Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Keywords: RNAi, PTGS, model organism, cancer research, dog, miRNA, siRNA

Biogenesis and function of small RNAs in mammals

In 2006, Andrew Fire and Craig C. Mello were awarded with the Nobel Prize in medicine for their work on RNA interference (RNAi). Since the discovery of post-transcriptional gene silencing (PTGS) mechanism in various species, the interest in using small RNA molecules and its endogenous mechanisms as a new pharmacological approach to human diseases is constantly rising (Fire et al., 1998; Elbashir et al., 2001). Micro RNAs (miRNA) and small interfering RNAs (siRNA) are two small non-protein-coding RNA molecule types which play a leading role in PTGS. Thereby, contrary to siRNAs, miRNAs appear to act as “fine tuner” of gene regulation (Sevignani et al., 2006).

miRNAs are endogenously expressed as primary miRNA (pri-miRNA) transcripts composed of up to several thousand nucleotides (Mondol and Pasquinelli, 2012) which are processed by the nuclear enzyme Drosha to precursor miRNAs (pre-miRNA) (Zeng and Cullen, 2006). Following the enzymatic processing, the cytoplasmic enzyme Dicer cleaves the pre-miRNAs generating the mature double-stranded miRNAs (Bohnsack et al., 2004; Lund et al., 2004). Dicer not only processes pre-miRNAs, it also cleaves long double stranded RNA molecules and generates the second class of small RNAs, named siRNAs, which show a miRNA-similar size of ~20 base pairs (Tomari and Zamore, 2005).

Mature miRNAs and siRNAs are chemically and physiologically indistinguishable, apparently only differing in their respective origins (Ambros et al., 2003). Further comparison of these molecules shows that the “guide strand” of miRNAs seen in mammals, is in most cases significantly but not obligatory completely complementary to the 3′-untranslated region of the respective target mRNA. In the case of siRNAs the “guide strand” shares absolute complementarity to a small region in the target structure. After “guide strand” incorporation into the RNA-induced silencing complex (RISC), the respective target mRNA stability and/or translation are modulated (Tomari and Zamore, 2005). Interestingly many miRNAs, their biogenesis and functions are conserved among several organisms of higher and lower complexity as fungi, worms, Drosophila, and mammals confirming the general importance of the PTGS mechanism (Filippov et al., 2000; Pasquinelli et al., 2000; Wu et al., 2000; Fortin et al., 2002; Yan et al., 2003; Han et al., 2004; Ibanez-Ventoso et al., 2008).

miRNA genes itself show to be very versatile, they were described to be polycistronic or monocistronic and occasionally located in intron- as well as exon-regions of protein-coding genes. Some miRNAs are co-expressed with their target-mRNAs as one transcript (Bartel, 2004; Kim et al., 2009). Different miRNAs target a single mRNA, and a single miRNA can also be regulating the expression of many different targets. Additionally, miRNAs were also reported to be able to regulate other miRNAs by direct interactions (Winter et al., 2009; Chen et al., 2011) and in some cases miRNAs were described to be regulated by proteins translated from their respective target mRNA, constituting a regulatory negative feedback loop (Bracken et al., 2008; Rybak et al., 2008).

In general, long double stranded RNA molecules are not common in mammals, suggesting that the RNAi mechanism mediated by siRNAs evolved for defense of viral infections and transposable elements (Obbard et al., 2009).

Despite their different origin these small non-coding RNA molecules have many things in common including the small size, specificity of inhibition, and potency and considering therapeutic applications a diminished risk to induce unspecific effects as immune responses. Due to these properties, these molecule types are considered to be potential key players in the development of next generation therapeutics for treatment of a variety of major diseases including cancer (Barh et al., 2010).

miRNA transcription and maturation is not the only process regulating functional miRNA levels. Stability of functional miRNAs is a further key factor in miRNA regulation. Molecule stability was reported to be dependent on several cis- and trans-acting factors varying considerably between miRNAs and their spatiotemporal expression (Kai and Pasquinelli, 2010). Exosomal release of miRNAs into the extracellular environment (Ohshima et al., 2010) and long non-coding RNAs (lncRNA) mimicking target mRNA sites and thereby acting as decoys, were also shown to decrease functional miRNA levels in cells (Cesana et al., 2011). Interestingly, numerous lncRNAs were reported to be deregulated in human cancer (Shore et al., 2012).

