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Novel cadmium responsive microRNAs in Daphnia pulex Shuai Chen, Garrett Justin McKinney, Krista M. Nichols, John K Colbourne, and Maria S Sepulveda Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03988 • Publication Date (Web): 09 Nov 2015 Downloaded from http://pubs.acs.org on November 12, 2015
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Environmental Science & Technology
Novel cadmium responsive microRNAs in Daphnia pulex
1 2 3
Shuai Chen1, Garrett J. McKinney2, Krista M. Nichols1,3, John K. Colbourne4 and Maria S. Sepúlveda1*
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
1
Department of Forestry and Natural Resources, Purdue University, West Lafayette, Indiana, USA 2 School of Aquatic and Fishery Sciences, University of Washington, Seattle, Washington State, USA 3 Conservation Biology Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Ocean and Atmospheric Administration, Seattle, Washington State, USA 4 Environmental Genomics Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK *Corresponding Author: Maria S. Sepúlveda Address: FORS Room 103 195 Marsteller St West Lafayette, IN 47907-2033 Phone: 765.496.3428 Fax: 765.496.2422 Email:
[email protected]
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Abstract
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Daphnia pulex is a widely used toxicological model and is known for its sensitivity to
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cadmium (Cd). Recent research suggests microRNAs (miRNAs) play a critical role
36
in animal responses to heavy metals.
37
miRNAs under Cd exposure, we analyzed the miRNA profiles of D. pulex after 48
38
hours using miRNA microarrays and validated our findings by q-PCR.
39
dpu-let-7 was identified as a stably expressed gene and used as a reference. We
40
identified 22 and 21 differentially expressed miRNAs under low (20 µg/L CdCl2) and
41
high (40) exposure concentrations compared to controls, respectively.
42
functions of predicted miRNA target Cd-responsive genes included oxidative stress,
43
ion transport, mitochondrial damage and DNA repair.
44
also identified in relation to several Cd-responsive miRNAs. The expression of three
45
predicted target genes for miR-71 and miR-210 were evaluated and expression of two
46
of them (SCN2A and SLC31A1) was negatively correlated with the expression of their
47
regulator miRNAs.
48
propose Cd and hypoxia induce miR-210 via a same HIF1α modulated pathway.
49
Collectively, this research advances our understanding on the role of miRNAs in
50
response to heavy metal exposure.
To investigate the functions of D. pulex
miRNA
Cellular
An insulin-related network was
We show miR-210 is hypoxia-responsive in D. pulex and
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Introduction
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Cadmium (Cd) is highly toxic to aquatic organisms1 and ranks seventh on the
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substance priority list developed by the Agency for Toxic Substances and Disease
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Registry (ATSDR)2. In aquatic animals, Cd ions are up-taken through passive or
58
facilitated diffusion3.
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therefore perturb Ca+2 uptake, transport and homeostasis4,5,6. Furthermore, Cd ions
60
increase the production of reactive oxidative species (ROS) leading to oxidative
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stress, DNA damage, enhanced lipid peroxidation and apoptosis7,8.
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research also suggests ROS inducers such as hypoxia might also lead to cellular
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stress via similar pathways9.
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Within cells, they act as calcium (Ca+2) antagonists and
Previous
MicroRNAs (miRNAs) are a family of short non-coding endogenous RNAs that
65
play a critical role in response to heavy metals including Cd10,11.
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post-transcriptional regulators, miRNAs are involved in a variety of cellular processes
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via negative regulation of their target mRNA12,13.
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gene expression by miRNAs requires the binding of miRNA to the target gene’s
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3’-untranslated region (3’-UTR), resulting in cleavage or suppressed translation14. A
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number of heavy metal responsive miRNAs have been identified in vertebrates; their
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functions have been associated with regulatory transcription factors, metabolic
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processes and stress responses15,16,17.
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miRNAs and heavy metals makes miRNAs promising molecules for elucidating the
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mechanisms involved in heavy metal response, given its role in gene regulation.
