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MiRNA presentation
1. Mir193b–365 is essential for
brown fat differentiation
Lei Sun, Huangming Xie, Marcelo A.
Mori, Ryan Alexander, Bingbing Yuan, Shilpa
M. Hattangadi, Qingqing Liu, C. Ronald Kahn
and Harvey F. Lodish
3. History of miRNA Research
• Their importance was first recognized in
1993 in C. Elegans when a mutation
caused loss of function in lin-4 and let-7.
• Caused disruption of developmental
timing and revealed a previously unknown
level of complexity in genetic regulation.
5. Biogenesis of a miRNA
• miRNA gene is transcribed
• Pri-miRNAPre-miRNAmature miRNA
• Mature miRNA Duplex is split in two and
the single stranded molecule is
incorporated into the RISC (Ribosome
Induced Silencing Complex) assembly
7. Profiling miRNAs
• Reverse transcription of miRNAS and total
RNAs for cDNA.
• Helps to use a variety of tissues.
• With the cDNA you can then analyze
expression via microarray or real time PCR.
• Important to note that this only tells you if
something is there.
12. Function Analysis of miRNAs
• If you want to see a miRNA:mRNA
relationship and/or establish a miRNA’s role in
a certain phenotype, there are really 2
different methods
• Reporter Assay
• “Round-a-bout” Method
14. Diabetes and Obesity
• According to the NIDDK diabetes is a
chronic lifelong disease which is
characterized by chronic high levels of
sugar in the blood.
• In reality the picture is much, much bigger
18. All FAT was not created equally.
• Fat, or adipose tissue, is not just the
body’s way to store excess calories.
• It functions as a major metabolic organ
which helps regulate glucose
homeostasis.
23. Their Method
• Identified lineage rich miRNAs by comparing
genome wide miRNA expression patterns in
WAT, BAT, and skeletal muscle microarrays
• Looked for miRNAs expressed in at least three
samples with different expression in all 3
tissues.
• CAGE analysis which measures expression
levels of transcription starting (start points)
24. • Use LNA modified inhibitors to block miR-193
expression and see what happens.
• Check brown fat markers with Western
Blotting which pointed to miR-193 being
critical to certain steps in development
25.
26.
27.
28.
29.
30. Conclusions
• Determine that miR-193b-365 complex is
necessary for BAT differentiation
• Determine that its relationship to Prdm16 and
Pparα helps upregulate this microRNA during
differentiation, inducing adipogenic
factors, while suppressing myogenic ones.
• miR-193b also associates closely with the
Runx1t1 mRNA, and other mRNAs
32. The Future
• Examine function within a mouse model using
adenoviruses to specifically introduce
inhibitors and mimics
• Examine their therapeutic potential
33. MiRNA Profiling and Function
Analysis in a Diet Induced Mouse
Model of Obesity and Diabetes
34. What my lab does
• Diabetes and Obesity focus in addition to
regenerative biology
• Specifically the crucial roles of Cdk4, Smad3,
and TGF-β in cell cycle regulation and
metabolic function.
• The miRNA component comes in when
considering why certain genes are turned
on/off in the obese and overweight.
36. My Experiment
• Microarray profiling of human and mouse
miRNAs in Liver, Adipose, and Muscle tissue
• qPCR of total cDNA from samples looking for
changes in expression of genes related to
glucose homeostasis
• Narrow down list of miRNAs using additional
qPCR and target scan software
• Look for phenotypic affects.
37. Real Time and a Microarray
• Microarray Cards were • Real time was then
used to profile used to look for genes
everything we could important in
look for in muscle and WAT/BAT, metabolism, a
adipose tissue. nd glucose homeostasis
• Over 500 hits. • PC, Lxr, Akt2, Creb3, Lca
• Narrowed down the list d, IR, FAS, PEPCK, Scd1,
to things that behaved Ggpale, Srebp2, Pgc1a,
similarly in mice and Pgc1b, Ppara, etc.
human tissues.
38. Target Scan
• Online database for
predicting miRNA
targets.
• Looks at 8mer and
7mer sites of mRNA
which match up with
“Seed region” of miRNA
• There is a lot of overlap.
40. Changes in Gene Expression during differen a on for BAT
16
14
12
pparalpha
prdm16
10
smad3
cox8b
Fold Change
8
UCP1
ppargamma
6
pgc1a
FAS
4 Serpin3AK.
resis n
2 ap2
0
0 1 2 3 4 5 6 7 8 9
-2
Day No.
41.
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43.
44. Results?
• miRNAs: 425, 501, 574, 10b, 339, 17, 107, 32,
191, 708, 346, 494, 145, 489, 449b, 194,
302a, 200a, 181c, 346
• Gene expression is clearly different between
tissue types and normal, overweight, and
obese models.
• miRNAs are heavily regulated
45. Future Experiments
• Looking at miRNA expression in first 24hrs.
• Want to examine expression in various
tissues: liver, muscle, hypothalamus, pancreas
• Use lento/adeno viruses to insert LNA-
miRNAs into the mice and see what happens
Notes de l'éditeur
Change from a very linear view of how genetic information is processed to a complex network that proceeds in various directions to create the necessary degree of regulatory specificity.
-Make sure to point out the ones that are most widely used. Northern blotting, microarry, RT-PCR, and surface enhance Raman spectrometry.
Round about method is what I am doing in my lab, so talk about it then. Luciferase assay is the only concrete way of establishing a direct relationship between a certain miRNA and mRNA.
Heat map of the expression of miRNAs enriched in BAT compared to WAT & Skeletal muscleRelative quantitation of each mRNA in various mouse tissues, miR-223 is the controlExpression changes during differentiation
Test of miRNA inhibitors effectivenessShows accumulation of lipid droplets in BAT at day 4Mimics increase expressionrtPCR of adipogenesis markers
-In the microarray we wanted to find changes in expression that were similar in both the mouse and human tissue samples.-qPCR, obviously can’t look at changes in expression for everything so we focused on genes important for glucose homeostasis and adipocyte differentiation-
Result was I then had ~15 miRNAs that were of potential interest. So I then ran them through Target Scan, online database/algorithm to help identify targets. miRNAs that regulate a certain protein.
Predictions are ranked based on:-site type contribution-3’pairing-local AU relationships-position
Just targets related to glucose homeostasis.miR-425#, miR-501-5p, miR-574-3p, miR-10b#, miR-339-3p, miR-17, miR-107, miR-32, miR-191#, miR-708, miR-346, miR-494, miR-145#, miR-489, miR-449bmiR-194, miR-302a, miR-200a, miR-181c, miR-346