Epigenetic alterations of miRNA in neuropathic pain reveal novel

40 Pages·2010·0.5 MB·English

Epigenetic alterations of miRNA in neuropathic
pain reveal novel regulatory mechanisms
Seena Ajit PhD
Pharmacology & Physiology Department
Drexel University College of Medicine
Philadelphia
MicroRNAs
Single-stranded RNA molecules of 21-23 nucleotides in length

Encoded as independent genes or processed from introns of mRNAs

Modulate gene expression post-transcriptionally by binding to the 3’-

untranslated regions (3’-UTR) of specific mRNAs
 Translational repression
 miRNA induced cleavage of mRNA
Potential regulatory circuitry involving miRNA is enormous

 Each miRNA has multiple targets
 Each 3’-UTR may be targeted by many miRNAs
Deregulation of miRNA well-established in a number of human

diseases
Neuropathic pain
Pain resulting from damage or disease of the nervous

system
Heterogeneous conditions that cannot be explained

by a single etiology or specific lesion
Patients suffer from spontaneous pain, allodynia

and hyperalgesia
(abnormal pain to normally innocuous stimuli),
(exaggerated response to noxious stimuli)
Difficult to treat with only some 40-60% of patients

achieving partial relief
Widely used animal models involve partial injury of the

sciatic nerve or its components.
Spinal nerve ligation (SNL)
Involves tight ligation of the L5 (and

L6) spinal nerve(s)
Enables study of changes in both

injured primary and neighboring
intact sensory neurons and their
relative contribution to the
pathophysiology of pain
Fukuoka and Noguchi (2002)
Contribution of injured vs spared neurons to
neuropathic pain
Two views
SNL induced changes are initiated and

maintained by activity originating from the
injured afferents
SNL
The alternative or uninjured/intact afferent

hypothesis suggests changes in the
uninjured and not the injured afferents may
underlie the development and maintenance
Adapted from
of neuropathic pain
Decosterd and
Woolf, Pain, 2000
SNL model
SNL injury created by tight ligation of L5 spinal

nerve
Sham rats underwent same surgery but without

damage or manipulation to spinal nerves
Rats tested for tactile sensitivity immediately

prior to surgery (baseline) and before tissue
extraction 4 weeks post-surgery to ensure SNL SNL
rats were hypersensitive
Total RNA isolated from individual DRG

Adapted from
Decosterd and
Woolf, Pain, 2000
SNL Sham
L4 L5 L4 L5
ipsi 5 5 5 5
contra 5 5 5 5
microRNA and mRNA levels measured from
the same RNA
miRNA expression profiling:

Quantitative RT-PCR on ABI TaqMan Human low density array
(TLDA), which monitors 369 miRNAs
Single DRG per TLDA card, 5 animals per group, 40 TLDA cards
total
mRNA expression profiling:

Labeling and cRNA generated using Nugen Technologies Ovation
amplification kit
Hybridization to Affymetrix Rat Genome Genechip 430 2.0
Data normalization using MAS 5 algorithm, statistical analysis using
GeneData Expressionist system
Pairwise correlation of raw Ct values
SNL group Sham group
SNL-L4 ipsi
SNL-L5 ipsi
SNL-
L4+L5
contra
Heat map of sample-to-sample (Pearson) correlation of raw Ct values
from 248 detectable miRNAs in 40 DRG samples from the 8
experimental groups
Column and row labels are symmetric
Unbiased normalization
RNU6B (ncRNA, involved in spliceosome, 45 nt) has been

suggested in literature as potential housekeeping RNA
Identified the most “rank-preserved” TaqMan products and

selected the 11 least changing miRNAs across all 40 tissues
Not dependent on one transcript for normalization
L4 SNL L5 SNL L4 Sham L5 Sham
Post normalization Ct-distributions show
same separation of sample groups
SNL group Sham group
n n
o o
it it
u L4+L5 u
b b
ir Contra ir
t t
s s
id id

e e
v L4 Ipsi v
it it
a a
lu lu
m m
L5 Ipsi
u u
C C
SNL+Sham
Ct Ct
n
o
it Sham & SNL contra
u
b
ir
t
s
id
e L4
v Ipsi
it
a L5 (SNL)
lu
m Ipsi
u (SNL)
C
Ct