lymphoblastoid leukemia cells containing the translocation induces apoptosis. Oncogene 24:5396–5400
Kemp G, Willems E, Abdo S, Lambiv S, Leyns L (2005) Expression of all Wnt genes and their secreted antagonists during mouse blastocystand postimplantation development. Dev Dynam 233:1064–1075
Spater D, Hill TP, O’sullivan RJ, Gruber M, Conner DA, Hartmann C (2006) Wnt9a signaling is required for joint integrity and regulation of Ihh during chondrogenesis. Development 133:3039–3049
Good PD, Krikos A, Li SX, Bertrand E, Lee NS, Giver L, Ellington A, Zaia JA, Rossi JJ, Engelke DR (1997) Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther 4:45–54
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494–498
Acknowledgments
This work was supported by grants from the National Nature Science Foundation of China (No. 30371493) and the Major State Basic Research Development Program of China (973 program) (No. G1999055901).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Xiang, Y., Lin, G., Zhang, Q. et al. Knocking down Wnt9a mRNA levels increases cellular proliferation. Mol Biol Rep 35, 73–79 (2008). https://doi.org/10.1007/s11033-007-9055-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11033-007-9055-9