MicroRNA-122 Regulates Postnatal Polyploidization in the Murine Liver

A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion: Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease. Livers from C57Bl/6 mice were isolated at defined ages: embryonic day 15.5 (n=3; mixed gender), 2 weeks (n=3; male), 3 weeks (n=3, male) and 7 weeks (n=3; male). Differential miRNA expression was assessed using the nCounter Mouse miRNA Expression Assay Kit (nanoString).哺乳动物肝脏的标志性特征之一是多倍体化，即整套染色体的数目发生改变。多倍体化的第一步涉及胞质分裂失败的细胞分裂过程。尽管多倍体化较为普遍，约占小鼠肝细胞的90%、人类肝细胞的50%，但多倍体细胞在肝脏稳态与疾病中所扮演的特殊角色仍有待深入解析。本研究旨在鉴定调控肝脏多倍体化的新型信号通路，并将研究焦点聚焦于微小RNA（microRNAs, miRNAs）。 首先，为验证微小RNA是否可调控肝脏多倍体化，我们检测了Dicer1肝脏特异性敲除小鼠的肝脏组织——此类小鼠体内缺乏成熟的微小RNA。实验结果显示，微小RNA的缺失导致双核肝细胞数量减少3倍，证实微小RNA确实参与调控肝脏多倍体化过程。 其次，我们对野生型小鼠体内微小RNA的年龄依赖性表达谱进行了分析，鉴定出一组在2~3周龄时差异表达的微小RNA（包括miR-122），而该时期正是肝脏广泛发生多倍体化的关键阶段。 随后，我们对Mir122敲除小鼠进行了实验检测，观察到多倍体肝细胞出现永久性的显著耗竭，证明miR-122是肝脏完成完全多倍体化所必需的调控因子。此外，通过腺病毒介导的miR-122过表达可有效改善Mir122敲除小鼠的多倍体化缺陷，进一步凸显了miR-122在多倍体化进程中的核心作用。 最后，我们鉴定出miR-122的6个直接靶基因（Cux1、Rhoa、Iqgap1、Mapre1、Nedd4l及Slc25a34），这些基因均参与调控胞质分裂过程。抑制任一靶基因均可诱导胞质分裂失败，并促进肝脏细胞形成双核结构。 研究结论：本研究数据证实，miR-122在肝脏多倍体化过程中既是必需的，也是充分的调控因子。在已报道的与肝脏多倍体化相关的各类信号通路中，miR-122是首个被鉴定出的肝脏特异性信号。本研究将为后续探索miR-122在肝脏成熟、稳态维持及疾病发生发展中的作用奠定坚实的研究基础。 实验样本与方法：本研究从C57Bl/6小鼠体内分离肝脏组织，采样时间涵盖明确的发育阶段：胚胎第15.5天（n=3，雌雄混合）、2周龄（n=3，雄性）、3周龄（n=3，雄性）及7周龄（n=3，雄性）。采用nCounter小鼠微小RNA表达分析试剂盒（nanoString公司）对微小RNA的差异表达水平进行检测。