Interesting miR-71 targets in
MicroRNAs (miRNAs) found in animals, plants, and some viruses belongs to the heterogeneous class of non-coding RNAs (ncRNAs), which posttranscriptional regulates gene expression. They are linked to various cellular activities such as cell growth, differentiation, development and apoptosis. Also, they have been involved in cancer, metabolic diseases, viral infections and clinical trials targeting miRNAs has shown promising results. This chapter provides an overview on Taenia solium and Taenia crassiceps miRNAs, their possible biological functions, their role in host–parasite communication and their potential role as biomarkers and drug targets.
- small noncoding RNAs
- drug target
1.1 Overview of miRNAs: definition, biogenesis, and functions
MicroRNAs (miRNAs) are a major class of small noncoding RNAs (ncRNAs) found in animals, plants, and some viruses, which negatively regulate gene expression at the messenger RNA (mRNA) level . The first known miRNA (lin-4) was found in the free-living nematode
It is well established that miRNA biogenesis is a complex process classified into canonical and non-canonical pathways. The canonical biogenesis is the dominant pathway by which miRNAs are processed . This pathway includes three main steps: (i) In the nucleus, miRNA genes are transcript by RNA polymerase II as part of much longer RNAs called pri-miRNAs which contain one or a few stem-loop structures composed of approximately 70 nucleotides each (Figure 1). Sometimes miRNAs are transcribed as one long transcript called clusters, which may have similar seed regions, and in which case they are considered a family . About half of all currently identified miRNAs are intragenic and processed mostly from introns and relatively few exons of protein coding genes, while the remaining are intergenic, transcribed independently of a host gene and regulated by their own promoters  (ii) Then, the microprocessor complex, consisting of an RNA binding protein DiGeorge Syndrome Critical Region 8 (DGCR8) and a ribonuclease III enzyme, Drosha trims the pri-miRNA to liberate a pre-miRNA hairpin which is actively transported to the cytoplasm by 5 (XPO5)/RanGTP complex (Figure 1) (iii) Its final maturation is processed in the cytoplasm where Dicer RNase III endonuclease cleaves the pre-miRNA into a single-stranded mature miRNA, removing the terminal loop (Figure 1) . The directionality of the miRNA strand determines the name of the mature miRNA form . Subsequently, with assistance from chaperone proteins (HSC70/HSP90) the mature miRNA is loaded into proteins of the Argonaute (Ago) family and assembles the RNA induced silencing complex (RISC) together to exert its further physiological functions (Figure 1). We defined the unloaded strand as passenger or star strand. The start strand that contains no mismatches are cleaved by AGO2 and degraded (Figure 1). Also, miRNA duplexes with central mismatches or non-AGO2 loaded miRNA are unwound and degraded. After being incorporated into the RISC, the mature miRNA induces posttranscriptional gene silencing by tethering RISC to be partially complementary to the target mRNA predominantly found within the 3′-untranslated regions (UTR). Targeting can also be facilitated by additional sequence elements, such as an unpaired Adenosine in the mRNA target sequence, corresponding to the nucleotide 1 in 5′ end of the mature miRNA. On the other hand, non-canonical miRNA biogenesis pathways are grouped into Drosha/DGCR8-independent and Dicer-independent pathways. In general, these pathways make use of different combinations of the proteins involved in the canonical pathway, mainly Drosha, Dicer, exportin 5, and AGO2 .
It has been estimated that miRNAs regulate the expression of approximately one-third of the protein-coding genes . Each miRNA can have many target mRNAs and a single mRNA can be regulated by multiple miRNAs . Given this vast majority of mRNA targets regulated by miRNAs, aberrant miRNA expression profoundly influences a wide variety of cell regulation pathways important to cell proliferation, apoptosis, and stress responses.
Single miRNA gene can generate multiple miRNA isoforms (isomiRs). For example, the inconsistent choice of the strand loaded into AGO can generate two different functional miRNAs from both strands of the pre-miRNA duplex . Imprecise cleavage of pri-miRNA by Drosha or pre-miRNA by Dicer can generate heterogeneous 5′ or 3′ ends. Another way to generate isomiRs is by RNA editing which may have a functional impact when the seed nucleotide is altered . Exonuclease activity could remove nucleotides from miRNA 3′ end, and terminal nucleotide-transferase could add nucleotides to the miRNA 3′ end generating different isomiRs.
For almost a decade, some of the miRNA genes have been categorized into different groups, named miRNA families, based on the mature miRNA sequence and/or structure of pre-miRNAs . Thus, means that microRNAs are grouped into families based on their targeting properties, which depend primarily on the identity of their extended seed region (miRNA nucleotides 2–8) . Interestingly, it has been observed that miRNA genes in the same miRNA family are non-randomly co-localized and well organized around genes involved in infectious, immune system, sensory system and neurodegenerative diseases, development and cancer . As with paralogous proteins, members of the same seed families often have at least partially redundant functions, with severe loss-of-function phenotypes apparent only after multiple family members are disrupted .