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However, the role of miRNAs in response to heavy metal exposure in aquatic
As key
Generally, the inhibition of target
The rapidly growing evidence linking
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invertebrates is largely unknown, despite the fact that heavy metals are persistent in
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aquatic environments and can bioaccumulate and biomagnify in the food chain18,19.
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A better understanding on the regulatory role of miRNAs in these organisms after
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heavy metal exposure is needed.
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Daphnia pulex is a branchiopod crustacean that resides in continental waters and
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are widely used as a toxicological model organism. This species is also the first
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crustacean to have its genome sequenced and annotated20, thereby represents an
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ideal candidate for aquatic invertebrate Cd-responsive miRNA research.
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cornerstone species in freshwater ecosystems, Daphnia are important sentinels and
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have much accumulated knowledge on their acute responsiveness and rapid
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acclimation to Cd21,22,23. In daphiids, acclimation to Cd is thought to be mostly driven
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by upregulation of metallothionein genes24.
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miRNAs play critical roles during D. pulex development25, however, no studies have
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examined the role of miRNAs on Cd acclimation in daphniids.
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work are two-fold: 1) identify Cd-responsive D. pulex miRNAs; and 2) study the
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potential regulatory mechanisms of these miRNAs under Cd exposure.
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studies are the first to report on the role of miRNAs in aquatic invertebrates in
93
response to heavy metals.
As a
Our previous research revealed that
The objectives of this
These
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Materials and Methods
95 96 97
Animal culture D. pulex were obtained from Carolina Biological (Burlington, NC, USA).
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Cultures were maintained at a density of approximately 35 individuals/L in
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reconstituted moderate hard water (RMHW)26 in 3 L plastic tanks in an environmental
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chamber (20°C on a 16/8 light/dark cycle).
Culture water was changed twice a week
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and D. pulex were fed 15 ml YCT mix (yeast, cereal, trout chow) after every water
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change. In order to prevent potential maternal or grand maternal co-variance, D.
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pulex were grown in the environmental chamber for four generations before the start
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of exposures and only neonates (< 24 h) from the third through eighth brood were
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used for exposures.
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Daphnia tests and sample preparation
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Cadmium exposure
Animals were not fed during the exposure test.
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Cadmium exposures were conducted by exposing 70 neonates to 2 L of either 20
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µl/L CdCl2 (Cd-low, ~48 h LC01 value), 40 µl/L CdCl2 (Cd-high, ~48 h LC10 value), or to
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RMHW (control) for 48 h24.
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were diluted from a stock solution of 100 mg/L CdCl2 (Sigma-Aldrich, St. Louis, MO,
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USA) using RMHW.
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(ThermoFinnigan, Bremen, Germany) mass spectrometer in the medium resolution
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mode27 and all measured concentrations varied within 10% of the nominal
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concentrations.
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Hypoxia exposure
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Each treatment included six replicates and Cd solutions
Cd concentrations were tested using an ELEMENT-2
Predicted targets of Cd-responsive miR-210 suggest it should also be involved in
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D. pulex response to hypoxia.
Because both hypoxia and Cd can induce ROS
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production28,29 and mammalian miR-210 is known to be up-regulated by
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hypoxia-induced ROS production30, we tested the hypothesis that miR-210 is
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hypoxia-inducible in D. pulex.
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our hypothesis.
A follow up hypoxia test was then carried out to test
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Hypoxic exposures were carried out by introducing 70 neonates to 2 L of either
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hypoxic RMHW (dissolved oxygen, DO, 1.0-1.5 mg/L), or normoxic RMHW (DO,
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8.0-9.0 mg/L) for 48 h. Hypoxic or normoxic RMHW was produced by aerating N2 or
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fresh air into RMHW31, respectively.
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optical dissolved oxygen meter (YSI, Yellow Springs, Ohio, USA).
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had three replicates.
DO was measured hourly using a YSI ProODO Each treatment
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Immediately after exposure, 50 neonates from each replicate were randomly
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selected and pooled for small RNA (including miRNAs) extraction using protocols
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already published25.
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sample was extracted using a Purelink Micro-to-Midi total RNA purification kit
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(Invitrogen, Carlsbad, CA, USA) following the manufacture’s protocol. Small RNA
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and total RNA extracts were treated with DNAse I (ThermoScientific, Waltham, MA,
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USA) following manufacture’s protocols.
