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Comparing the blockage of the S1PR1 receptor to silencing m Comparing the blockage of the S1PR1 receptor to silencing m

Comparing the blockage of the S1PR1 receptor to silencing m - PowerPoint Presentation

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Uploaded On 2017-11-30

Comparing the blockage of the S1PR1 receptor to silencing m - PPT Presentation

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

experiment s1pr1 cell step s1pr1 experiment step cell fingolomid s1p 155 mir measure expression results micrornas crohn

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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?