Normal liver function includes building biomolecules for export to consumer tissues of the body (Yokoyama et al, 2005
). This catabolism requires energy and liver tissue is rich in mitochondria. Loss of mitochondrial function may in turn be associated with loss of liver function. Dysregulation of normal mitochondrial functions may also contribute to cancer metabolism and hepatocarcinogenesis, as the connection between mitochondrial dysfunction and cancer is well known (Brandon et al, 2006
). It is possible that loss of miR-122 expression in HCC may contribute to loss of liver function, a contributor to morbidity and mortality in HBV-associated HCC. We have shown that loss of expression of miR-122 positively correlated genes predicts poor survival for HCC patients. In our patient cohort, expression levels of miR-122-regulated pathways in both tumor and adjacent non-tumor tissue appear to be independently functioning as good markers of patient prognosis.
Although miR-122 negatively correlated genes were enriched for cell-cycle function in HCC (E
-value=8e−18), the miR-122 seed-matched transcripts showed only random overlap with cell-cycle transcripts (single-test hypergeometric P
-value=0.78). These observations suggest that there is likely no direct connection between up-regulation of potential miR-122 targets and up-regulation of the proliferative apparatus in tumor tissue. Further, we did not find strong prognostic power for patient survival in miR-122-expression levels, although a smaller study has (Coulouarn et al, 2009
). We also did not find strong prognostic power in expression levels of proposed miR-122 direct targets, whereas expression of the putative secondary targets, transcripts positively correlated with miR-122, is predictive of patient survival. Two possible implications of these observations are that the downstream target pathways are more connected to clinical outcome than the primary targeting mechanisms and that downstream pathways may form a more sensitive measure of miRNA activity than direct measurements of miRNA-expression levels (Davis et al, 2009
Genes that were up- and down-regulated in HCC and in anti-miR-122-treated mice (putative primary and secondary miR-122 targets), appear to function as a biological network. A number of these genes are connected by functional similarity as determined by the total ancestry method of functional classification analysis (Yu et al, 2007
). In addition, a number of proposed primary and secondary target genes found in this work have been previously reported to be associated in various functional networks. The networks associated with statin (an HMG-CoA reductase inhibitor to lower cholesterol levels) treatment, high-fat feeding or fasting, expression of the obesity causal gene Zfp90, and with cohesive gene expression in normal liver overlap significantly with miR-122 primary and secondary targets (Schadt et al, 2005, 2008
,Schadt et al, 2005, 2008
; Chen et al, 2008
PPARGC1A is the miR-122 secondary target most connected by functional similarity to genes up-regulated by loss of miR-122 in both human HCC and anti-miR-122-treated mice. PPARGC1A transcription is activated by cAMP-response element-binding proteins (CREB) (Wu et al, 2001
). Among those genes connected to PPARGC1A in this study, PPP1CC negatively regulates CREB, and LCMT1 (Leulliot et al, 2004
) activates PPP2A, which inhibits CaM-kinase activation of CREB and MEK/ERK signaling in the MAP-kinase pathway (Wu et al, 2001
). The MAP-kinase pathway represented by MAP3K3 and MAPKAP2 among genes up-regulated by loss of miR-122 leads to phosphorylation of PPARGC1A that both activates the protein and enhances SCF/Cdc4-mediated degradation of the protein (Olson et al, 2008
). These published observations suggest that multiple miR-122 targets may contribute to the down-regulation of PPARGC1A observed with loss of miR-122.
PPARGC1A is proposed to be the master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008
), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. PPARGC1A-over-expressing mouse strains show uncontrolled mitochondrial biogenesis (Lehman et al, 2000
), whereas PPARGC1A-knockout mouse strains show decreased expression of mitochondrial genes with strain-dependent compensatory phenotypes (Benton et al, 2008
). PPARGC1A and HNF-4 are thought to act together to stimulate cholesterol biosynthesis (Rodgers and Puigserver, 2007
), so loss of PPARGC1A may contribute to the reduction in plasma cholesterol seen in anti-miR-122-treated animals (Jopling et al, 2005
; Esau et al, 2006
; Elmén et al, 2008a, b
,Elmén et al, 2008a, b
). SMARCD1(BAF60a), which stimulates fatty-acid oxidation in conjunction with PPARGC1A without changing its expression level (Li et al, 2008
), is proposed to be a primary target of miR-122 in this study and in a recent publication (Gatfield et al, 2009
), suggesting that increased fatty-acid oxidation seen with miR-122 depletion (Esau et al, 2006
; Gatfield et al, 2009
) may be a direct effect. Another study found that reduction of miR-122 levels in non-alcoholic steatosis was associated with increased expression of lipogenic genes (Cheung et al, 2008
). In anti-miR-122-treated mice in this study, expression levels for genes encoding cholesterol biosynthesis and lipid metabolism are not tightly anti-correlated with expression levels of miR-122 seed-matched genes (data not shown), suggesting that the observed effects of anti-miR-122 on cholesterol and lipid synthesis may be further downstream of miR-122. Microarray studies of anti-miR-122 in high-fat-fed mice may be of interest in elucidating the connection between miR-122 and cholesterol biosynthesis.
Interestingly, down-regulation of SDH subunits A and B is associated with loss of miR-122 in both mouse and human tissues in this study. Increased expressions of both the genes were detected in cell culture after treatment with miR-122 mimetic. Loss-of-function mutations in genes encoding subunits B, C, or D of SDH lead to loss of mitochondrial function and to hereditary paraganglioma or in the case of SDHB, also to phaeochromocytoma or renal cell carcinoma (King et al, 2006
). A decline in SDH function concomitant with the loss of miR-122 may thus increase the risk of oncogenesis.