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were quantified using NanoDrop (Invitrogen, Carlsbad, CA, USA) and stored at -80°C
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until microarray or q-PCR analysis as described below.
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miRNA microarray analysis
Total RNA (with exception of small RNAs) from the same
After DNAse treatment, all RNA samples
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Affymetrix GeneChip 4.0 (Affymetrix, Santa Clara, CA, USA) miRNA microarrays
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(catalog number: 902412) were used to determine differences in miRNA expression
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upon exposure to Cd.
Microarray analyses were conducted at the Center for
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Medical Genomics at Indiana University School of Medicine, Indianapolis, IN.
Small
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RNA was extracted as already described and 300 ng from each of 3
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samples/treatment was labeled using the Affymetrix FlashTag Bioton HSR kit.
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Labeled samples were hybridized to the arrays for 17 hours and stained and washed
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using standard protocols32.
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used to scan the arrays and generate CEL files containing information on the intensity
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values of the individual probes. Arrays were visually scanned for abnormalities or
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defects.
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USA) to generate RMA (robust multi-array average) expression levels using the RMA
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normalization option32. Only miRNAs with a Log2 average expression level (Log2
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AE) >2 were further investigated. Significant changes in miRNA expression were
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identified by one-way ANOVA followed by Tukey’s HSD post-hoc test (Cd-low vs
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Control, Cd-high vs Control) with Benjamini and Hochberg correction33 (FDR
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4).
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example, cte-miR-279 (log2AE = 13.34) and dwi-miR-279 (log2AE = 5.90) were
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grouped in the miR-279 family.
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sequence of miRNA with highest log2AE as a novel D. pulex miRNA sequence (e.g.
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cte-miR-279 had the highest log2AE level in the miR-279 family, thus the sequence of
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cte-miR-279 was selected as the miR-279 sequence in D. pulex. The probes on the
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microarray differ from the annotated D. pulex locus for this gene by only 3 of 21
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aligned nucleotides in the 3’ end.
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miRNA cDNA preparation
To identify robustly expressed D. pulex miRNAs, we
Secondly, we grouped the robustly expressed miRNAs by family name. For
And lastly, from each miRNA family, we selected the
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miRNA-specific stem-loop primers and forward primers (Table S2) were designed
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following the method described by Chen et al.36. miRNAs were reversed-transcribed
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by mixing 2 pmol of stem-loop primer, 0.5 mM dNTPs, 2 µg of RNA template and
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incubated at 65°C for 5 min.
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of RNase OUT and 2.5 units of SuperScript III were then added and incubated on ice
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for 2 min.
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30°C for 30 sec, 42°C for 30 sec and 50°C for 1 sec and then incubated at 85°C for 5
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min.
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-20°C until q-PCR analysis.
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q-PCR reference gene selection and validation
Another mix of 1X first-strand buffer, 5 mM DTT, 2 units
The final mix was incubated at 16°C for 30 min followed by 60 cycles at
The reverse transcribed products were quantified by NanoDrop and stored at
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The protocol for miRNA q-PCR was adapted from Chen et al.25. Each q-PCR
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reaction contained 4 µM SYBR Green I master mix (Bio-Rad, Hercules, CA, USA), 1
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µM forward primer, 1 µM reverse primer and 2 µl RT product. q-PCR was conducted
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on a Bio-Rad real-time PCR system (Bio-Rad, Hercules, CA, USA) using the following
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program: 95°C for 5 min, 40 cycles of 95°C for 5 sec, 60°C for 10 sec and 72°C for 1
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sec.
A melting curve analysis was performed to check for primer-dimers.
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Although our previous research identified U6 ribosomal RNA as a stable
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expressed reference gene during D. pulex development25, this gene is not stably
194
expressed under Cd exposure (data not shown).
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gene selection process to identify stably expressed miRNAs under Cd exposure.
