Multi-walled carbon nanotubes-induced alterations in microRNA let-7 and its targets activate a protection mechanism by conferring a developmental timing control
© The Author(s). 2017
Received: 27 March 2017
Accepted: 14 July 2017
Published: 20 July 2017
Multi-walled carbon nanotubes (MWCNTs) have been produced and applied for diverse purposes. MWCNTs can potentially cause the adverse effects on organisms. MicroRNA let-7 and its targets of HBL-1 and LIN-41 play a central role in regulating developmental timing by acting as a developmental switch. The sequence of let-7 and the underlying mechanisms for let-7 in the control of developmental timing are conserved among different species. In this study, we investigated the potential effect of MWCNTs exposure on the molecular basis for developmental timing mediated by let-7 and its targets of HBL-1 and LIN-41.
MWCNTs exposure decreased let-7 expression, and increased expressions of hbl-1 and lin-41. let-7 mutant was resistant to MWCNTs toxicity, whereas hbl-1 or lin-41 mutant was susceptible to MWCNTs toxicity. hbl-1 or lin-41 mutant suppressed the resistance of let-7 mutant to MWCNTs toxicity. HBL-1 acted upstream of TIR-1, an adaptor protein, and SYM-1, a protein containing leucine-rich repeats, and ALG-1 and ALG-2, two RDE-1 proteins related to RNA interference (RNAi) pathway, acted upstream of LIN-41 to regulate MWCNTs toxicity. Moreover, we identified a feedback loop between let-7 and its targets of HBL-1 and LIN-41 in the regulation of MWCNTs toxicity. The observed increase in let-7::GFP expression in MWCNTs exposed nematodes with mutation of hbl-1 or lin-41 implied that a feedback mechanism may exist to strengthen the function of let-7 suppression in protecting the animals from MWCNTs toxicity.
Our results demonstrate the protection function of let-7 suppression for animals from MWCNTs toxicity by conferring a robust developmental timing control. Our results highlight the importance of molecular basis for developmental timing in influencing MWCNTs toxicity.
Multi-walled carbon nanotubes (MWCNTs), one member of the carbon-based engineered nanomaterials (ENMs), have numerous unique physicochemical properties, such as high electrical conductivity and thermal conductivity . MWCNTs have been produced in bulk for diverse purposes, and can be potentially used in several fields, including drug delivery, heterogeneous catalysis to optoelectronic device development, and environmental remediation [2–4]. With the increase in MWCNTs manufacture, the exposure possibility of human and environmental organisms to MWCNTs is also increased [5, 6]. A series of in vitro and in vivo studies have demonstrated several aspects of MWCNTs toxicity on organisms, such as the pulmonary toxicity, through induction of oxidative stress and/or inflammation [7, 8].
Caenorhabditis elegans is a classic non-mammalian model animal . Meanwhile, largely due to its sensitivity to environmental toxicants [10–12], C. elegans has already been widely used for toxicity assessment and toxicological study of various environmental toxicants, including carbon-based ENMs [13–17]. In nematodes, MWCNTs exposure could cause the damage on the functions of both primary targeted organs (such as intestine) and secondary targeted organs (such as neurons and reproductive organs) [18–21]. After exposure, MWCNTs could be potentially translocated into the secondary targeted organs, such as reproductive organs, through the intestinal barrier in nematodes [18, 21–23].
microRNAs (miRNAs) are a class of short noncoding RNAs, and can exhibit their biological functions by inhibiting the expression of targeted genes post-transcriptionally . In nematodes, miRNAs have been shown to play important functions in regulating the toxicity of certain carbon-based ENMs, such as MWCNTs and graphene oxide (GO) [25–29]. For example, mir-355 may regulate the MWCNTs toxicity by inhibiting the function of its target of DAF-2, an insulin receptor . The activated mir-259 may protect the nematodes from MWCNTs toxicity by inhibiting the function of its target of RSKS-1, a putative ribosomal protein S6 kinase . In C. elegans, let-7 is one of the founding members of the miRNA family firstly identified via forward genetic screen . It has been shown that let-7 regulates the timing of larval and adult transition by acting as a developmental switch, and the sequence and functions of let-7 are conserved among different species [30, 31]. During the control of transition of developmental timing, let-7 suppresses the expression and function of its direct targets of HBL-1 and LIN-41 [30, 31]. In this study, we investigated the potential effect of MWCNTs exposure on the molecular basis for developmental timing mediated by let-7 and its targets of HBL-1 and LIN-41 using in vivo assay system of C. elegans. Our results demonstrated that the miRNA let-7 could be decreased by MWCNTs exposure, and was required for the regulation of MWCNTs toxicity. let-7 further regulated MWCNTs toxicity by suppressing the functions of HBL-1 signaling and LIN-41 signaling. Our data imply that the let-7-mediated molecular basis for the control of developmental-timing transition is dysregulated by MWCNTs exposure, and this molecular basis may be required for the control of MWCNTs toxicity in organisms.