Recently, miR-122 has been suggested to act as a tumor suppressor in HCC (Tsai et al, 2008
; Coulouarn et al, 2009
). In the study of Tsai et al
, a combination of bioinformatics and tumor profiling was used to identify 45 genes as potential miR-122 targets. About half of the proposed targets were anti-correlated with miR-122 in the tumor and non-tumor tissues profiled in our study. A total of 11 of the genes were also up-regulated in the anti-miR-122-treated mouse livers we profiled, suggesting cross-species conservation of the regulations these authors identified. However, ADAM17, followed up in more depth by Tsai et al
, was not significantly anti-correlated with miR-122 in our profiles. The study of Coulouarn et al
found gene-expression clusters associated with high and low miR-122 levels in 32 HCC tissue samples. Genes whose increased expression was associated with lower miR-122 levels in these samples included predicted miR-122 targets and genes up-regulated in anti-miR-122-treated mice, whereas genes whose increased expression was associated with higher miR-122 levels were enriched for lipid metabolism functions and were more likely to be well expressed in control mice. This study emphasized the function of HNF1A and HNF3, transcription factors mediating hepatocyte differentiation and liver functions, in potential regulation of miR-122 expression. Our study is unable to support a primary or secondary function for these genes. HNF3 components were uncorrelated with miR-122 in tumor and non-tumor tissues profiled in our study and were not consistently regulated in anti-miR-122-treated mouse livers. HNF1A showed no significant relationship to miR-122 in tumor and non-tumor profiles; we have no data on mouse expression.
Taken together, our results imply that normal mitochondrial function in liver, including expression of mitochondrion-associated metabolic pathways, may be maintained in part by miR-122 expression. Impaired mitochondrial functions are observed in many tumor types, suggesting an alternate possibility that the observed decline in mitochondrial function in HCC may be tumor related rather than miRNA related (Jopling et al, 2005
). Our observations that mitochondrial function pathways and miR-122 levels also decline coordinately in cirrhotic liver and in anti-miR-122-treated mouse livers argue against this explanation.
Other connections between loss of miR-122 expression and changes in liver function have been proposed. CAT-1 (SLC7A1) was shown to be a direct target of miR-122 (Chang et al, 2004
; Jopling et al, 2006
), and although it is negatively correlated with miR-122 levels in HCC in this study, it is unregulated in anti-miR-122-treated mouse livers profiled herein. Bcl-w, recently found to be targeted by miR-122 (Lin et al, 2008
), was negatively correlated with miR-122 levels in HCC in this study and is up-regulated in anti-miR-122-treated mouse livers, supporting a pro-apoptotic function for miR-122 in HCC and indicating a survival advantage to its down-regulation in HCC. Expression of miR-122 precursors is known to be circadian; in a recent study, eight genes were identified as showing circadian accumulation in microarray experiments, showing up-regulation in mouse livers treated with anti-miR-122 and having 3′UTRs down-regulated by miR-122 mimetics (Gatfield et al, 2009
). In total, 11 other genes were identified as showing up-regulation by anti-miR-122 and having 3′UTRs down-regulated by miR-122 mimetics, but without circadian accumulation. In this study, 13 of these 19 genes were up-regulated by anti-miR-122 treatment in mice, including 7 of the 8 genes showing circadian accumulation. However, only one gene, SMARCD1 (BAF60a), also showed expression negatively correlated with miR-122 expression in our HCC samples, emphasizing the importance of cross-species analysis. Cyclin G1, found by others (Gramantieri et al, 2007
) to be anti-correlated with miR-122 in HCC, was not significantly correlated or anti-correlated with miR-122 in our samples. Similarly, although N-myc has been suggested as a target of miR-122 (Girard et al, 2008
), its expression levels are not significantly correlated or anti-correlated to miR-122 levels in this study.
Other published studies indicate that miR-122 is a host factor for HCV replication (Jopling et al, 2005, 2006
,Jopling et al, 2005, 2006
; Shan et al, 2007
; Chang et al, 2008
; Henke et al, 2008
; Lupberger et al, 2008
) and show that HCV-infected HCC patients usually do not show a reduction in and may in fact show an increase in miR-122 expression (Varnholt et al, 2008
), although higher miR-122 levels have also been shown to predict better response of HCV patients to standard therapy (Sheikh et al, 2008
). HCV appears to target mitochondria directly causing liver dysfunction (Sarasin-Filipowicz et al, 2009
) by a route not dependent on decreasing miR-122 expression.
The primary targets of an miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007
). In our study, we found increased lactate production in tissue culture cells after treatment with anti-miR-122. Reduced mitochondrial oxidative phosphorylation is commonly observed in cancer cells, and we postulate that the reduced expression of miR-122 may contribute to this effect in HCC. In light of the observed connection between miR-122 expression and mitochondrial function pathways in liver, we speculate that increasing miR-122 expression may possibly improve mitochondrial function in liver and perhaps in liver tumor tissues. Therefore, it is of great interest to determine the phenotypic changes of HCC after miR-122 targeting delivery in our established HCC mouse models (Zender et al, 2008
; Liu et al, 2009
Our findings reveal potential new biological functions of miR-122 in liver physiology. We have observed the decrease of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 appears to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC.