Understanding miRNA Turnover: A Study of miRNA Half-Life

Dominic McDonaldMassachusetts Institute of Technology

Mentor: Jun Lu, Ph. DTodd Golub Laboratory

Broad Institute of MIT and Harvard

What are miRNAs?

miRNAs are short non-coding RNAs

Post-transcriptional regulators of gene expression

miRNAs regulate diverse biological and disease processes

miRNA Biogenesis

Zamore PD & Haley B. Science 2005

How do miRNAs turnover?

Approach to Measure miRNA t1/2

Culture Cells in Triplicates

Treat Cells with Actinomycin D

Harvest RNA at 6 time points throughout the day

Measure miRNA and control gene expression levels using Luminex

NextGen and RT-PCR

Cell Lines

Maintained 26 different cancer cell lines.

Due to time constraints only able to collect data from 9 different cancer cell lines: HD-MY-Z, 697, HL60, K562, Ramos (RA1), RS4;11, SEMK2, U937, and HEL cell lines.

Triplicates of every cell line done at 6 time points each for a total of 162 samples.

Control Gene Degradation c-myb

U937 c-myb

0

20

40

60

80

100

120

140

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Time (hr)

% E

xp

res

sio

n

Control Gene Degradation c-myb

U937 c-myb

-19

-18

-17

-16

-15

-14

-13

-12

-11

-10

0 2 4 6 8 10 12 14

Time (hr)

log

2 e

xp

res

sio

n

Control Gene Degradation c-myc

SEMK2 c-myc

-22

-21

-20

-19

-18

-17

-16

-15

-14

-13

0 2 4 6 8 10 12 14

Time (hr)

log

2 e

xpre

ssio

n

MicroRNA Profiling

Lu, J Nature 2005

K562 hsa-miR-18a

8

9

10

11

12

0 5 10 15 20 25

Time (hr)

log

2 e

xp

res

sio

n

HL60 hsa-miR-18a

7

8

9

10

11

0 5 10 15 20 25

Time (hr)

log

2 e

xp

res

sio

n

K562 hsa-miR-125a

7

8

9

10

11

0 5 10 15 20 25

Time (hr)

log

2 e

xp

res

sio

n

RT-PCR Validation

Use additional methodology to validate results

Use RT-PCR for validation Measure miR-125a in K562 cells Measure u6 small RNA as a control

RT-PCR Validation

K562 miR-125a

3

4

5

6

7

8

9

0 5 10 15 20 25

Time (hr)

log

2 e

xp

res

sio

n

RT-PCR Validation

K562 miR-125a

20

21

22

23

24

25

26

27

28

29

30

0 5 10 15 20 25

Time (hr)

Raw

Exp

ress

ion

Ct

RT-PCR Validation

K562 u6

28

29

30

31

32

33

34

35

36

37

38

0 5 10 15 20 25

Time (hr)

Raw

Exp

ress

ion

Ct

What Does This All Mean?

Most miRNAs have long t1/2 (over 24 hours)

We have not observed any fast degrading miRNAs (t1/2 less than 8 hours)

Why would nature create a class of stable cellular regulators?

Acknowledgements

Our Team: Jun Lu Hao Zhang Judy Wang

Golub Lab: Todd Golub Jinyan Du

Broad Institute: Shawna Young Bruce Birren Lucia Vielma Maura Silverstein