Effect of MWCNTs exposure on let-7 expression
Mutation of let-7 induced a resistance to MWCNTs toxicity
let-7 mutation increased the expressions of its targets of HBL-1 and LIN-41 in MWCNTs exposed nematodes
During the development, HBL-1 and LIN-41 have been identified as the direct targets for let-7 in the control of developmental-timing transition [30, 31]. Under normal conditions, let-7 mutation increases the expression of its targets of HBL-1 and LIN-41 [30, 31]. Moreover, let-7 mutation significantly increased the expressions of hbl-1 and lin-41 in MWCNTs exposed nematodes (Additional file 1: Figure S1A). Additionally, MWCNTs could significantly increase the expressions of hbl-1 and lin-41 (Additional file 1: Figure S1B).
Mutation of hbl-1 or lin-41 induced a susceptibility to MWCNTs toxicity
Genetic interaction between let-7 and hbl-1 or lin-41 in the regulation of MWCNTs toxicity
Identification of downstream targets for HBL-1 in the regulation of MWCNTs toxicity
In MWCNTs exposed nematodes, mutation of tir-1 or sym-1 resulted in the more severe induction of intestinal ROS production compared with that in MWCNTs exposed wild-type nematodes (Fig. 5b). In contrast, mutation of lpr-4 did not significantly affect the MWCNTs toxicity in inducing intestinal ROS production (Fig. 5b). Moreover, mutation of tir-1 or sym-1 led to the more severe decrease in locomotion behavior in MWCNTs exposed nematodes compared with that in MWCNTs exposed wild-type nematodes (Fig. 5c). These results suggest that mutation of tir-1 or sym-1 may induce a susceptibility to MWCNTs toxicity.
Genetic interaction between LIN-41 and ALG-1 or ALG-2 in the regulation of MWCNTs toxicity
A feedback loop between let-7 and HBL-1 or LIN-41 in the regulation of MWCNTs toxicity
In this study, we examined the potential effect of MWCNTs exposure on the molecular basis for developmental timing using the in vivo assay system of C. elegans. In C. elegans, the miRNA let-7 and its direct targets of HBL-1 and LIN-41 regulate the transition of developmental timing [30, 31]. After MWCNTs exposure, we observed the decreased expression of let-7::GFP (Fig. 1a). Moreover, we detected the increased expressions of hbl-1 and lin-41 in MWCNTs exposed nematodes (Additional file 1: Figure S1B). Therefore, MWCNTs may affect the molecular basis for developmental timing in animals.
Meanwhile, we investigated the potential role of let-7 in the regulation of MWCNTs toxicity. We found that loss-of-function of let-7 resulted in the formation of resistance to MWCNTs toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 2). Therefore, after MWCNTs exposure, the decreased let-7 may mediate a protection mechanism for nematodes against the MWCNTs toxicity. More recently, it was also reported that Pseudomonas aeruginosa infection could suppress the let-7 expression, and loss-of-function mutation of let-7 enhanced the innate immune response of nematodes to P. aeruginosa infection [36, 37]. The induced decrease in let-7 expression may be helpful for nematodes against the adverse effects from exposure to environmental toxicants or environmental pathogens.
For the normally identified targets of let-7, HBL-1 and LIN-41 [30, 31], we found that mutation of hbl-1 or lin-41 caused the formation of a susceptibility to MWCNTs in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 3). In C. elegans, hbl-1 encodes a C2H2-type zinc finger transcriptional factor, and lin-41 encodes a Ring finger-B box-Coiled coil (RBCC) protein. Both HBL-1 and LIN-41 are heterochronic proteins that play an essential role in the regulation of developmental timing during the postembryonic development [31, 38]. Our results imply that let-7 and its targets may affect the MWCNTs toxicity by conferring a robust developmental timing control.
In C. elegans, hbl-1 or lin-41 translation is negatively regulated by the let-7 that binds to the site(s) in the hbl-1 3’UTR or lin-41 3’UTR [30, 31, 38]. In this study, we further observed that both the hbl-1 and the lin-41 were increased by let-7 mutation in MWCNTs exposed nematodes (Additional file 1: Figure S1A). Moreover, the resistance of let-7(mg279) mutant to MWCNTs toxicity in inducing intestinal ROS production and in decreasing locomotion behavior could be suppressed by hbl-1 mutation or lin-41 mutation (Fig. 4). Similarly, the resistance of let-7 mutants to P. aeruginosa infection could also be suppressed by hbl-1 mutation or lin-41 mutation . Therefore, the miRNA let-7 may regulate both the MWCNTs toxicity and the innate immunity by suppressing the expressions and functions of its direct targets of HBL-1 and LIN-41.