Biological functions of individual miRNAs have been extensively explored and have revealed the important role of miRNAs in many biological functions such as developmental timings, cell differentiation, embryogenesis, metabolism, organogenesis, and apoptosis . Thus, miRNAs have been introduced as therapeutics or as targets of therapeutics for the treatment of disease . Also, the existence of extracellular miRNAs has been widely reported these molecules as potential biomarkers for a variety of diseases. At present, miRNAs-mediated therapies for treatment of cancer and chronic hepatitis C virus (HCV) infection have shown promising results in human Phase I clinical trial .
2. miRNAs in
Taenia soliumand Taenia crassiceps
The presence of homologs to Drosha, Dicer, and Pasha (as identified in the
The expression profile of
The identification and characterization of miRNA targets is essential for understanding the function of these ncRNAs at molecular level. MiR-10 is the most expressed miRNA in
|TsM_001055500||Zinc finger, type RING / FYVE / PHD||—||bantam; miR-4989|
|TsM_001107400||Ubiquitin carboxyl terminal hydrolase (inferred by orthology to a protein from ||Thiol-dependent ubiquitinyl hydrolase activity||miR-36a; miR-36b|
|TsM_000718200||Ceramide glucosyltransferase (inferred by orthology to a human protein)||Transferase activity, transfer of glycosyl groups||let-7|
|TsM_000983300||Casp, putative (inferred by orthology to a protein from S. mansoni)||—||miR-71|
|TsM_000787600||Protein kinase-like domain. Tyrosine-protein kinase, active site.||Protein kinase activity, protein serine / threonine kinase activity, ATP binding, phosphotransferase activity, alcohol group as acceptor||miR-9|
|TsM_000964500||IUAA Family Transporter||—||miR-10|
|TsM_000502500||Fibroblast growth factor receptor homolog 1 (inferred by orthology to a ||Protein kinase activity, protein tyrosine kinase activity, ATP binding||miR-124a; miR-124b|
|TsM_000692000||Mitochondrial coenzyme transporter A SLC25A42 (inferred by orthology to a human protein)||—||miR-61|
Parasite miRNAs that are absent in the host, such as miR-71 or highly divergent (e.g let-7) from their host orthologs may be considered as selective therapeutic targets for treatment and control of helminth parasite infections. In addition, miR-71 is highly expressed in
2.1 miRNAs and immune response
Helminth’s parasites modulate immune responses in their host to prevent their elimination and establish chronic infections. Neurocysticercosis (NCC) implicates chronic parasitic disease with different variety of host and parasite interactions . Clinical manifestations are mainly the result of inflammatory response to degeneration of parenchymal cysticercus .
Already knowing that the more abundant miRNAs (miR-10-5p, let-7-5p) putatively have target genes of immune response and that macrophages in murine cysticercosis promote Th1 or Th2 responses it was demonstrated that synthetic miR-10-5p and let-7-5p were internalized into the cytoplasm of murine peritoneal macrophages in vitro . Interestingly, the down regulation of the expression of pro-inflammatory cytokines, such as Il6, Il1b, and TNF, IL-12, was reported when activated macrophages were incubated with IFN-γ and miR-10-5p or let-7-5p. Moreover, in macrophages activated with IL-4 these miRNAs reduced the expression of cytokines involved in M2/Th2 differentiation. These results were important, because murine resistant to cysticercosis display high levels of TNF, IL-12, IL1-β, and NO during early infection (Th1 response), which is associated with the elimination of larvae . On the other hand, high levels of pro-inflammatory cytokines (IL-6 and TNF) cause damage to the microglia promoting autoimmune and neurodegenerative diseases [45, 46]. This tissue damage is also observed in human NCC at the beginning of larvae degeneration and in pig NCC when they are treated with praziquantel . In contrast, viable larvae are associated with a long initial asymptomatic phase that correlate with undetectable inflammation in the SNC, presumably due to
The striking ability of helminth parasites in conferring protection from diseases of immune dysregulation has increased the attention into the immunomodulatory mechanisms evoked by these parasites. Administration of E-S antigens of
3. miRNAs in drug response
In 2010 Devaney and collaborators  speculated that the link between changes in miRNA levels and drug resistance in cancer cells may also be a feature of drug resistance in parasitic nematodes. On the other hand, few data have been published in connection with drug resistance in cestodes . Our group study the miRNA expression profile of
