Previously, we and others observed a surge in miR-21 expression in G1 phase in hepatocytes after 2/3 PH (4
). Here, we investigated the function of induced miR-21 expression in liver regeneration by specifically antagonizing the miR-21 surge, but not baseline miR-21 expression, with miR-21–ASO, a short LNA-stabilized antisense oligonucleotide inhibitor of miR-21. This partial knockdown strategy revealed that increased miR-21 expression facilitates cyclin D1 translation in early liver regeneration. Cyclin D1 translation is mediated by the eIF-4F translation initiation complex, which is activated by Akt1/mTORC1 signaling (24
). Our results showed that miR-21 activates Akt1/mTORC1-mediated cyclin D1 translation by inhibiting Rhob. While Rhob has been shown to inhibit Akt1 (22
), to our knowledge, it was not previously known that this effect of Rhob results in inhibition of cyclin D1 translation.
Our results further showed that promotion of cyclin D1 translation by miR-21 is important for rapid G1 phase progression and S phase entry of hepatocytes after 2/3 PH. In mice in which the miR-21 surge was antagonized by miR-21–ASO, the number of hepatocytes in S phase at 36 hours after 2/3 PH was markedly reduced compared with controls. However, hepatocyte cell cycle progression was not permanently blocked in miR-21–ASO–injected mice, and the cells eventually entered S phase and restored the lost liver mass. Impaired hepatocyte proliferation, particularly if it is caused by single gene deficiencies, is typically compensated for in liver regeneration. Compensation is the result of redundant signaling pathways providing the missing function, which leads to delayed rather than failed liver mass restoration (32
). A previous study suggested that overexpression of cyclin E can compensate for lack of cyclin D1 in Ccnd1–/–
mice, thereby facilitating normal hepatocyte proliferation (14
). In accordance with this result, we found that cyclin E1 and downstream cyclins involved in liver regeneration were overexpressed as early as 18 hours after 2/3 PH, when cyclin D1 protein deficiency was most prominent in miR-21–depleted hepatocytes. In addition, we observed that cyclin D1 translation was slowly improving between 18 and 36 hours after 2/3 PH. Because miR-21 was still depleted at 36 hours after 2/3 PH, the finding suggests miR-21–independent cyclin D1 translation as another mechanism that helps rescue liver regeneration in miR-21–ASO–injected mice.
Our finding that miR-21 promoted cyclin D1 translation in liver regeneration by activating Akt1/mTORC1 prompted us to investigate whether PTEN, a miR-21 target known to inhibit Akt1 (30
), is also involved in this process. After performing 2/3 PH on normal mice, we found that PTEN protein accumulated during G1 phase despite the surge in miR-21 expression induced by 2/3 PH. The finding revealed that accumulation of PTEN does not prevent Akt1 activation in normal liver regeneration. Moreover, the finding showed that suppression of PTEN by miR-21 is not involved in Akt1 activation and thus cyclin D1 translation in early liver regeneration. Nevertheless, miR-21’s previously reported ability to activate Akt1 by inhibiting PTEN in cancer cells (30
), taken together with our present results, suggests the intriguing possibility that miR-21 acts as a central regulator of Akt1/mTORC1 signaling in other contexts.
Because miRNAs can target many genes, the function of miR-21 in liver regeneration is likely not limited to promotion of cyclin D1 translation. In fact, we and others have previously identified Btg2 and Pellino 1, which are regulators of FoxM1b and NF-κB activity, respectively, as targets of miR-21 during liver regeneration after 2/3 PH (4
). In addition, our present results suggest that Pdcd4 is moderately suppressed at 18 hours after 2/3 PH, and it is possible that in the future, other miR-21 targets will be identified as playing a role in liver regeneration.
However, our finding that the hepatocyte cell cycle defect in miR-21–ASO–injected mice phenocopied that of mice in which cyclin D1 was suppressed with Ccnd1
-ASO suggests that miR-21’s effect on hepatocyte cell cycle progression in early liver regeneration is mainly mediated by cyclin D1. Importantly, derepression of Btg2, Pellino 1, or Pdcd4 cannot explain the lack of cyclin D1 expression in hepatocytes of miR-21–ASO–injected mice. Hepatocyte-specific deficiency of the Btg2 target FoxM1b leads to impaired activity of cyclin-dependent kinase complexes involving cyclin E1, A2, and B1, but not D1, after 2/3 PH in mice (33
). Moreover, Btg2 overexpression in human hepatoma cells decreases mRNA and protein levels of cyclin B1, but not cyclin D1 (34
). Similarly, Pdcd4 overexpression fails to suppress cyclin D1 mRNA or protein levels (19
). Because Pellino 1 activates NF-κB signaling (35
), its derepression in hepatocytes of miR-21–ASO–injected mice would be expected to accelerate, not delay, cell cycle progression (32
). Thus, the evidence points to Rhob as the critical target of miR-21 in promotion of Akt1/mTORC1-mediated cyclin D1 translation and cell cycle progression of hepatocytes in early liver regeneration.
Our finding that miR-21–ASO can be used to partially inhibit miR-21 and derepress its target genes within 8 hours of intravenous injection has important implications. First, it highlights a method of miRNA inhibition in hepatocytes that avoids unspecific changes due to compensation or adaptation, which can mask or confound the phenotype caused by deficiency of the miRNA. Compensation of impaired hepatocyte proliferation by redundant signaling pathways occurs not instantaneously, but typically within a day after 2/3 PH (32
). The rapid onset of miR-21 inhibition by miR-21–ASO limits the time available for activation of redundant pathways and may therefore be more effective than genetic deletion for studies of liver regeneration. In addition, limiting the extent of miRNA inhibition in hepatocytes may help to maintain homeostasis and avoid secondary changes and adaptation. Second, our study showed that the rapid onset of miR-21–ASO’s effect allows the dynamic processes that occur during liver regeneration to be dissected: miR-21 and Ccnd1
expression are concomitantly induced after 2/3 PH, and our results revealed that promotion of cyclin D1 translation is critical for the early phase of liver regeneration, when Ccnd1
mRNA levels are still low, but not for later phases. Finally, our results raise the possibility of using oligonucleotide mimics of miR-21 for therapy of liver failure. Provided that miR-21 mimics enter hepatocytes and exert their function as quickly as miR-21–ASO, they may be effective in accelerating progression of hepatocytes through G1 and into S phase, which is critical for survival from liver injury (36
In conclusion, our results revealed that the increased expression of miR-21 during liver regeneration functions to kick-start cyclin D1 translation in hepatocytes. This effect of miR-21 was mediated by a mechanism that integrated inhibition of Rhob by miR-21 with a function of Rhob that we believe to be previously unrecognized, the suppression of eIF-4F–mediated translation initiation through suppression of Akt1/mTORC1 signaling. Our results, together with miR-21’s ability to activate Akt1 by inhibiting PTEN (30
), suggest miR-21 as a key regulator of mTORC1. Moreover, our finding that miR-21 derepressed eIF-4F, which mediates the rate-limiting ribosomal binding step of translation initiation, revealed its profound impact on cell proliferation. In liver regeneration, acceleration of cyclin D1 translation by miR-21 facilitated rapid progression of hepatocytes through G1 and into S phase. This effect of miR-21 may also be involved in the rapid proliferation of other regenerative cell types and cancer cells.