The gene-silencing phenomenon of RNAi is triggered by double-stranded (ds) RNA, and guides the mRNA destruction . In C. elegans, alg-1 and alg-2 are two genes related to the RNAi pathway . In this study, we found that alg-1 or alg-2 mutation induced a resistance to MWCNTs toxicity in inducing intestinal ROS production and in decreasing locomotion behavior (Fig. 7), implying the further involvement of molecular basis for RNAi in the regulation of MWCNTs toxicity. Moreover, we observed that lin-41 mutation suppressed the resistance of alg-1 or alg-2 mutant to MWCNTs toxicity (Fig. 7), indicating that LIN-41 may also regulate the MWCNTs toxicity by acting as the downstream target of ALG-1 or ALG-2 (Fig. 9). The genetic assay has further indicated the genetic suppression of alg-1/alg-2(RNAi) by lin-41 in regulating the heterochronic phenotypes .
In this study, we also observed that both HBL-1 and LIN-41, the let-7’s direct targets, are responsible for the inhibition of transcription of let-7. Moreover, we found that mutation of hbl-1 or lin-41 significantly increased the expression of let-7::GFP in MWCNTs exposed nematodes (Fig. 8), suggesting a feedback loop may be formed between let-7 and its targets of HBL-1 and LIN-41 in the regulation of MWCNTs toxicity. That is, in MWCNTs exposed nematodes, the suppressed let-7 could be further inhibited by its targets of HBL-1 and LIN-41 (Fig. 9). Such a feedback mechanism will further strengthen the function of let-7 suppression in protecting the animals from the MWCNTs toxicity in animals.
In conclusion, we here investigated the potential effect of MWCNTs exposure on the molecular basis for developmental timing using in vivo assay system of C. elegans. We found that MWCNTs exposure could dysregulate the molecular basis for developmental timing as indicated by the altered expression patterns of let-7 and its targets in MWCNTs exposed nematodes. Meanwhile, we observed the resistance of let-7 mutant and the susceptibility of hbl-1 or lin-41 mutant to MWCNTs toxicity, suggesting the potential protection function of let-7 suppression in being against the MWCNTs toxicity. We further found the formation of a feedback loop between let-7 and its targets of HBL-1 and LIN-41 in the regulation of MWCNTs toxicity, which will further strengthen the function of let-7 suppression in protecting the nematodes from the MWCNTs toxicity. Moreover, we identified the signaling cascades of HBL-1-TIR-1/SYM-1 and ALG-1/2-LIN-41 in the regulation of MWCNTs toxicity. Our results highlight the important link between the molecular basis for developmental timing and the regulation of MWCNTs toxicity in organisms.
Characterization of MWCNTs
MWCNTs (diameter: 10–20 nm, length: 5–15 μm) were from Shenzhen Nanotech. Port Co. Ltd. (Shenzhen, China). MWCNTs morphology in K-medium was examined by transmission electron microscopy (TEM, JEM-200CX, JEOL, Japan) (Additional file 1: Figure S2A). Length distribution of MWCNTs was shown in Additional file 1: Figure S2B based on the TEM assay. The presence of Ni (0.077%) and Fe (0.017%) impurities was determined by elemental inductively coupled plasma mass spectrometer (ICPMS). After prolonged exposure, it was found that both 0.077% Ni and 0.017% Fe in 1000 μg/L MWCNTs could not induce the toxicity, such as the induction of intestinal ROS production, on nematodes . Zeta potential of MWCNTs was analyzed by Nano Zetasizer (Nano ZS90, Malvern Instrument, UK). Zeta potential of MWCNTs (1 mg/L) in K-medium was −32.9 ± 2.9 mV.
C. elegans Strains and culture
Nematodes used in this study were wild-type N2, mutants of MT7626/let-7(mg279), CT11/hbl-1(mg285), CT8/lin-41(ma104), RF54/alg-1(gk214), RB574/alg-2(ok304), RB1085/tir-1(ok1052), ZD101/tir-1(qd4), SP2163/sym-1(mn601), hbl-1(mg285)let-7(mg279), lin-41(ma104);alg-1(gk214), lin-41(ma104);alg-2(ok304), and lin-41(ma104);let-7(mg279), and transgenic strains of CT12/zaEx5[let-7::GFP], lin-41(ma104);zaEx5, Ex(Phbl-1-hbl-1), tir-1(ok1052);Ex(Phbl-1-hbl-1), sym-1(mn601)Ex(Phbl-1-hbl-1), and hbl-1(mg285)zaEx5. Some of these strains were from Caenorhabditis Genetics Center (funded by NIH Office of Research Infrastructure Programs (P40 OD010440)). Nematodes were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 °C , and lysed with a bleaching mixture (0.45 M NaOH, 2% HOCl) after washing off the plates into the centrifuge tubes. The age synchronous L1-larvae population was prepared as described previously .
