RNA Interference (RNAi) Ryan Duval Endodontics
May 31, 2015
RNA Interference (RNAi)
Ryan Duval Endodontics
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
Goals of RNA interference
• Defending cells against parasitic genes• Defense from viruses
• Defense from transposons• Directing development and gene expression in
general
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
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
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
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
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
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
Recognizing the dsRNA
• Detected and bound by effector protein
• RDE-4 in nematodes (C. elegans)
• R2D2 in Drosophila
• Both stimulate DICER
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
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]
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]
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
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?
Materials/Methods
• Mouse hematopoetic cells osteoclasts
• siRNA transfection (silencing NFATc1
• ELISA for TNF-α/IL-6• Immunocytochemistry • Staining of nuclei
Materials/Methods
• Primers for detection of– Cathepsin K gene CSK– IL-6– TNF-α
• Compare osteoclasts transfected w/control vs NFATc1 siRNA
Results
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
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
Any Questions?