It was estimated that HCV infection causes the liver-related death in more than 300000 people, annually (
9). Once exposure to HCV occurs, only a minority of patients clear the acute infection by HCV-specific interferon-γ responses ( 10), whereas about 80% persist with life-long chronic viremia, if not successfully treated ( 11). Up to date, there is no vaccine for prevention of HCV infection. Interferon/ribavirin therapy is currently the mainstay in the management of HCV infection. However, the drug has been plagued with excessive toxicity, therapeutic limitations, and restricted patient availability ( 12). Recently, an oral antiviral agent sofosbuvir has been approved by the US food and drug administration (FDA) and European Union, for the treatment of chronic HCV infection. Although the new anti-HCV drug looks promising, the development of new therapy or preventative vaccines that definitely reduce the global HCV burden remains a formidable challenge.
Chimpanzee is the only animal model for studies of HCV infection and related innate and adaptive host immune responses (
9). However, their large adult body size and long reproductive cycle create challenges for in-depth study on mechanism of HCV infection and pathogenicity. Most efforts have been directed towards the study of development of small animal models of HCV infection. Although T-cell and B-cell deficient mice grafted with human hepatocytes could support HCV infection robustly, it could not be used in studies of adaptive immunity ( 13). Recent studies in developing genetically humanized mice are in process, although these animal models only permit studies of specific steps in the HCV life cycle and have limited or no viral replication ( 14).
Genome sequencing of the Chinese tree shrew and comparison with 14 other species, including six primate species, showed that the tree shrew was clustered with primate species (
5). Our previous studies had proved that CD81, scavenger receptor class B1, claudin-1 and occludin, the considered HCV receptors of tree shrew, have the mediating function to support HCV infection ( 15). Therefore, multiple attempts are made to employ tree shrew to create animal models for studying HCV infection ( 16, 17). However, although the tree shrew has the ability to be infected, the low infection rate and instable infection effect are still complicated issues, urgently needing to be addressed. Here, three second filial generation tree shrews were used to generate the liver miRNA data, which could help us in a future study to investigate the mechanisms of HCV infection in tree shrew.
MicroRNAs are important players in the establishment of HCV infection replication and its propagation in infected hepatocytes. Deregulated expression of miRNAs has been linked to the pathogenesis associated with HCV infection, by controlling the process of cell proliferation, apoptosis and migration (
4). Therefore, the miRNA data will provide us more information for the utilization of tree shrew, as an animal for HCV research. The current study was designed to profile the general expression of miRNAs in the liver of tree shrew. A total of 2060 conserved miRNAs and 80 novel microRNAs were identified, and miR-122 was the most abundant miRNA in the liver of tree shrew. The miR-122 was also identified as a liver-specific, well-conserved and the most abundant microRNA in liver, accounting for about 70% in the adult liver. Many studies have previously demonstrated that miR-122 is bind to two different sites in the 5’ untranslated region (UTR) of the HCV genome and promotes viral RNA accumulation. The miR-122 plays an important positive role in the regulation of HCV replication and is a crucial target for anti-HCV therapy ( 18). Therefore, several miR-122 inhibitors become potential drug candidates for the treatment of HCV infection. However, miR-122 also has an essential role in maintaining liver homeostasis and differentiation, while a downregulated expression of miR-122 has been associated with liver disease. Recent studies demonstrated that decreased miR-122 have been associated with poor prognosis and metastasis of liver cancer and can even promote hepatocarcinogenesis ( 19). The findings mentioned above indicate that the role of miR-122 is controversial. To clarify the role of miR-122 in liver disease, it is imperative to find an applicable animal-model of HCV infection, to further research the molecular mechanisms of miR-122 in liver disease. In the current study, we demonstrated that the miRNA data generated in tree shrew was consistent with previous studies in humans. Further comparison of the secondary structures of miR-122 precursors, among tree shrew, human, mouse and rat, revealed the close relationship between tree shrew and human. Therefore, based on the evidences of the high expression level of miR-122 in the liver of tree shrew, the similarity of the secondary structure of miR-122 between tree shrew and human, and the link between miR-122 and HCV processing, it further supported tree shrew as an potential animal model for HCV infection. In addition, our findings also suggest that tree shrew may be a potential animal model to better understand the molecular mechanisms of miR-122 in liver disease caused by HCV infection in vivo.
Another interesting finding in our study was that half of the top 20 small RNAs, sorting by expression level, belonged to
let-7 family. Previous studies found that the let-7 family was also strongly associated with HCV infection. Several members of let-7 family ( has-let-7a, has-let-7b, has-let-7c, has-let-7d, has-let-7e, has-let-7f, has-let-7g, has-let-7i) had the potential to repress the expression of the HCV core protein in vitro ( 20). Another research found that Let-7b repressed HCV replication by interaction with the coding sequences of NS5B and 5’-UTR of HCV genome that were conserved among various HCV genotypes ( 21). Therefore, further studies on the roles of the let-7 family in HCV replication, in tree shrew, are helpful for the research on HCV infection.
Taken together, tree shrew has been proposed as a valid experimental animal to replace primates for studying HCV infection. However, there are limited usages of this animal in the field, because of the unclear mechanism on HCV infection. The miRNA data we generated and the recently annotated genome sequence of the Chinese tree shrew offer an opportunity to decipher the genetic basis of the tree shrews’ suitability, as an animal model in HCV research.
The second filial generation tree shrew, used in our study, underwent elaborate biological evolution. The indexes of microbiology, virology and parasitology can be precisely controlled. In addition, they have the advantages of relatively clear genetic background and strong disease resistance. However, there are several limitations in our current study. Due to the poor breeding potential, only three livers of tree shrews, which were 6-month-old, were used to generate the relatively limited miRNA data. To accurately characterize the expression profiles of miRNA in tree shrew, further studies, involving more liver tissue samples of different growth and development periods of tree shrew, would be necessary.