Autophagy-mediated reduction of miR-345 contributes to hepatic cystogenesis in polycystic liver disease.
Autophagy-mediated reduction of miR-345 contributes to hepatic cystogenesis in polycystic liver disease.
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DOI：
10.1016/j.jhepr.2021.100345
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发表时间：
2021-10
期刊：
影响因子：
--
通讯作者：
LaRusso N
中科院分区：
文献类型：
--
作者：
Masyuk T;Masyuk A;Trussoni C;Howard B;Ding J;Huang B;LaRusso N
多囊肝病(PLD)的特征是胆管细胞自噬增加和miRNA水平降低。鉴于自噬与miRNA调控有关，我们检验了自噬增加可以解释PLD胆管细胞(PLDC)中miRNA减少和加速肝脏囊变的假设。我们用miRNA测序技术(miRNA-seq)评估了培养的正常人胆管细胞(NHCs)、PLDC和分离的PLDC自噬小体中miRNA的水平，并用mRNAseq方法检测了miRNA靶标。采用原位杂交、定量聚合酶链式反应、免疫印迹和荧光共聚焦显微镜等方法，研究miR-345和miR-345靶向蛋白在PLD动物和人类肝脏、NHC和PLDC中的表达水平。我们还评估了体外细胞增殖和囊性生长，以及体内肝脏囊性形成。总共有81%的miRNAs在PLDC中被下调，其中10个miRNAs的水平下降了10倍以上，miR-345是下降最严重的miRNA。在计算机分析和荧光素酶报告分析中发现miR-345靶基因包括与细胞周期和细胞增殖相关的基因[即细胞分裂周期25A(CDC25A)、细胞周期蛋白依赖性激酶6(CDK6)、E2F2和增殖细胞核抗原(PCNA)]；4个所研究的miR-345靶基因在mRNA和蛋白水平均在PLDC中上调。转染前miR-345的PLDC在体外增加miR-345，降低miR-345靶蛋白的表达，细胞增殖和囊泡生长。MIR-345在PLDC的自噬体内蓄积，而在NHC中不积聚。抑制自噬增加miR-345的水平，降低miR-345靶向蛋白的表达，并在体外和体内减少肝脏囊变。自噬介导的pldCs中miR-345的减少(即miRNAutophagy)加速了肝脏的囊变。抑制自噬可以恢复miR-345水平，减少囊泡生长，对PLD有利。多囊肝病(PLD)是一种无法治愈的遗传性疾病，其特征是肝囊肿的进行性生长。我们发现，当PLD胆管细胞(PLDC)中miR-345的水平通过自噬降低时，肝脏的囊性形成就会增加。通过抑制自噬来修复PLDC中的miR-345可以减少肝脏囊变，从而有利于PLD的发生。在PLD中，miRNA的图谱发生了变化。MiR-345是PLDC中表达最少的miRNA。MiR-345的表达下调促进了PLDC的增殖和肝脏的囊变。PLDC中的miR-345受自噬调节，称为“miRNAutophagy”。在PLDC中修复miR-345对PLD是有利的。
Polycystic liver disease (PLD) is characterised by increased autophagy and reduced miRNA levels in cholangiocytes. Given that autophagy has been implicated in miRNA regulation, we tested the hypothesis that increased autophagy accounts for miRNA reduction in PLD cholangiocytes (PLDCs) and accelerated hepatic cystogenesis. We assessed miRNA levels in cultured normal human cholangiocytes (NHCs), PLDCs, and isolated PLDC autophagosomes by miRNA-sequencing (miRNA-seq), and miRNA targets by mRNA-seq. Levels of miR-345 and miR-345-targeted proteins in livers of animals and humans with PLD, in NHCs and PLDCs, and in PLDCs transfected with pre-miR-345 were assessed by in situ hybridisation (ISH), quantitative PCR, western blotting, and fluorescence confocal microscopy. We also assessed cell proliferation and cyst growth in vitro, and hepatic cystogenesis in vivo. In total, 81% of miRNAs were decreased in PLDCs, with levels of 10 miRNAs reduced by more than 10 times; miR-345 was the most-reduced miRNA. In silico analysis and luciferase reporter assays showed that miR-345 targets included cell-cycle and cell-proliferation-related genes [i.e. cell division cycle 25A (CDC25A), cyclin-dependent kinase 6 (CDK6), E2F2, and proliferating cell nuclear antigen (PCNA)]; levels of 4 studied miR-345 targets were increased in PLDCs at both the mRNA and protein levels. Transfection of PLDCs with pre-miR-345 increased miR-345 and decreased the expression of miR-345-targeted proteins, cell proliferation, and cyst growth in vitro. MiR-345 accumulated in autophagosomes in PLDCs but not NHCs. Inhibition of autophagy increased miR-345 levels, decreased the expression of miR-345-targeted proteins, and reduced hepatic cystogenesis in vitro and in vivo. Autophagy-mediated reduction of miR-345 in PLDCs (i.e. miRNAutophagy) accelerates hepatic cystogenesis. Inhibition of autophagy restores miR-345 levels, decreases cyst growth, and is beneficial for PLD. Polycystic liver disease (PLD) is an incurable genetic disorder characterised by the progressive growth of hepatic cysts. We found that hepatic cystogenesis is increased when the levels of miR-345 in PLD cholangiocytes (PLDCs) are reduced by autophagy. Restoration of miR-345 in PLDCs via inhibition of autophagy decreases hepatic cystogenesis and thus, is beneficial for PLD. The miRNA profile is altered in PLD. MiR-345 is the most-reduced miRNA in PLDCs. The reduction of miR-345 increases PLDC proliferation and hepatic cystogenesis. MiR-345 in PLDCs is regulated by autophagy, termed ‘miRNAutophagy’. Restoration of miR-345 in PLDC is beneficial for PLD.
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