This document summarizes research modulating the protein TRIM21 to potentially treat autoimmune diseases like Sjögren's syndrome and systemic lupus erythematosus. Preliminary results found that certain drug compounds increased TRIM21's ability to reduce inflammatory responses. MicroRNA analysis identified differentially expressed miRNAs between disease patients and controls, targeting TRIM21, though further validation is needed. Overall, modulating TRIM21 and characterizing miRNA involvement shows promise for developing personalized miRNA-based therapies for autoimmune conditions.

1. MODULATING TRIM21 TO
AMELIORATE DISEASE
SYMPTOMS IN PATIENTS WITH
AUTOIMMUNE DISEASES
PHILIP ALLEN DY

2. AUTOIMMUNE DISEASES
Sjögren’s Syndrome (SS):
 Is an inflammatory disease that can
affect many parts of the body but most
often affects the tear and saliva glands.
 Dry eyes and dry mouth are the
hallmark of the disease(1).
 SS mostly affects women with a ratio of
9:1 to men.
 SS can exist either as a primary
disorder (pSS) or in patients with other
rheumatologic disorders such as SLE
and RA.
 The patho-aetiology of SS is still
unknown.
(2)Adapted from Kallen, 2014. Sjogrens Syndrome
Effects on the Body Image. [Online] Available at:
http://autoimmunesupport.com/category/sjogrens
-syndrome/ [Accessed 19 March 2015].
(2)

3. Systemic Lupus Erythematosus (SLE):
 Is a chronic disease causing
inflammation such as pain and
swelling.
 SLE affects many organs of the body.
 Similar to SS, SLE affects women more
than men.
 Branded as a ‘great imitator’ (4) due to
its wide range of symptoms that
mimics other diseases.
(5) Adapted from The Lupus Effect, (n.d.). Symptoms.
[Online] Available at:
http://thelupuseffect.com/about-lupus/symptoms/
[Accessed 19 March 2015].
(5)

4. PROJECT AIMS
Objectives:
 To validate compounds that regulate the activity
of Trim21
 To identify miRs whose expression is altered in
patients with autoimmune conditions compared
to healthy controls
Hypothesis:
 The treatment of drug compounds that will
essentially increase the activity of Trim21
leading to the increase of its ability to reduce
type I IFN production thus reducing
inflammatory response
 Differential expression of microRNAs may
play a role in the in the pathogenesis and
progression of pSS
(6) Adapted from Jefferies, C., Wynne, C. &
Higgs, R. Antiviral TRIMs: friend or foe in
autoimmune and autoinflammatory disease?
Nat Rev Immunol 2011; 11: 617-25.
(6)

5. LUCIFERASE REPORTER ASSAY
Luciferase Reporter Assay:
 Useful method for studying gene expression.
 Uses two reporter enzymes, Firefly and
Renilla.
 Both of these enzymes are situated within
specific light emitting plasmids.
 The amount of light given off directly
correlates to the activity of the system.
 The Renilla enzyme acts as an internal
control and the Firefly enzyme correlates to
the effect of the specific experimental
conditions.
(7) Adapted from Ní Gabhann, J. (n.d.). Luciferase
Reporter Assay Plate Setup Protocol.
(7)

6. LUCIFERASE ASSAY
PROTOCOL
1x105 cells/ml
seeded in a 96-
well plate
Add Luciferase
Assay Mix
Reagent or
Renilla Mix
Read
Luminescence
Plasmid DNA
added to
each well
Transfection
Mix

7. 0
50
100
150
Foldexpression
CD Vehicle Control
Controls
0
50
100
150
Foldexpression
PEG
EV
TRIF
TRIM21
TRIF+TRIM21
50
5
0.5
0.05
0
50
100
150
200
Drug Concentration (μM)
Foldexpression
NaOH
A
B
C
TESTING INDIVIDUAL
COMPONENTS OF VC
 Individual components of VC: CD, PEG, and
NaOH.
 Positive control: TRIF which drives IFN-β
promoter activity or Trim21 which is
inhibiting IFN-β promoter activity.
 Negative control: EV
 Therefore, TRIF+TRIM21 together provides
our threshold level.

8. A
B
0
20
40
60
80
100
120
Foldexpression
4A CD Vehicle Control
Drug Compound
Controls
EV
TRIF
TRIM21
TRIF+TRIM21
VC
PBS
MEDIA
0
20
40
60
80
100
120
Diluents
Foldexpression
4A PEG
A
B
0
20
40
60
80
100
120
Foldexpression
3A CD Vehicle Control
Drug Compound
Controls
EV
TRIF
TRIM21
TRIF+TRIM21
VC
PBS
MEDIA
0
20
40
60
80
100
120
Diluents
Foldexpression
3A PEG
DRUG COMPOUNDS @
50NM CONCENTRATION
 Single concentration at 0.05μM with different diluents including VC itself, PBS, and media based of
previous results.
 Further analysis resulted in the conclusion in mycoplasma infection = false interpretation of
experimental data.

