Alterations in the lung miRNA
profile of newborns and adult
onset lung cancer in mice
transplacentally exposed to the
environmental contaminant DBC
David A. Sampson1, 2, Christiane V. Löhr6, Kate G. Fields5, David Hendrix4,
David E. Williams1,2,3 1Department of Environmental and Molecular Toxicology,
2Linus Pauling Institute, 3Superfund Research Program, 4Department of Biochemistry
and Biophysics, 5Department of Microbiology, 6College of Veterinary Medicine,
Overview
1. Introduction
2. RNA-seq method development
3. Discussion
4. Future research
Overview
1. Introduction
2. RNA-seq method development
3. Discussion
4. Future research
What is DBC?
• Group of compounds
• Polycyclic Aromatic Hydrocarbon (PAH)
• Formed through incomplete combustion
• Persistent Organic Pollutant
• Dibenzo[def,p]chrysene
• Unique bay and fjord regions
Katz, A. K., Carrell, H. L., & Glusker, J. P. (1998). Dibenzo[a,l]pyrene (dibenzo[def,p]chrysene): fjord-region distortions. Carcinogenesis, 19(9), 1641–1648
PAH exposure: mixtures
• Inhalation – through atmosphere
• Oral– through cooked foods
• Dermal – through direct physical contact
Barker hypothesis
• Environment in womb may determine gene expression
• Epigenetics
• Maternal exposure
• Fetal exposure
Transplacental study design
• Pregnant wild-type mice gavaged with 12 mg DBC /
kg body weight (n = 4) or corn oil (n = 4) on gestation
day 17
• Mice weaned at 21 days postpartum or sac’ed at birth
• Housed until 10 month terminal time point
• Lungs excised, individual tumors separated, and flash
frozen in liquid nitrogen and stored at -80 °C
In utero exposure outcomes
•
Castro, D. J., Baird, W. M., Pereira, C. B., Giovanini, J., Löhr, C. V., Fischer, K.
A., … Williams, D. E. (2008). Fetal Mouse Cyp1b1 and Transplacental
Carcinogenesis from Maternal Exposure to Dibenzo(a,l)pyrene. Cancer
Prevention Research, 1(2), 128–134. http://doi.org/10.1158/1940-6207.CAPR-070004
•
Yu, Z., Mahadevan, B., Lohr, C. V., Fischer, K. A., Louderback, M. A., Krueger,
S. K., … Williams, D. E. (2006). Indole-3-carbinol in the maternal diet provides
chemoprotection for the fetus against transplacental carcinogenesis by the
polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene. Carcinogenesis, 27(10), 2116–
2123. http://doi.org/10.1093/carcin/bgl072
•
Yu, Z., Loehr, C. V., Fischer, K. A., Louderback, M. A., Krueger, S. K.,
Dashwood, R. H., … Williams, D. E. (2006). In utero exposure of mice to
dibenzo[a,l]pyrene produces lymphoma in the offspring: Role of the aryl
hydrocarbon receptor. Cancer Research, 66(2), 755–762.
http://doi.org/10.1158/0008-5472.CAN-05-3390
Central dogma of
molecular biology
What is miRNA?
• Types of non-coding RNA
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•
•
•
•
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Transfer RNAs
Ribosomal RNAs
siRNA
piRNA
lncRNA
miRNA
History of miRNA
• Discovered in 1993
• lin-4
• Discover of homologs in 2000
• let-7
• miRNA implicated in carcinogenesis in 2003
• No data on transplacental DBC induced lung cancer
Kamanu, T. K. K., Radovanovic, A., Archer, J. A. C., & Bajic, V. B. (2013). Exploration of miRNA families for hypotheses generation.
Scientific Reports, 3. http://doi.org/10.1038/srep02940
Potential as biomarker
• Aberrant expression of miRNAs in cancer
• Tumor classification
• Disease advancement
• Stability
• miRNA reliably extracted from fixed tissues
• Protection from RNase A activity
• Circulating miRNA
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Blood
Saliva
Breast milk
Urine
Hypothesis
• Transplacental DBC-induced lung cancer
yields a unique miRNA signature relative to
control
• Specific aims
• Develop quality control and miRNA
isolation methods for sequencing
• Test precision and accuracy of sequencing
instrument (Illumina miRNA beta test)
Overview
1. Introduction
2. RNA-seq method development
3. Discussion
4. Future research
Developing a miRNA
sequencing core services
• Isolation quality
• Yield
• Purity
• Reliability
• Instrument performance
• Reproducibility
• Technical replicate
• Biological replicate
• Read quality
Developing a miRNA
sequencing core services
• Isolation quality
• Yield
• Purity
• Reliability
• Instrument performance
• Reproducibility
• Technical replicate
• Biological replicate
• Read quality
RNA isolation workflow
1.
