In this study, we investigated the biologic function of miR-122 through the generation and characterization of mice with liver-specific (LKO) and germline (KO) deletion of this miRNA locus. Analysis of these mice revealed that loss of miR-122 does not lead to overt defects in liver development. This finding suggests that a previously described positive feedback loop involving hepatocyte nuclear factor 6 (HNF6) and miR-122 that has been proposed to regulate hepatocyte differentiation (37
) is not required for grossly normal liver development. Furthermore, HNF6 expression was unaltered at the RNA or protein level in adult LKO and KO mice (data not shown). While this suggests that endogenous miR-122 does not regulate HNF6 expression in adults, it remains possible that HNF6 is controlled by this miRNA during embryogenesis and HNF6–miR-122 reciprocal regulation influences the kinetics of hepatocyte differentiation in a manner that is no longer evident at birth.
Transient inhibition of miR-122 using antisense oligonucleotides is well tolerated in adult mice and results in reduced serum cholesterol (2
). While genetic deletion of miR-122 also lowers serum cholesterol, our study revealed that prolonged loss of function of this miRNA leads to accumulation of hepatic TGs in young mice. This abnormality is associated with the upregulation of several gene products that catalyze TG biosynthesis and storage, including the newly identified direct miR-122 targets Agpat1
. As Mir122
-LKO/KO mice aged, hepatic inflammation ensued, preceding the progressive onset of fibrosis and, eventually, tumors resembling HCC. Although a tumor suppressor role for miR-122 has previously been proposed based on in vitro studies and expression analyses of human HCC samples, these findings provide the first in vivo evidence to our knowledge that loss of this miRNA is sufficient to initiate highly penetrant HCC development. It is well established that liver damage and inflammation potently promote the development of HCC, and, in humans, this tumor type nearly always arises in the setting of underlying liver injury (26
). Thus, chronic steatohepatitis in Mir122
-LKO/KO mice is an important component of the pathology that leads to HCC in these animals (38
). In addition, upregulation of the chemokine Ccl2
, both directly through loss of targeting by miR-122 and indirectly as a consequence of liver injury, resulted in the intrahepatic recruitment of CD11bhi
inflammatory cells that locally produce pro-tumorigenic cytokines including IL-6 and TNF-α.
The importance of underlying inflammation in HCC development in these mice is further highlighted by the greater tumor incidence and higher tumor burden and grade in male LKO and KO mice, respectively. In humans, HCC exhibits a similar sex bias, with 2- to 4-fold greater incidence in males (7
). This bias is believed to be partially attributable to the greater susceptibility of males to injury-induced hepatic inflammation and hepatocyte proliferation mediated by IL-6, whose production is suppressed by estrogens in females (39
). Indeed, circulating IL-6 is increased in tumor-bearing male, but not female, LKO/KO mice, likely contributing to the increased severity of hepatocarcinogenesis in male mice in these animals. Nevertheless, it will be important for future studies to examine the contribution of additional factors that are known to play a role in sexual dimorphism of HCC, such as Foxa1/a2 (40
), in the phenotype of miR-122–deficient animals.
The ability of miR-122 to suppress tumorigenesis when delivered to a non-inflammatory MYC
-driven HCC model establishes that this miRNA performs a tumor suppressor function that is independent of its role in reducing inflammation and maintaining hepatocyte integrity. This activity is likely mediated by the ability of miR-122 to directly and indirectly control a broad program of gene expression, which includes key factors that influence HCC pathogenesis such as Igf2
, and Rhoa
. miR-122 also appears to be essential for maintenance of the mature hepatocyte gene expression program as manifested by the reactivation of fetal genes including Afp
, and Igf2
in LKO/KO mice. In the setting of tonically increased signaling through canonical oncogenic pathways, the expansion of immature hepatocytes in an inflammatory microenvironment likely results in a state of highly increased susceptibility to cellular transformation and tumorigenesis. It is also noteworthy that the cancer phenotype is more severe in germline Mir122
-KO mice compared with those with liver-specific deletion of the miRNA. While this may simply reflect the earlier and more uniform Mir122
deletion in KO mice, another intriguing possibility is that a non-hepatocyte function of miR-122 might contribute to the more severe phenotype. Indeed, low-level expression of miR-122 in fibroblasts has been demonstrated (41
Beyond contributing to our understanding of the physiologic functions of miR-122, the findings reported herein have important therapeutic implications. First, our results demonstrate that delivery of miR-122 using AAV is sufficient to suppress tumor progression in a highly aggressive HCC model. We have previously reported similar results using miR-26a, another miRNA that is frequently downregulated in human HCC (35
). Both of these miRNAs, therefore, represent promising candidates for miRNA replacement therapy in patients with this tumor type. Additionally, since miR-122 is downregulated in NASH (9
), the efficacy of miR-122 delivery for reducing progression to cirrhosis and HCC in relevant animal models of this disease is worthy of investigation. However, given the essential role of miR-122 in HCV replication (4
) the treatment of HCC or other liver diseases arising in the context of HCV infection would not be an appropriate setting for miR-122 delivery.
Finally, miR-122 inhibition therapy using an LNA-modified antisense oligonucleotide, SPC3639, is currently in phase II clinical trials for the treatment of HCV infection (5
). Treatment of HCV-infected chimpanzees with SPC3639 reduced viremia and hepatitis without causing any adverse effects during the 12-week period of the study (5
). However, our examination of Mir122
-LKO and -KO mice has demonstrated that the chronic loss of miR-122 causes steatohepatitis and altered liver function that ultimately leads to liver cancer. Multiple explanations may account for the apparent discrepancy between the phenotypes observed after transient inhibition versus genetic deletion of miR-122. First, the more severe phenotype in LKO and KO mice might arise due to a developmental defect resulting from the absence of miR-122 throughout gestation, or it may result from the effects of complete deletion of the miRNA as opposed to the partial loss of function achieved with chemical inhibitors. Alternatively, the development of liver damage and the resulting sequelae might simply require a longer period of miR-122 depletion beyond that which has been examined using injected inhibitors. Distinguishing between these possibilities will be a priority for future research. Nevertheless, the insights gained through the study of mice lacking miR-122 should aid the design of safe therapeutic strategies based on miR-122 inhibition.