The experiments showed that the overall miRNA profile remained unchanged under PZQ treatment, except for miR-7b that showed a sixfold enhanced expression  under PZQ treatment. One of the predicted miR- 7b targets was calponin, a calcium binding protein that inhibits myosin. This may be related to the expected alteration of intracellular calcium concentration produced by PZQ , a drug binding and inhibiting voltage-gated calcium channels, a key molecule for the regulation of calcium level inside the cell. Also, other targets of miR-7b are involved in several pathways such as amino acid and nucleotide metabolism, vesicular transport, signaling pathways, cell adhesion, cell growth, cell death and interaction with neuroactive ligands, suggesting the importance of this miRNA in parasite biology . Calponin is one of miR- 7b predicted targets linked to calcium binding protein that inhibits myosin and may be related to the intracellular calcium concentration produced by PZQ . Other predicted targets of miR-7b are involved in several pathways such vesicular transport, signaling pathways, cell adhesion, cell growth, cell death and interaction with neuroactive ligands, suggesting the importance of this miRNA in parasite biology . In these experiments other miRNAs showed differences in expression levels during treatment with PZQ , such as miR-31. This miRNA showed a decrease in the level of expression in cysticerci treated with PZQ and therefore the genes regulated by the miRNA would be overexpressed compared to the cysticerci that did not receive treatment with PZQ . Predicted target genes for this miRNA include: ABC transporters (transporters responsible for expelling different drugs out of the cell), thioredoxins (involved in drug metabolism), and the voltage-gated L-type calcium channel subunit alpha-1D, which is a probable site of action for praziquantel (PZQ)  Table 2. Additionally, other
|Voltage-gated calcium channel 2C alpha-2 / delta||miR-307|
|Drug metabolism / enzyme conjugation||TsM_000648500|
|Cytochrome b5-heme type / steroid binding|
Selenoprotein type 2C Rdx
Glutathione S-transferase 2C class S
Glutathione S-transferase 2C similar to C-terminal
|ABC type conveyors|
Type 2 ABC conveyors
Type 1 2C ABC conveyors
These results prepare the way for continue with more studies in order to understand the response of miRNAs to drug treatment and the influence that these ncRNAs may have on drug action and/or drug resistance.
4. miRNAs as potential biomarkers
A biomarker is described as a feature that is objectively measured and evaluated as an indicator of many biological processes. Hunting for helminths biomarkers capable of providing diagnostic, prognostic, or therapeutic information has become a necessary but challenging work in cestodes research. MiRNAs were reported in blood – plasma, serum and other fluids like urine and saliva. This attribute has raised the interest of their use as potential biomarkers and diagnostic tools . In the case of cestodes diseases the use of pathogen miRNAs as biomarkers promises the advent of highly specific and non-invasive diagnostic tools, since the miRNA repertoire of
The small size and the stability of miRNAs are two important features that permit the circulation of these molecules in biological fluids. The formation of protein–miRNA allows circulating miRNAs escape of degradation . Also, the majority of miRNAs detectable in serum and saliva are found inside extracellular vesicles (EV) that could avoid miRNA degradation and serve as transport particles to facilitate miRNA actions in neighboring cells . The term EV groups includes several types of vesicles among which microvesicles and exosomes are the most thoroughly characterized. In helminths parasites EVs are the preferred extracellular compartment under study and miRNAs as the most thoroughly characterized RNA biotype . The identification and sequencing of
The extensive use of next generation of technologies such as miRNA microarrays and high-throughput deep sequencing techniques, translating biomarker into practice with increased diagnostic and therapeutic sensitivity and specificity would be less of a problem . With respect to the use of ex-RNAs as biomarkers in NCC, to date, no laboratory assay from plasma, serum or cerebrospinal fluid has been performed. Furthermore, patient samples from different geographic regions together with specificity assessment with samples of patients would also provide a more realistic view of the potential of ex-RNAs as biomarkers of NCC.
5. miRNAs as potential drug target
The hypothesis that many
For the better understand of the pathophysiology of parasitic diseases at the molecular level is crucial identify and characterize parasite-specific miRNAs and their targets in hosts. The significant advance in biomedical research of miRNAs as target drugs and biomarkers is expected to be widely translated in the field of parasitology in the coming years. Why not think about miRNAs as a profitable approach to better diagnose and properly treat NNC? There is an increasing number of studies that are being done in
Regarding the neglected diseases, researchers have dedicated decades to the development of new drugs and identifying new biomarkers of disease progression but most researches are limited to academia indicating a gap between basic science and clinical application. Also, the use of miRNAs as a biomarker or potential drug target are poorly explored compared with cancer, neurological disorders, metabolic, cardiac and circulatory diseases.
It is expected that in future years the biological knowledge acquired on miRNAs, especially in biomedical research, could be widely translated into NNC since miRNAs could hold great potential as therapeutic and diagnosis targets for the control of diseases.