Typically one strand of the mature miRNA hybrid, the “guide strand” is maintained during interaction with RISC proteins while the “passenger strand” is degraded. This dichotomy is generally known to be caused by the stabilization of the “guide strand” by RISC loading, while the “passenger strand” stays unprotected (Kai and Pasquinelli, 2010). miRNA methylation, uridylation, and adenylation are some of the modifications having an influence on small RNAs half-life as well (Burroughs et al., 2010; Kai and Pasquinelli, 2010). However, miRNA stability-enhancing proteins were also described to be actively involved in miRNA half-life, suggesting that the miRNA-mediated gene regulation processes are more complex and as variable as these genes are itself (Kai and Pasquinelli, 2010).

 

The dog as model organism

In recent years, the role of the domestic dog as model organism for various human diseases constantly gained increasing importance. Many canine inherited diseases were described, including Alzheimer's disease (Rofina et al., 2003), narcolepsy (Lin et al., 1999), diabetes (Ionut et al., 2008), epilepsy (Lohi et al., 2005), atrial fibrillation (Shan et al., 2009), Duchenne muscular dystrophy (Mizuno et al., 2011), heart diseases (Eaton et al., 1995), and cancer (Mueller et al., 2007; Boggs et al., 2008; Noguchi et al., 2011; Uhl et al., 2011). All these disorders occur in dogs, just as in humans, spontaneously during their lifetime and many of them show similar clinical manifestations (Ostrander et al., 2000; Sutter and Ostrander, 2004).

Cancer is a complex, polygenic disease spontaneously occurring in man and dog (~1 million diagnosed pet dog cancer cases per year in the United States), whereas tumors in most laboratory animals must be artificially induced (Mueller et al., 2007; Karlsson and Lindblad-Toh, 2008; Paoloni and Khanna, 2008). Indeed, mans best friend shares many features, including tumor genetics, molecular targets, histological appearance, and response to conventional therapies (Paoloni and Khanna, 2008). Additionally dogs often cohabitate with their owners, are exposed to similar environmental stresses, which may have a big impact on cancer development, and enjoy the best medical care among pets (Sutter and Ostrander, 2004; Rowell et al., 2011). Furthermore, dogs show a higher genetic variability than inbred laboratory mice and enable an easier and faster surgical intervention and imaging due to body size (Mueller et al., 2007). The five- to eight-fold faster aging of dogs in comparison to humans facilitates long-term studies of cancer treatments (Rowell et al., 2011). In 2005, the sequenced canine genome was published (Lindblad-Toh et al., 2005). Having a less different genome from humans than rodents and sharing a similar metabolism, according to their body size, the dog classifies as a very good model organism for molecular studies on human diseases (Sutter and Ostrander, 2004; Karlsson and Lindblad-Toh, 2008; Pinho et al., 2012).

In contrast to investigations on human miRNAs in cancer, the research on canine miRNAs is often limited by the lack of specific canine assays. Up to now only a limited number of studies were done on canine non-neoplastic and tumor tissues. In 2008, Zhou et al. identified 357 canine miRNA-genes by computational analysis, 300 of these were homologs of known human miRNAs (Zhou et al., 2008). Currently 1527 human and 323 canine miRNA matches of hairpin precursors are registered in the web-database, miRBase (Sanger Institute, version 16.0) (Kozomara and Griffiths-Jones, 2011).

Due to high homology of mature miRNAs between human and dog (Table (Table1),1), many of the human miRNA assays can be used for analyses of canine miRNA expression. In Table Table11 canine miRNAs are listed, which share absolute homology to the human counterparts. Homologous canine miRNAs with overhangs or major sequential differences are not listed.

 

Table 1: Comparison of canine and human mature miRNAs. 207 of the canine mature miRNAs listed in miRBase (Sanger Institute, version 16.0) show absolute sequence complementarity to the human counterparts. The sequence identity of the canine mature miRNA sequences with the corresponding human homologs was confirmed by miRBase, single sequence search (http://www.mirbase.org/search.shtml).

miRNAs in non-neoplastic diseases

Recent studies showed that nicotine can induce atrial structural remodeling and increase atrial fibrosis vulnerability in dogs. Shan et al. reported a decreased miR-133 and miR-590 expression in smoking individuals with atrial fibrosis and showed that an ectopic over-expression of miR-133 and miR-590 resulted in a post-transcriptional suppression of TGF-β 1 and TGF-β RII in cultured canine atrial fibroblasts (Shan et al., 2009).

Another disease affecting man as well as dogs is Duchenne muscular dystrophy. It is a lethal X-chromosome linked disorder caused by mutations in the dystrophin gene, which encodes a cytoskeletal protein. Mizuno et al. studied serum miRNA expression in the X-linked muscular dystrophy in Japan dog model (CXMD_J) and found, as in humans, increased miR-1, miR-133a, and miR-206 levels (Cacchiarelli et al., 2010, 2011; Mizuno et al., 2011). The study indicates that serum miRNAs might be a reliable biomarker for muscular dystrophy (Mizuno et al., 2011).