Exposure and toxicity assessment
MWCNTs were dispersed in K medium to prepare a stock solution (1 mg/mL). The stock MWCNTs solution was further sonicated for 30 min (40 kHz, 100 W), and diluted to the working concentrations (10 μg/L) with K medium just prior to exposure. Prolonged exposure to MWCNTs was performed from L1-larvae to young adults in 12-well sterile tissue culture plates at 20 °C in the presence of food (OP50). After MWCNTs exposure, the examined nematodes were used for the toxicity assessment using endpoints of intestinal ROS production and locomotion behavior.
The endpoint of intestinal ROS production was used to reflect the functional state of intestinal cells . Intestinal ROS production was analyzed as described previously [44, 45]. After exposure, the examined nematodes were transferred to 1 μM 5′,6′-chloromethyl-2′,7′-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA; Molecular Probes) solution to incubate for 3 h in the dark. After labeling, the nematodes were mounted on a 2% agar pad for the observation and examination at 488 nm of excitation wavelength and 510 nm of emission filter under a laser scanning confocal microscope (Leica, TCS SP2, Bensheim, Germany). Relative fluorescence intensity of ROS signals in the intestine was semi-quantified and expressed as the relative fluorescence units (RFU). Sixty nematodes were examined per treatment.
The endpoint of locomotion behavior was used to reflect the functional state of motor neurons . Head thrash and body bend were selected to evaluate the locomotion behavior. The head thrash and the body bend were analyzed under a dissecting microscope by eyes as described previously [47, 48]. In C. elegans, a head thrash is defined as a change in the direction of bending at the mid body, and a body bend is defined as a change in the direction of the part of the nematodes corresponding to the posterior bulb of the pharynx along the y axis, assuming that nematode was traveling along the x axis. Sixty nematodes were examined per treatment.
Reverse-transcription and quantitative real-time polymerase chain reaction (qRT-PCR) assay
Total RNAs of nematodes were extracted using an RNeasy Mini kit (Qiagen), and reverse transcribed using a PrimeScript ™ RT reagent kit (Takara, Otsu, Shiga, Japan). After the cDNA synthesis, real-time PCR was performed using the SYBR Premix Ex Taq™ (Takara) for the amplification of certain PCR products. Real-time PCR was run at an optimized annealing temperature of 58 °C. The relative quantification of targeted genes in comparison to a reference tba-1 gene, encoding a tubulin, was determined. The final results were expressed as relative expression ratio between the targeted genes and the reference gene. All the reactions were performed for three biological replicates. For each biological replicate, three technical replicates were performed further. The related primer information for qRT-PCR is shown in Additional file 1: Table S1.
RNAi was performed by feeding nematodes with E. coli strain HT115 (DE3) expressing double-stranded RNA that is homologous to a target gene as described . E. coli HT115 (DE3) was prepared onto NGM containing ampicillin (100 μg/mL) and isopropyl 1-thio-β-D-galactopyranoside (IPTG, 5 mM). L1 larvae were transferred onto RNAi plates for 2 days at 20 °C until they became gravid. Gravid adults were transferred onto the fresh RNAi-expressing bacterial lawns to lay eggs so as to obtain the second generation of RNAi population.
DNA constructs and transformation
Promoter region for hbl-1 amplified by PCR from wild-type C. elegans genomic DNA was inserted into the pPD95_77 vector in the sense orientation. hbl-1/F13D11.2b cDNA was amplified by PCR, and inserted into corresponding entry vector carrying the hbl-1 promoter sequence. Germline transformation was performed as described by coinjecting a testing DNA (30 μg/mL) and a marker DNA (Pdop-1::rfp, 60 μg/mL) into the gonad of nematodes . The related primer information for DNA constructions were shown in Additional file 1: Table S2.
Data in this article were expressed as means ± standard deviation (SD). Statistical analysis was performed using SPSS 12.0 software (SPSS Inc., Chicago, USA). Differences between groups were determined using analysis of variance (ANOVA), and probability levels of 0.05 and 0.01 were considered statistically significant. Graphs were generated using Microsoft Excel software (Microsoft Corp., Redmond, WA).
Availability of data and materials
All the data and materials are available.
DW conceived and designed the experiments. LZ and HW performed the experiments. LZ and HW analyzed the data. DW wrote the paper. All authors read and approved the final manuscript.
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