The Expression of miRNAs Involved in Long-Term Memory Formation in the CNS of the Mollusk Helix lucorum.

Mollusks are unique animals with a relatively simple central nervous system (CNS) containing giant neurons with identified functions. With such simple CNS, mollusks yet display sufficiently complex behavior, thus ideal for various studies of behavioral processes, including long-term memory (LTM) formation. For our research, we use the formation of the fear avoidance reflex in the terrestrial mollusk Helix lucorum as a learning model. We have shown previously that LTM formation in Helix requires epigenetic modifications of histones leading to both activation and inactivation of the specific genes. It is known that microRNAs (miRNAs) negatively regulate the expression of genes; however, the role of miRNAs in behavioral regulation has been poorly investigated. Currently, there is no miRNAs sequencing data being published on Helix lucorum, which makes it impossible to investigate the role of miRNAs in the memory formation of this mollusk. In this study, we have performed sequencing and comparative bioinformatics analysis of the miRNAs from the CNS of Helix lucorum. We have identified 95 different microRNAs, including microRNAs belonging to the MIR-9, MIR-10, MIR-22, MIR-124, MIR-137, and MIR-153 families, known to be involved in various CNS processes of vertebrates and other species, particularly, in the fear behavior and LTM. We have shown that in the CNS of Helix lucorum MIR-10 family (26 miRNAs) is the most representative one, including Hlu-Mir-10-S5-5p and Hlu-Mir-10-S9-5p as top hits. Moreover, we have shown the involvement of the MIR-10 family in LTM formation in Helix. The expression of 17 representatives of MIR-10 differentially changes during different periods of LTM consolidation in the CNS of Helix. In addition, using comparative analysis of microRNA expression upon learning in normal snails and snails with deficient learning abilities with dysfunction of the serotonergic system, we identified a number of microRNAs from several families, including MIR-10, which expression changes only in normal animals. The obtained data can be used for further fundamental and applied behavioral research.

Expression of the miR-190 family is increased under DDT exposure in vivo and in vitro.

A non-genotoxic insecticide dichlorodiphenyltrichloroethane (DDT), can affect mRNA and microRNA levels, however, its precise mechanism of action remains poorly understood. Using in silico methods we found that the rat miR-190 family is potentially regulated by CAR and ER receptors activated by DDT. We showed that exposure to DDT results in a dose- and organ-dependent increase in the expression of miR-190a, -190b in the liver, uterus, ovaries and mammary gland of female Wistar rats. Additionally, we demonstrate a decrease in protein product level of Tp53inp1, the target gene of these microRNAs, in the rat uterus. It is known that miR-190 is probably regulated by ER in humans, thus we measured the level of miR-190a, -190b in primary cultures of malignant and normal human endometrial cells treated with different doses of DDT. We detected an increase in miR-190b level in normal endometrial cells under DDT exposure. Thus, our results indicate that DDT exposure lead to change in the expression of oncogenic miR-190 family and its target gene Tp53inp1 which may be due to activation of CAR and ER.

EV-transported microRNAs of Schistosoma mansoni and Fasciola hepatica: Potential targets in definitive hosts.

Trematodes are widespread parasitic flatworms that significantly affect mankind either directly as human parasites, or indirectly via the infection of livestock and the related economic damage. The two most important trematode taxa are the blood flukes Schistosoma and the liver flukes Fasciola, but detection and differentiation of these parasites remains a challenge. Recently, microRNAs (miRNAs) were described from extracellular vesicles (EV) for both parasites secreted into respective hosts. These molecules have been proposed as mediators of parasite-host communication, and potential biomarkers for the detection of parasitic infections from host blood. Our aim here was to study similarities and differences in the miRNA complements of Schistosoma mansoni and Fasciola hepatica, EV-load in particular, to predict their targets and potential functions in the parasite-host interaction. We reanalyzed the known miRNA complements of S. mansoni and F. hepatica and found 16 and 4 previously overlooked, but deeply conserved miRNAs, respectively, further moving their complements closer together. We found distinct miRNA enrichment patterns in EVs both showing high levels of flatworm miRNAs with potential for the detection of an infection from blood. Two miRNAs of the protostome specific MIR-71 and MIR-277 families were highly expressed in EVs and could, therefore, have potential as biomarkers for trematode infection. Curiously, we identified nucleotide differences in the sequence of Mir-277-P2 between S. mansoni and F. hepatica that hold great promise for the distinction of both parasites. To test whether the EV-miRNAs of S. mansoni and F. hepatica could be modulating the expression of host genes, we predicted miRNA targets in 321 human and cattle messenger RNAs that overlapped between both hosts. Of several predicted targets, wnt signaling pathway genes stood out and their suppression likely leads to changes in the glucose concentration in host blood and the reduction of inflammatory and immune responses.