 

miRNAs in mammary tumors

Mammary tumors are among the most common neoplasias of female dogs, with an estimated lifetime risk for malignant tumors varying from 2 to more than 20%. The risk for malignant mammary tumors in dogs spayed before and after their first estrus, is in comparison to intact dogs 0.05 and 8%. In dogs spayed after their second estrus, the risk rises up to 26% (Withrow and Vail, 2007). Data from a Swedish study, based on 80,000 insured female, mostly sexually intact dogs, reported a rate of 111 mammary tumors (benign and malignant) per 10,000 dog-years risk (Egenvall et al., 2005). The age-standardized incidence rate for human breast cancer estimates 66.4 per 100,000 in more developed areas and is thus the most common cancer (Jemal et al., 2011).

According to a recent study, nine miRNAs, let-7f, miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-155, and miR-181b involved in human mammary cancer, appear to follow the same expression pattern in the canine counterpart. In this study, only the investigated miR-145 was not shown to be differently expressed comparing non-neoplastic and neoplastic canine tissues (Boggs et al., 2008).

miRNAs linked to lymphomas

Besides mammary cancer, canine lymphomas show the highest estimated annual incidence with 13 to 24 cases per 100,000 accounting for up to 24% of all canine neoplasias (Withrow and Vail, 2012). In human, chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world with an annual incidence of approximately 10,000 new cases in the United States (Calin and Croce, 2009).

In a recent study, the relative expression pattern of 12 canine miRNAs (cfa-let-7a, cfa-miR-15a, cfa-miR-16, cfa-miR-17-5p, cfa-miR-21, cfa-miR-26b, cfa-miR-29b, cfa-miR-125b, cfa-miR-150, cfa-miR-155, cfa-miR-181a, and cfa-miR-223) in CLL was analyzed. Due to stable expression between the investigated samples four of the 12 miRNAs (cfa-let-7a, cfa-miR-17-5p, cfa-miR-26b and cfa-miR-223) were used as endogenous controls. miR-15a, miR-16, and miR-181a were reported to be downregulated in canine and human CLL (Calin et al., 2002; Gioia et al., 2011; Zhu et al., 2012). Four of the investigated miRNAs (cfa-miR-21, cfa-miR-125b, cfa-miR-150, cfa-miR-155) were described to be upregulated in human and canine B-CLL as well (Pekarsky et al., 2006; Fulci et al., 2007; Wang et al., 2008; Bousquet et al., 2010; Palamarchuk et al., 2010; Rossi et al., 2010). Only miR-29b, which was shown to be downregulated in human B-CLL, was upregulated in canine CLL (Pekarsky et al., 2006; Fulci et al., 2007; Wang et al., 2008; Bousquet et al., 2010; Palamarchuk et al., 2010; Rossi et al., 2010).

It was also observed by Gioa et al. that in comparison to canine B-CLL the miR-125 expression was significantly downregulated in canine T-CLL. On the basis of the mature miRNA expression ratio between miR-150/miR-125b, and miR-150/miR-155, it was reported that it is also possible to distinguish among normal blood, B-CLL and T-CLL samples (Gioia et al., 2011). This illustrates the potential of miRNA expression analyses not solely as tumor marker but as an instrument to distinguish between different but similar cell or cancer types.

miRNAs associated with melanomas

Melanomas are very aggressive malignant skin cancers in man and dog (Noguchi et al., 2011). Accounting for 5–7% of canine skin tumors. Tumors of the melanocytes and melanoblasts are relatively common in dogs (Withrow and Vail, 2007; Uhl et al., 2011). In human melanoma cell lines A2058, Mewo, and canine melanoma LMeC cells as well as malignant melanoma tissues the miR-145, miR-203, and miR-205 expression was reported to be downregulated. An ectopic expression of each of these miRNAs-induced in vitro growth inhibition in A2058, Mewo, and LMeC cells (Noguchi et al., 2011, 2012a,b), indicating their potential for treatment of human and canine malignant melanoma.

miRNAs involved in epithelial to mesenchymal transition

Furthermore, aberrant activation of the epithelial to mesenchymal transition (EMT) has been observed to promote invasion and metastasis in several human cancers. The EMT inducers ZEB1 and ZEB2 have been shown to be direct targets of the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, and miR-429) in the human breast cancer cell line MDA-MB-231 and in Madin Darby canine kidney epithelial cells (MDCK). Lost expression of these miRNAs was detected in human metaplastic breast cancer specimens, indicating that downregulation of miR-200 family members may be an important step in tumor progression (Bracken et al., 2008; Gregory et al., 2008; Adam et al., 2009).