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Fourteen highly expressed (log2AE > 4) but not differentially expressed miRNAs (let-7,
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miR-1, miR-2, miR-7, miR-8, miR-9, miR-10, miR-12, miR-33, miR-34, miR-100,
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miR-153, miR-283, and miR-317) identified by microarray were selected as candidate
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reference genes.
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Cd-high and control groups following the method described above.
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included three biological replicates and each biological replicate included three
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technical replicates. Two algorithms, geNorm37 and NormFinder38, were performed
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to identify stably expressed reference gene under Cd exposure.
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candidate genes were transformed to ∆Ct values and imported into geNorm.
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geNorm calculates an M value for each gene, which represents the combined
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variation within treatment group and between candidate genes.
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expressing the smallest M values have the highest expression stability.
Thus, we conducted a reference
q-PCR was conducted using miRNA samples from the Cd-low,
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The Ct values of
Transcripts The Ct
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values were transformed into relative quantities and imported to NormFinder.
The
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NormFinder algorithm calculates a stability (S) value for each gene based on the
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inter- and intra-group variance.
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expression stability.
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of determination.
The gene with the lowest S value has the highest
The correlation between M and S was measured by coefficient
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Seven differentially expressed miRNAs were selected for q-PCR validation, and
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included six significantly upregulated miRNAs (miR-71, miR-92, miR-204, miR-210,
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miR-252, and miR-279), and one significantly down-regulated miRNA (miR-7444).
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q-PCR was performed using three miRNA samples from each treatment group with
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three technical replicates for each sample following the method already described.
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Fold-changes (Cd-low vs Control, Cd-high vs Control) were calculated based on the
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expression of controls using ∆∆Ct method39 and normalized to the expression of
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Dpu-let-7, the gene identified as the most stably expressed.
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test, q-PCR was performed using three miRNA samples from hypoxia and normoxia
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groups with three technical replicates.
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calculated based on the expression of the normoxia group (control) using the ∆∆Ct
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method39 and normalized to the expression of Dpu-let-7.
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performed
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Cd/hypoxia-exposed animals (p 4) in
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D. pulex (Table S4), which included all previously predicted D. pulex miRNAs25 plus
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the highly conserved dpu-let-7, which had not been predicted earlier.
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miRNA clusters such as the miR-71/2 cluster were identified, supporting prior
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research that the miR-71/2 cluster is Protostome-specific51.
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Reference gene selection and q-PCR validation of microarray results
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Dpu-let-7 was identified as the most stably expressed reference gene by both
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geNorm and NormFinder, having both the smallest M and S values, respectively
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(Figure S2).
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therefore considered as stably expressed), which further confirmed microarray results
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that the expression of these miRNAs is no affected under Cd exposure. Let-7 has
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also been identified as a stably expressed reference gene in other species52, which
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further confirms its expression stability.
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were able to validate microarray results by q-PCR; for all genes tested, the q-PCR
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expression patterns matched the microarray results (Figure 2).
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Predicted miRNA target genes and their relation to Cd toxicityf
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All 14 candidate reference genes had M values of less than 1.5 (and
Using dpu-let-7 as a reference gene, we
We focused on highly conserved miRNAs related to Cd-induced cellular stress for
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target gene prediction and validation of expression.
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during computational target prediction, we compared miRNA targets predicted by both
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the thermodynamic method and the seed complementary method. Only genes that
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were 1) predicted by both methods; or 2) had multiple seed binding sites on its 3’UTR
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for one specific miRNA were selected as promising miRNA targets.
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inspection of predicted targets for the Cd-responsive miRNAs illustrates their function
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is related with Cd-induced cellular stress that includes 1) alteration of ion hemostasis;
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2) mitochondrial damage; and 3) DNA damage (Table S5).
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To diminish false positives
A detailed
Alteration of ion hemostasis: Cd is a known Ca+2 antagonist that can block Ca+2
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channels and modulate ion homeostasis5.
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and miR-71 are likely involved in Cd-induced alteration of ion homeostasis by
Predicted target genes indicate miR-210
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targeting
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calcium-transporting ATPase type 2C (ATP2C2) and Ca+2-transporting ATPase
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sarcoplasmic/endoplasmic reticulum type (Ca-P60A).