Mouse
2.
Excise lung
3.
Isolate RNA
4.
Prepare library
5.
Sequence
6.
Analysis
Tissue homogenization
• Tissue Tearor
• 2 mL frosted Pyrex mortar and pestle
Quality assessment
• NanoDrop 1000 (Thermo Fisher Scientific)
• RNA quantification
• A260/A280 ratio
• Qubit Fluorometer (Thermo Fisher Scientific)
• RNA quantification
• Bioanalyzer 2100 (Agilent Technologies)
• RNA Integrity Number (RIN)
• Electropherograms
RNA isolation
• Spin column-based methods
• Roche high pure miRNA isolation kit
• Qiagen RNeasy mini kit
• Qiagen RNeasy micro kit
• Phenol-chloroform method
• Trizol
Spin column-based methods
• Add homogenate to silica filter
• Nucleic acids bind to solid phase silica
• Treat with DNase
• Wash filter
• Elute RNA
Roche high pure miRNA
isolation kit
• Pros
• High specificity for miRNA
• Cons
• Low yield
• Unable to determine RIN
• A260/A280 ratio
• 1.39-1.76
• Concentration
• 2-5 ng/μL
Qiagen RNeasy mini kit
• Pros
• Able to produce RIN
• Higher yield
• Cons
• Yield not high enough
• A260/A280 ratio
• ~2.1
• Concentration
• 53.66-108.94 ng/μL
Qiagen RNeasy micro kit
• Pros
• Very high quality
• Cons
• Even lower yield than
RNeasy mini kit
• A260/A280 ratio
• ~2.1
• Concentration
• 12.12-19.47 ng/μL
Phenol-chloroform method
• Homogenize in Trizol
• DNA partitions to
organic phase
• Protein partitions to
interphase
• RNA partitions to
aqueous phase
• Precipitate RNA and
wash pellet
• Re-suspend in water
Trizol
• Pros
• Extremely high yield. 10 – 100 fold higher than previous
methods.
• Preserves small RNAs including miRNAs
• Cons
• Drying step leaves sample open for contamination
• Reduced 28S peak
Ovary
Small lung
Large lung
Developing a miRNA
sequencing core services
• Isolation quality
• Yield
• Purity
• Reliability
• Instrument performance
• Reproducibility
• Technical replicate
• Biological replicate
• Read quality
Illumina HiSeq
• ~1 Billion reads per lane
• Sequencing by synthesis technology
• 50 bp single-end sequencing
Sequencing beta test
• Biological replicates
• RNA extracted from 2 mouse lungs
• Technical replicates
• RNA from each mouse split in 2
• Sequenced on MiSeq
• 15 million reads
• Compare read abundances of known mature miRNA
sequences
Sequencing beta test
• Regression analysis outputs coefficient of determination
• Technical replicates
• Mouse #1: R2 = 0.999
• Mouse #2: R2 = 0.998
• Biological replicate
• Mouse #1 vs. mouse #2: R2 = 0.995
Overview
1. Introduction
2. RNA-seq method development
3. Discussion
4. Future research
Final RNA-prep method
• Frosted Pyrex homogenizer
• Better for fibrous tissue
• Trizol
• Sufficient yield
• High quality
• Preserves small RNAs
• Qubit Fluorometer
• Bioanalyzer 2100
Overview
1. Introduction
2. RNA isolation method development
3. Discussion
4. Future research
Status of current research
• RNA has been isolated and sequenced
• Awaiting data analysis
• Specific aims
• Develop quality control and miRNA isolation methods for
sequencing
• Test precision and accuracy of sequencing instrument
(Illumina miRNA beta test)
• Transplacental DBC-induced lung cancer yields a
unique miRNA signature relative to control
Tumor vs. healthy lung tissue
Neonatal lung tissue
Neonatal lung tissue
Acknowledgments
Erin Madeen
Tod Harper
Beth Siddens
Sharon Krueger
Kate Field
Christiane Löhr
David Hendrix
Mark Dasenko
David Williams
Brett Tyler