9. DRUG COMPOUNDS
DOSE RESPONSE
 3A compounds did not yield promising results since the drug compounds does not seem to be
inhibiting IFN-β promoter activity.
 On the other hand, the treatment with 4A compounds show a significant increase in the inhibition
of IFN-β promoter activity (p<0.05).
A
B
C
0
5
10
15
20
25
Foldexpression
3A CD Vehicle Control
Drug Compound
Controls
0
5
10
15
20
25
Foldexpression
3A PEG
EV
TRIF
TRIM21
TRIF+TRIM21
5
0.5
0.05
0.005
0
5
10
15
20
25
Drug Concentration (μM)
Foldexpression
3A TFA
*
A
B
C
0
50
100
150
200
250
Foldexpression
4A PEG
0
50
100
150
200
250
Foldexpression
4A CD
Vehicle Control
Drug Compound
Controls
*
*
*
*
*
EV
TRIF
TRIM21
TRIF+TRIM21
5
0.5
0.05
0.005
0
50
100
150
200
250
Drug Concentration (μM)
Foldexpression
4A TFA
*

10. DRUG COMPOUNDS
DOSE RESPONSE
 Increased concentration of our drug compounds for both 3A and 4A.
 However, no inhibition of IFN-β promoter activity exists.
 Also, due to miscalculations, only one VC was employed at 25μM.
A
B
C
0
20
40
60
80
100
Foldexpression
3A CD
Vehicle Control
Drug Compound
Controls*
*
0
20
40
60
80
100
Foldexpression
3A PEG
*
*
EV
TRIF
TRIM21
TRIF+TRIM21
25
5
1
0.2
0
20
40
60
80
100
Drug Concentration (μM)
Foldexpression
3A TFA
*
A
B
C
0
20
40
60
80
100
Foldexpression
4A CD
Vehicle Control
Drug Compound
Controls*
*
0
20
40
60
80
100
Foldexpression
4A PEG
*
*
EV
TRIF
TRIM21
TRIF+TRIM21
25
5
1
0.2
0
20
40
60
80
100
Drug Concentration (μM)
Foldexpression
4A TFA
*
*

11. THERMAL DENATURATION ASSAY
Differential Scanning Fluorimetry (DSF):
 Used as as screening method for Trim21 and IRF3 proteins.
 Measures the thermal stability of a target protein.
 Binding of low Mw ligands can increase the thermal stability of a
protein.
 The thermal stability change is measured a fluorescent dye, Sypro
Orange (8).
 The shifting of the curve decides the binding of the ligands.
 A shift towards the left = destabilisation of the protein whereas a
shift to the right = binding of ligand to protein.
 2-degree shift to the right is significant.

12. THERMAL SHIFT ASSAYS
 Determination of the optimal concentration for our proteins: Trim21 and IRF3 as well as the
optimal concentration for DMSO, in which the compounds were solubilised in.
 DMSO serves as our reference control.

13. THERMAL SHIFT ASSAY OF
3A AND 4A COMPOUNDS
 With the DMSO control in yellow, both
3A and 4A compounds with Trim21
protein have shifted to the right
greater than 2-degrees.
 In contrast, both 3A and 4A
compounds with IRF3 protein have
shifted to the left to our DMSO control.

14. PATHWAY OF MICRORNA
RNA Pol II
Exportin-5
Dicer
miRNA
gene
Drosha-
DGCR8 Pre-miRNA
Nucleus
Cytoplasm
miRISC
miRNA/miRISC
Target
mRNA
miRISC
Target Degradation
(9) Adapted from Pilson, Q. (2015).
Profiling microRNA in Primary
Sjögren’s Syndrome Related Dry Eyes.

15.  miRs are small, non-coding RNA molecule (~22nucleotides).
 miRs have been shown to regulate inflammation
 Trim21 (Ro52) functions in the ubiquitination process.
 Trim21: common target of circulating autoantibodies in AID.
 Trim21 has been identified as a key negative regulator of
inflammation.
(10) Adapted from Oke, V. & Wahren-
Herlenius, M. The immunobiology of Ro52
(TRIM21) in autoimmunity: a critical
review. J Autoimmun 2012; 39: 77-82.

16. BIOINFORMATICS

17. MIRS OF INTEREST
 Two upregulated and two downregulated miRs were picked for further analysis and their
respective gene targets were searched.