The search of CAR, AhR, ESRs binding sites in promoters of intronic and intergenic microRNAs.

MicroRNAs (miRNAs) play important roles in the regulation of gene expression at the post-transcriptional level. Many exogenous compounds or xenobiotics may affect microRNA expression. It is a well-established fact that xenobiotics with planar structure like TCDD, benzo(a)pyrene (BP) can bind aryl hydrocarbon receptor (AhR) followed by its nuclear translocation and transcriptional activation of target genes. Another chemically diverse group of xenobiotics including phenobarbital, DDT, can activate the nuclear receptor CAR and in some cases estrogen receptors ESR1 and ESR2. We hypothesized that such chemicals can affect miRNA expression through the activation of AHR, CAR, and ESRs. To prove this statement, we used in silico methods to find DRE, PBEM, ERE potential binding sites for these receptors, respectively. We have predicted AhR, CAR, and ESRs binding sites in 224 rat, 201 mouse, and 232 human promoters of miRNA-coding genes. In addition, we have identified a number of miRNAs with predicted AhR, CAR, and ESRs binding sites that are known as oncogenes and as tumor suppressors. Our results, obtained in silico, open a new strategy for ongoing experimental studies and will contribute to further investigation of epigenetic mechanisms of carcinogenesis.

Extreme conservation of miRNA complements in opisthorchiids.

MicroRNAs (miRNAs) are important gene regulators that are key players in animal development and diseases. They are excreted in extracellular vesicles and because they were shown to be taken up by host cells they have been proposed as mediators of parasite-host communication, and potential biomarkers for the detection of parasitic infections from host blood. Consequently, it is crucial to precisely know the miRNA complements of medically important agents such as the liver flukes of the Opisthorchiidae. Using publicly available and new datasets we curated and reannotated the surprisingly small and variable miRNA complements previously described for Opisthorchis viverrini, O. felineus and Clonorchis sinensis. We find three highly similar miRNA complements with 53 identical and two miRNA genes with species specific sequences that signify a set of potential biomarkers and promising candidates for further investigations.

Identification of microRNA genes in three opisthorchiids.

BACKGROUND: Opisthorchis felineus, O. viverrini, and Clonorchis sinensis (family Opisthorchiidae) are parasitic flatworms that pose a serious threat to humans in some countries and cause opisthorchiasis/clonorchiasis. Chronic disease may lead to a risk of carcinogenesis in the biliary ducts. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression at post-transcriptional level and are implicated in the regulation of various cellular processes during the parasite- host interplay. However, to date, the miRNAs of opisthorchiid flukes, in particular those essential for maintaining their complex biology and parasitic mode of existence, have not been satisfactorily described. METHODOLOGY/PRINCIPAL FINDINGS: Using a SOLiD deep sequencing-bioinformatic approach, we identified 43 novel and 18 conserved miRNAs for O. felineus (miracidia, metacercariae and adult worms), 20 novel and 16 conserved miRNAs for O. viverrini (adult worms), and 33 novel and 18 conserved miRNAs for C. sinensis (adult worms). The analysis of the data revealed differences in the expression level of conserved miRNAs among the three species and among three the developmental stages of O. felineus. Analysis of miRNA genes revealed two gene clusters, one cluster-like region and one intronic miRNA in the genome. The presence and structure of the two gene clusters were validated using a PCR-based approach in the three flukes. CONCLUSIONS: This study represents a comprehensive description of miRNAs in three members of the family Opistorchiidae, significantly expands our knowledge of miRNAs in multicellular parasites and provides a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites. Results of this study also provides novel resources for deeper understanding the complex parasite biology, for further research on the pathogenesis and molecular events of disease induced by the liver flukes. The present data may also facilitate the development of novel approaches for the prevention and treatment of opisthorchiasis/clonorchiasis.