Rachel Miller BNFO 300 Spring 2017 Crohns Disease Autoimmune Disease Classified by the Inflammation of the GI tract 400000 people affected in the United States General Question What causes Crohns disease ID: 611453
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Slide1
Comparing the blockage of the S1PR1 receptor to silencing microRNA miR-155
Rachel Miller
BNFO 300 Spring 2017Slide2
Crohn’s Disease
Autoimmune Disease
Classified by the Inflammation of the GI tract
400,000 people affected in the United StatesGeneral Question:What causes Crohn’s disease?Slide3
Possible Causes of Crohn’s Slide4
S1P and S1PR1
Sphingosine-1-phosphate (S1P)
Sphingosine-1-phosphate receptor 1 (S1PR1)
Involved in regulation of lymphocyte migration, maturation and trafficking
Only three S1PRs are present in immune systemSlide5
MicroRNAsSlide6
BACKGROUND EXPERIMENTSlide7
The Results from Background Experiment
When Fingolomid was present, the number of microRNAs
decreased
in both T cells and exosomes
Which leads us to our big question....Slide8
Big Question
Is the reduction of microRNAs in T cells caused by the blockage of S1PR1 using Fingolomid or is it correlated?Slide9
Experiment:Step 1
Fingolomid
T cell
S1PR1Slide10
Experiment:Step 1
Fingolomid
T cell
S1PR1Slide11
Experiment:Step 1
Fingolomid
T cell
S1PR1Slide12
Experiment:Step 1
T cell
S1PR1Slide13
Experiment:Step 1
T cell
S1PR1Slide14
Experiment:Step 1
S1P
T cell
S1PR1Slide15
Experiment:Step 1
S1P
T cell
S1PR1
miR-155
MeasureSlide16
Experiment:Step 1
S1P
Cytokines:
IL-2
IL-17
IFN-gamma
Measure
T cell
S1PR1
miR-155
MeasureSlide17
Experiment:Step 2
S1P
T cell
S1PR1Slide18
Experiment:Step 2
S1P
T cell
S1PR1Slide19
Experiment:Step 2
S1P
T cell
S1PR1
miR-155
siRNA
MeasureSlide20
Experiment:Step 2
S1P
Cytokines:
IL-2
IL-17
IFN-gamma
Measure
T cell
S1PR1
miR-155
siRNA
MeasureSlide21
How we are measuring our results: miRNAs
qPCR – quantitative polymerase chain reaction
Real time PCR with
Fluorescent dye binds to DNAFluorescent light will increase as qPCR continuesSlide22
Quantify microRNA ExpressionSlide23
How we are measuring our results: Cytokines
ELISA
Targets are pro-inflammatory cytokines
IL-2IL-17IFN-gammaSlide24
Quantify Cytokine ExpressionSlide25
Expected Results
When Fingolomid is blocking the S1PR1 receptor, microRNA-155 expression will decrease AND cytokine expression will decreased
When miRNA-155 is silenced with siRNA, cytokine expression will match the results with Fingolomid was present
What would these results tell us?Slide26
Recap
Is the reduction of microRNAs in T cells caused by the blockage of S1PR1 using Fingolomid or is it correlated
?
Compared silencing of miR-155 using siRNA to adding Fingolomid to block S1PR1Compared microRNA expressions between the two tests using qPCRCompared cytokine expression between the two tests using ELISASlide27
Why it Matters
One step closer in understanding Crohn’s disease and other autoimmune diseases
Shows evidence that S1PR1 and microRNAs are tied together
Possible treatment for patients with Crohn’s diseaseSlide28
Questions?