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

2. Why miRNAs?

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.

4. Revised Central Dogma of Molecular
Biology

5. Biogenesis of a miRNA
• miRNA gene is transcribed
• Pri-miRNAPre-miRNAmature miRNA
• Mature miRNA Duplex is split in two and
the single stranded molecule is
incorporated into the RISC (Ribosome
Induced Silencing Complex) assembly

6. Biogenesis of miRNAs

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.

8. miRNA Detection Methods

9. Microarray

10. RT-PCR with qPCR

11. miRNA Target Recognition Sites

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

13. Luciferase Assay

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

15. Diabetes is a Complex Beast

16. Pathophysiology of “Diabeetus”

17. Now what is obesity?

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.

19. Biogenesis of Adipose Tissue

20. Brown Adipose Tissue
• Does not primarily
perform lipogenesis
• Burns
calories, results in
decreased BMI

21. White Adipose Tissue
• Stores excess
calories as
triglycerides
• Secretes various
metabolic hormones

22. Brown vs. White

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

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

31. Visual Summary

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.

35. The DIO vs. DR Mouse Models

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.

39. miRNA Profile

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.

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