Gene expression changes in the respiratory epithelium of miR-34/449 knockout mice

MicroRNA ablation prevents multiciliogenesis via cell cycle deregulation MicroRNAs (miRNAs) have been implicated in various biological processes but have been most frequently described to inhibit proliferation and tumorigenesis. Here we describe an essential function of the miR-34/449 family during differentiation of multiciliated cells that is mediated by their well-described suppression of cell cycle progression. Constitutive deletion of all six members of this miRNA family triggers a derepression of multiple cell cycle-promoting proteins, thereby preventing epithelial cells from exiting the cell cycle and to maintain in a quiescent state. As a result, formation of motile multicilia is strongly inhibited in several tissues including the respiratory epithelium and the fallopian tube. Consequently, mice lacking miR-34/449 genes display infertility as well as severe chronic airway disease leading to postnatal death. These results show how miRNA-mediated repression of the cell cycle is required to allow multiciliogenesis to proceed during epithelial differentiation. We analyzed samples from the respiratory epithelium of the nose (NRE) of 5 wild-type (WT) and 5 miR-34/449 triple knockout (TKO) embryos (at E16.5), which were isolated using Laser Capture Microdissection (LCM).微小RNA敲除通过细胞周期失调阻断多纤毛发生。微小RNA（miRNAs）已被证实参与多种生物学过程，其中最广为报道的功能为抑制细胞增殖与肿瘤发生。本研究揭示了miR-34/449家族在多纤毛细胞分化过程中的核心调控功能，该功能通过其已知的抑制细胞周期进程作用实现。该miRNA家族全部6个成员的系统性敲除，会解除对多种细胞周期促进蛋白的抑制，从而阻碍上皮细胞退出细胞周期并维持静息状态。因此，包括呼吸道上皮与输卵管在内的多种组织中，可运动的多纤毛形成过程受到显著抑制。故而，缺失miR-34/449基因的小鼠会出现不育症，并伴随严重的慢性气道疾病，最终导致产后死亡。上述研究结果证实，在上皮细胞分化过程中，miRNA介导的细胞周期抑制是多纤毛发生顺利进行的必要条件。本研究通过激光捕获显微切割（Laser Capture Microdissection, LCM）技术，分离得到了5只野生型（WT）与5只miR-34/449三基因敲除（TKO）小鼠胚胎（胚胎发育第16.5天，E16.5）的鼻腔呼吸道上皮（NRE）样本并开展分析。