Long noncoding RNA Hoxb3os is dysregulated in autosomal dominant polycystic kidney disease and regulates mTOR signaling

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating disease that is characterized by the accumulation of numerous fluid-filled cysts in the kidney. ADPKD is primarily caused by mutations in two genes, PKD1 and PKD2. Long noncoding RNAs (lncRNA) - defined by a length >200 nucleotides and absence of a long open reading frame - have recently emerged as epigenetic regulators of development and disease; however, their involvement in PKD has not been explored previously. Here, we performed deep RNA sequencing to identify lncRNAs that are dysregulated in two orthologous mouse models of ADPKD (kidney-specific Pkd1 and Pkd2 mutant mice). We identified a kidney- specific, evolutionarily-conserved lncRNA called Hoxb3os that was downregulated in cystic kidneys from Pkd1 and Pkd2 mutant mice. The human ortholog HOXB3-AS1 was downregulated in cystic kidneys from ADPKD patients. Hoxb3os was highly expressed in renal tubules in adult wild- type mice, whereas its expression was lost in the cyst epithelium of mutant mice. To investigate the function of Hoxb3os, we utilized CRISR/Cas9 to knockout its expression in mIMCD3 cells. Deletion of Hoxb3os resulted in increased phosphorylation of mTOR and its downstream targets, including p70 S6 kinase, ribosomal protein S6, and the translation repressor 4E-BP1. Consistent with activation of mTORC1 signaling, Hoxb3os mutant cells displayed increased mitochondrial respiration. The Hoxb3os mutant phenotype was partially rescued upon re- expression of Hoxb3os in knockout cells. These findings identify Hoxb3os as a novel lncRNA that is downregulated in ADPKD and regulates mTOR signaling and mitochondrial respiration. Expression profiling by sequencing in primary kidney and cultured cells.常染色体显性遗传性多囊肾病（Autosomal dominant polycystic kidney disease, ADPKD）是一种致残性疾病，其特征为肾脏内大量充满液体的囊肿生成。ADPKD主要由PKD1与PKD2两个基因的突变所引发。长链非编码RNA（long noncoding RNAs, lncRNA）——定义为长度超过200核苷酸且不具备长开放阅读框的RNA分子——近年来被证实为发育与疾病进程中的表观遗传调控因子，但此前尚未有研究探讨其在多囊肾病中的作用。本研究通过深度RNA测序，在两种ADPKD同源小鼠模型（肾脏特异性Pkd1突变小鼠及Pkd2突变小鼠）中筛选失调的lncRNA。我们鉴定出一种肾脏特异性、进化保守的lncRNA Hoxb3os，其在Pkd1与Pkd2突变小鼠的囊性肾脏中表达下调。该基因的人类同源基因HOXB3-AS1在ADPKD患者的囊性肾脏中同样呈现表达下调。Hoxb3os在成年野生型小鼠的肾小管中高表达，而在突变小鼠的囊肿上皮细胞中表达缺失。为探究Hoxb3os的功能，我们利用CRISPR/Cas9技术在mIMCD3细胞中敲除其表达。敲除Hoxb3os会导致mTOR（哺乳动物雷帕霉素靶蛋白）及其下游靶点（包括p70 S6激酶、核糖体蛋白S6以及翻译抑制因子4E-BP1）的磷酸化水平升高。与mTORC1（哺乳动物雷帕霉素靶蛋白复合物1）信号通路激活的结果一致，Hoxb3os敲除细胞表现出线粒体呼吸作用增强。在敲除细胞中重新表达Hoxb3os可部分挽救该突变表型。本研究证实Hoxb3os是一种在ADPKD中表达下调的新型lncRNA，可调控mTOR信号通路与线粒体呼吸作用。本研究通过测序对原代肾细胞与培养细胞开展了表达谱分析。