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miR-71 should decrease ATP2C2 and Ca-P60A expression.
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supported by previous research showing that Cd can inhibit the activity of Ca+2
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ATPase53,54.
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sodium
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Ca+2-activated potassium channel protein (SK channel protein, KCNN1), sodium
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channel
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hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) are predicted to
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be the targets of miR-210.
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ion hemostasis.
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Cd-induced miR-71, and miR-210 could down-regulate the sodium channel protein by
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acting synergistically.
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can inhibit sodium channel activity55.
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channels, miR-210 and miR-71 might also co-regulate copper transport by targeting
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copper-transporting ATPase 1 gene (ATP7A) and the high affinity copper transporter
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gene (SLC31A1), respectively (Table S5).
336
a
and
number
of
key
ion
transport
proteins(Table
S5)
such
as
Cd up-regulated miR-210 and This finding is
Predicted targets also suggest that miR-210 and miR-71 regulate potassium
protein
ion
type
2
channels.
subunit
alpha
For
instance,
(SCN2A)
and
small
conductance
potassium/sodium
These ion channels play a critical role in maintenance of
SCN2A is also predicted as a target of miR-71, which suggests
This finding is in agreement with previous research that Cd In addition to Ca+2, sodium and potassium
Mitochondrial and DNA damage: Mitochondrial dysfunction is another important
337
aspect of Cd toxicity.
MiR-252 appears to be related to Cd-induced mitochondrial
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damage by targeting two mitochondrial ribosomal proteins (MRPs)(Table S5).
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Cd-induced miR-252 expression could decrease the expression of MRPs, which
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would cause inhibition of mitochondrial function. However, further investigation is
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needed to test this hypothesis.
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induce DNA damage by inhibiting mismatch repair56,57.
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miR-210 target is DNA mismatch repair protein (MLH3) which is known to be inhibited
344
by Cd57.
345
Target gene expression profile
346
As a mutagen and carcinogen, Cd is also known to Another notable predicted
To examine the negative correlation of miRNA-target expression, we quantified
347
the expression of three predicted target genes: SCN2A, KCNN1 and SLC31A1.
As
348
expected, the expression of SCN2A was negatively correlated with that of its regulator
349
miR-210 (Figure 3). Similarly, the expression of SLC31A1 was negatively correlated
350
with the expression of its regulator miRNA miR-71, but only in animals from the
351
Cd-low treatment.
352
promising targets for miR-210 and miR-71.
353
significantly changed under either Cd treatment, which could be explained by
354
involvement of other regulatory mechanisms (e.g. transcription factors and DNA
355
methylation)58 or a false positive computational prediction.
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validation such as luciferase assays are needed to prove these miRNA-target
357
relationships.
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Cd-responsive miRNAs regulatory network
This result further confirms that SCN2A and SLC31A1 are The expression of KCNN1 was not
Further experimental
359
Our IPA analysis revealed only one significant network (p-score=22, p=10-22)
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involving several Cd-responsive miRNAs in relation to insulin metabolism (Figure S3,
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Table S6).
For instance, Cd-induced miR-92 (synonym of miR-25) and miR-204 can
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block insulin production59,60 which results in an up-regulation of miR-210 and
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miR-61561. In addition, down-regulation of miR-144 decreases the activity of PGTS2
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(prostaglandin endoperoxide synthase 2), which can also reduce the secretion of
365
insulin.
366
growth factor, beta 1) which causes a down-regulation of miR-21662 and miR-21763.
367
The insulin pathway plays a critical role in predator-induced body size plasticity in D.
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pulex64,65.
369
development66,67.
370
reductions of D. pulex body length after Cd exposure24,68. In addition, IPA revealed
371
that the “Top Diseases and Bio Function” related with differentially expressed miRNAs
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is “organismal injury and abnormalities” which is related with Cd induced cellular
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stress69.
