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Understanding miRNA Turnover: A Study of miRNA Half-Life

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Title: Understanding miRNA Turnover: A Study of miRNA Half-Life


1
Understanding miRNA Turnover A Study of miRNA
Half-Life
  • Dominic McDonald
  • Massachusetts Institute of Technology
  • Mentor Jun Lu, Ph. D
  • Todd Golub Laboratory
  • Broad Institute of MIT and Harvard

2
What are miRNAs?
  • miRNAs are short non-coding RNAs
  • Post-transcriptional regulators of gene
    expression
  • miRNAs regulate diverse biological and disease
    processes

3
miRNA Biogenesis
Zamore PD Haley B. Science 2005
4
How do miRNAs turnover?
5
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
6
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), RS411, SEMK2, U937, and
    HEL cell lines.
  • Triplicates of every cell line done at 6 time
    points each for a total of 162 samples.

7
Control Gene Degradation c-myb
8
Control Gene Degradation c-myb
9
Control Gene Degradation c-myc
10
MicroRNA Profiling
Lu, J Nature 2005
11
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14
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

15
RT-PCR Validation
16
RT-PCR Validation
17
RT-PCR Validation
18
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)

19
  • Why would nature create a class of stable
    cellular regulators?

20
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
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