Genome-wide MicroRNA Downregulation as a Negative Feedback Mechanism in the Early Phases of Liver Regeneration

Liver is one of the few organs that have the capacity to regenerate in response to injury. We carried out genome-wide miRNA microarray studies during liver regeneration in rats after 70% partial hepatectomy (PH) at early and mid-time points to more thoroughly understand their role. At 3, 12 and 18 hrs post-PH ~ 40% of the miRNAs tested were up-regulated. Conversely, at 24 hrs post-PH, ~ 70% of miRNAs were down-regulated. Further, we established that the genome-wide down-regulation of miRNA expression at 24 hrs was also correlated with decreased expression of genes such as Rnasen, Dgcr8, Dicer, Tarbp2 and Prkra that are associated with miRNA biogenesis. To determine if a potential negative feedback loop between miRNAs and their regulatory genes existed, 11 candidate miRNAs which were predicted to target the above genes were examined and found to be up-regulated at 3 hrs post-PH. Using reporter and functional assays, we determined that expression of these miRNA-processing genes could be regulated by a subset of miRNAs and some miRNAs could target multiple miRNA biogenesis genes simultaneously. We also demonstrated that over-expression of these miRNAs inhibited cell proliferation and modulated the cell cycle in both Huh-7 human hepatoma cells and primary rat hepatocytes. From these observations, we postulated that selective up-regulation of miRNAs in the early-phase after PH was involved in the priming and commitment to liver regeneration, while the subsequent genome-wide down-regulation of miRNAs was required for efficient recovery of liver cell mass. Conclusion: Our data suggest that miRNA changes are regulated by negative feedback loops between miRNAs and their regulatory genes that may play an important role in the steady-state regulation of liver regeneration.肝脏是少数可对损伤产生再生响应的器官之一。为深入阐明微小RNA（miRNA）的作用机制，我们针对大鼠经70%体积肝部分切除术（PH）后的肝脏再生过程，在早期及中期时间节点开展了全基因组miRNA芯片研究。在肝部分切除术后3、12及18小时，约40%的检测miRNA呈现表达上调；与之相反，术后24小时约70%的检测miRNA呈现表达下调。进一步研究发现，术后24小时的全基因组miRNA表达下调，与Rnasen、Dgcr8、Dicer、Tarbp2及Prkra等与miRNA生物发生相关的基因表达降低存在显著关联。为探究miRNA与其调控基因之间是否存在潜在负反馈环路，我们对11个预测可靶向上述基因的候选miRNA进行了检测，发现其在术后3小时呈现表达上调。通过报告基因及功能实验，我们证实这些miRNA加工相关基因的表达可被一类miRNA调控，且部分miRNA可同时靶向多个miRNA生物发生相关基因。我们还证实，在Huh-7人肝癌细胞及原代大鼠肝细胞中，过表达这些miRNA可抑制细胞增殖并调控细胞周期。基于上述观察结果，我们提出假说：肝部分切除术后早期阶段miRNA的选择性上调参与肝脏再生的启动与定向过程，而后续的全基因组miRNA表达下调则是高效恢复肝脏细胞总量所必需的。结论：本研究数据表明，miRNA的表达变化受miRNA与其调控基因之间的负反馈环路调控，该环路可能在肝脏再生的稳态调控中发挥重要作用。