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Cd and hypoxia induce miRNA-210 via the HIFα pathway
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Further, reduced insulin levels decrease the activity of TGFB1 (transforming
TGFB1 regulates cell growth and proliferation as well as skeleton Thus, a reduction of insulin and TGFB1 levels might explain
Other than the Cd-related genes mentioned above, a hypoxia up-regulated gene
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HYOU1 was also identified as a target of miR-210.
This finding suggests that
377
miR-210 could also modulate cellular responses under hypoxia in D. pulex.
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miR-210 was previously identified as the predominantly up-regulated miRNA under
379
hypoxia in mammalian cell lines30, we speculated that miR-210 should also be
380
up-regulated in D. pulex under hypoxia.
381
miR-210 expression under hypoxic and normoxic conditions and observed a
382
significant up-regulation of miR-210 under hypoxia (Figure 4).
383
that hypoxia induces miR-210 via a conserved pathway across taxa.
Because
To test this prediction, we profiled the
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Our results indicate
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Hypoxia-induced
miR-210 1
expression α
(HIF1α)
occurs protein
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via
stabilization
which
binds
of
385
hypoxia-inducible-factor
to
386
hypoxia-responsive-elements (HRE) on the miR-210 promoter region70,71.
387
the HRE sequence ((A/G)CGTG) in the miR-210 promoter region is highly conserved
388
across mammalian species72.
389
HIF1α by increased ROS production28.
390
stabilization of HIF1α, which promotes miR-210 expression, we hypothesized that the
391
up-regulation of D. pulex miR-210 is due to Cd-induced HIF1α stabilization (Figure 4).
392
To address this hypothesis, we searched for the D. pulex homologs by BLAST73 of the
393
human HIF1α gene against the D. pulex genome and identified a homologous HIF1α
394
gene in D. pulex (E Value= 1e-44).
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documented in OrthoDB74 (protein ID EFX84860).
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miR-210 promoter region within the D. pulex genome and found a HRE motif ~400 bp
397
upstream from the coding region. We also analyzed the miR-210 promoter region of
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D. magna - a closely related species to D. pulex.
399
motifs on the D. magna miR-210 promoter region, which implicate this HRE motif is
400
also conserved in Daphnia.
401
responsive to hypoxia and indicates a conserved regulatory pathway of HIF1α
402
induced miR-210 expression, which further support our hypothesis.
Further,
Previous research has shown Cd can also induce Since hypoxia and Cd can both induce
This homologous HIF1α gene has also been Secondly, we searched the
Our search identified two HRE
Our investigation revealed that miR-210 is also
403
In conclusion, our research provides the basis for D. pulex miRNA research,
404
including identification of a stably expressed reference gene under Cd exposure and a
405
list of Cd-responsive miRNAs with potential targets. To our knowledge, this is the
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first study on miRNA responses in aquatic invertebrates after heavy metal exposure.
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Finally, we have shown that miR-210 is also responsive to hypoxia via a proposed
408
HIFα modulated pathway.
409
environmental stress.
Our results indicate miR-210 is a promising marker of
410 411
Acknowledgements The authors would like to thank Dr. Don Gilbert, Indiana University, IN for
412 413
elaborations on the D. pulex genome annotations.
The authors gratefully
414
acknowledge funding from the Department of Forestry and Natural Resources,
415
Purdue University, IN in the form of an assistantship for Shuai Chen. Microarray
416
studies were carried out at the Center for Medical Genomics, Indiana University
417
School of Medicine, IN which is partially supported by the Indiana Genomic Initiative
418
at Indiana University (INGEN); INGEN is supported in part by the Lilly Endowment,
419
Inc. Our work benefits from and contributes to the Daphnia Genomics Consortium.
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Figure and Table Legends Figure 1. (A) Microarray identified significantly altered miRNAs (p-value < 0.05 and fold change > 1.5 compared with control group) in Daphnia pulex neonates exposed to low (20 µg/L CdCl2) and high (40 µg/L CdCl2) cadmium (Cd) for 48 h. (B) Heatmap of significantly expressed miRNAs. Figure 2. Validation of miRNA microarray results by q-PCR. Bars plotted represent means and standard errors with black bars representing microarray results and gray bars representing q-PCR results. Asterisks denote significant differences from controls (p
