RNA Interference (RNAi) Ryan Duval
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  • 1. RNA Interference (RNAi) Ryan Duval Endodontics
  • 2. Discovery of RNAi
    • First observed in petunias
    • Napoli et al 1990
    • Observed “cosuppression”
    • Occurring at post-transcriptional level (plants and fungi)
    • Fire et al 1998 silencing of genes in nematodes (nice Prize ‘06)
    • Mammalian cells – chemically synth or expressed from plasmid or viral vector
  • 3. Goals of RNA interference
    • Defending cells against parasitic genes
    • Defense from viruses
    • Defense from transposons
    • Directing development and gene expression in general
  • 4. RNAi
    • Gene silencing mediated by double-stranded RNA
    • Silencing of gene expression
      • Results from cleavage and degreadation of a target gene’s mRNA
      • Also results from blocking translocation of intact mRNA
    • Usually about 20 – 25 base pairs long
    RNA Interference Howard Hughes Medical Institiute
  • 5. Workings of RNAi
    • Dicer recognizes and cuts the double-stranded RNA (dsRNA not common)
    • Results: short 21 to 25 base-pair molecules called “small interfering RNAs” (siRNAs)
    • siRNAs bind to several proteins (3’ overhangs)
    • Forming RISC (RNA-induced silencing complex)
    DICER
  • 6. RNAi contd.
    • RISC becomes activated when the siRNA its carying is unzipped (utilizing ATP)
    • Activated RISC bind to target mRNA
    • RISC subunits then cleave mRNA
    • Other proteins degrade mRNA & prevent protein production
  • 7. RISC
    • The two dsRNA pathways
      • Exogenous
        • Coming from infection by VIRUS w/RNA genome
        • By lab manipulation
      • Endogenous
        • Pre-microRNA expressed from RNA-coding genes in the genome
    • Both pathways converge at the RISC complex
  • 8. RISC
    • How the activated RISC complex locates complementary mRNAs within the cell is UNKNOWN?
    • Located in P-bodies (cytoplasmic bodies)
    • The active components of RISC are ARGONAUTE proteins
      • Endonuclease
      • Cleaves the the target mRNA strand complementary to bound siRNA
  • 9. Overview
    • 20-25nt length (siRNA)
    • siRNA separated into single strands
    • Single strands integrated into RISC
    • siRNA induce cleavage of the mRNA
    • Preventing it from being used as a translation template
  • 10. Recognizing the dsRNA
    • Detected and bound by effector protein
    • RDE-4 in nematodes (C. elegans)
    • R2D2 in Drosophila
    • Both stimulate DICER
  • 11. Gene knockdown
    • A drastic decrease in the expression of a targeted gene
    • Studying the effects of the decrease can show the physiologic role of the gene product
    • RNAi may not totally abolish expression of the gene = knockdown vs knockout
  • 12. RNAi applications
    • Silencing CD44 (hyaluronan receptor) gene in nasopharyngeal carcinoma =  malignant potential of the cells. [Jod et al 2007, Shi Oncol Rep 2007]
    • Expression of p27 (common protein in oral squamous cell carcinoma), siRNA inhibited the cell proliferation in vitro and in vivo of the p27. [Kudo et al Oral Oncol 2005]
    • siRNA used for the treatment of ankylosis and periodontal disease. [Yamada et al J Biol Chem 2001, 2007]
  • 13. Various RNAi uses
    • Topical microbicide treatments of HSV II [Jiang, Milner; Oncogene 2002]
    • Knockdown host receptors for HIV [Crowe; AIDS Supp 2003]
    • Silencing Hep A and Hep B genes [Kusov et al; J Virol 2006]
    • Silencing Influenza gene expression [Jia, Zhang, Liu; Biotechnol Lett 2006]
    • Inhibition of LPS induced osteoclast formation and cytokine stimulation [Fahid et al; JOE 2008]
  • 14. Application of Small Interfering RNA for Inhibition of LPS-Induced Osteoclast Formation and Cytokine Stimulation
    • Purpose: Suppression of NFATc1 (transcription factor) expression in monocytes and osteoclast cells using RNAi technique
  • 15. Background
    • Bone homeostasis
    • RANKL (TNF family)
    • Induces osteoclastogenesis
    • LPS  express RANKL = osteoclast formation
    • Final stage of osteoclast differ. NFATc1 is crucial part of osteoclast differentiation.
    • Inhibit NFATc1 pathway  inhibit bone destruction?
  • 16. Materials/Methods
    • Mouse hematopoetic cells  osteoclasts
    • siRNA transfection (silencing NFATc1
    • ELISA for TNF-α/IL-6
    • Immunocytochemistry
    • Staining of nuclei
  • 17. Materials/Methods
    • Primers for detection of
      • Cathepsin K gene CSK
      • IL-6
      • TNF-α
    • Compare osteoclasts transfected w/control vs NFATc1 siRNA
  • 18. Results
  • 19. Conclusion
    • Deliver siRNA into cytoplasm w/  efficiency
    • Significant  of TNF-α and IL-6 in response to LPS stimulation
    • Significant  in # of mature osteoclasts in response to LPS
    •  in osteoclast-specific gene expression to LPS stimulation
  • 20. RNAi Challenges
    • Systemic delivery obstacle (for RNAi drugs)
    • How to control the amount of siRNA being delivered (above or below therapeutic levels)
    • Possible stimulation of “off target” genes
    • Long-term effects
  • 21. Any Questions?