18. PRIMER OPTIMISATION
 Primers for miRs of interest and their predicted targets were
optimised at three different annealing temperatures.
 Optimal annealing temperatures were picked based on their
corresponding amplicon size.
 Mw ladder: 1kb DNA ladder visualised by SyproRed staining
on a 1% Agarose gel.

19. QPCR ANALYSIS OF PBMCS OF PSS
PATIENTS VS HEALTHY CONTROLS
A B
 miR-30e was found to be overexpressed in pSS versus healthy controls from screening.
 Real-time analysis confirms it is overexpressed in pSS patients.
 SOCS1 is a predicted target of miR-30e that is involved in cytokine signal transduction.
 SOCS1 gene expression in pSS patients vs healthy controls showed decreased
expression, as hoped.
n = 6 n = 8

20. A B
 miR-98 was found to be underexpressed in pSS versus healthy controls from screening.
 Real-time investigation demonstrates it is underexpressed in pSS patients.
 IL10 is a predicted target of miR-98 that plays a vital role in inflammatory and immune
reactions.
 IL10 gene expression in pSS patients vs healthy controls confirmed increased
expression, but not deemed to be statistically significant (p<0.05).
n = 6 n = 2

21. A B C
 miR-194 was found to be underxpressed in pSS versus healthy controls from screening.
 Real-time analysis confirms it is underexpressed in pSS patients.
 CXCL3 is a predicted target of miR-194 that regulates monocyte migration and adhesion.
 JAK3 is another predicted target of miR-194 that functions in signal transduction.
 Both predicted targets should be upregulated. CXCL3 was found to be overexpressed as
expected but JAK3 showed underexpression in pSS patients.
n = 8 n = 8 n = 2

22.  miR-125a was found to be underexpressed in pSS versus healthy controls from screening.
 Real-time investigation did not agree with initial bioinformatic analysis as miR-125a was
found to be overexpressed in pSS patients.
 No primers for miR-125a targets were ordered on time.
n = 8

23. SUMMARY
 Initial preliminary results of drug compounds look promising.
 4A compounds: significant increase in the inhibition of TRIF driven IFN-β promoter
activity.
 3A compounds: modifications in the structure of 3A compound could potentially
increase the activity of Trim21.
 Thermal melts
 In our case, the expected results were achieved following 3A and 4A compounds with
a positive shift when bound to Trim21 and a negative shift when bound to IRF3.
 miRs of interest shows promising results.
 Three miRs showed differential expression in SS vs SLE patients targeting Trim21.
 Extensive analysis is required to further validate the legitimacy of the miRs and
specifically, their gene targets in expression in pSS patients (more samples required).
 Finally, further investigation of miRs could potentially lead to the development of
personalised microRNA-base therapies as novel therapeutics.

24. ACKNOWLEDGEMENTS
SUPERVISORS
• Prof. Caroline Jefferies
• Prof. Conor Murphy
• Dr. Joan Ní Gabhann
• Dr. Qistina Pilson
• Dr. Siobhán Smith
DIT KEVIN ST.
• Dr. Sara Lynch
• Dr. Claire Wynne
MOLECULAR IMMUNOLOGY
RESEARCH GROUP, RCSI
• Dr. Joan Ní Gabhann
• Dr. Jay Chandanshive
• Dr. Marian Brennan
• Shane O’Grady
• Leah Arkins

25. REFERENCES
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109-14.
(2) Kallen, 2014. Sjogrens Syndrome Effects on the Body Image. [Online] Available at:
http://autoimmunesupport.com/category/sjogrens-syndrome/ [Accessed 19 March 2015].
(3) Kivity, S., Arango, M. T., Ehrenfeld, M., Tehori, O., Shoenfeld, Y., Anaya, J. M. & Agmon-Levin, N. Infection and autoimmunity
in Sjögren's syndrome: a clinical study and comprehensive review. J Autoimmun 2014; 51: 17-22.
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(5) The Lupus Effect, -. Symptoms. [Online] Available at: http://thelupuseffect.com/about-lupus/symptoms/ [Accessed 19
March 2015].
(6) Jefferies, C., Wynne, C. & Higgs, R. Antiviral TRIMs: friend or foe in autoimmune and autoinflammatory disease? Nat Rev
Immunol 2011; 11: 617-25.
(7) Ní Gabhann, J. (n.d.). Luciferase Reporter Assay Plate Setup Protocol.
(8) Kranz, J. K. & Schalk-Hihi, C. Protein thermal shifts to identify low molecular weight fragments. Methods Enzymol 2011;
493: 277-98.
(9) Pilson, Q. (2015). Profiling microRNA in Primary Sjögren’s Syndrome Related Dry Eyes.
(10) Oke, V. & Wahren-Herlenius, M. The immunobiology of Ro52 (TRIM21) in autoimmunity: a critical review. J Autoimmun
2012; 39: 77-82.