Understanding miRNA Turnover: A Study of miRNA Half-Life

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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

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What are miRNAs?

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

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miRNA Biogenesis
Zamore PD Haley B. Science 2005
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How do miRNAs turnover?
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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
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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.

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Control Gene Degradation c-myb
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Control Gene Degradation c-myb
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Control Gene Degradation c-myc
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MicroRNA Profiling
Lu, J Nature 2005
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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

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RT-PCR Validation
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RT-PCR Validation
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RT-PCR Validation
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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)

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

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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