Fbxo30在失重性肌萎缩形成中的作用与机制研究

Muscle atrophy caused by weightlessness seriously affects astronauts' resident time in space and accomplishment of missions with success or not. However, the molecular mechanism of weightlessness-induced muscle atrophy has not yet been fully revealed, which hinders the development of effective protective measures. By using the transcriptome sequencing screening, we identified an ubiquitin ligase Fbxo30, which was significantly up-regulated in the process of weightlessness-induced muscle atrophy. In this study, by means of in vivo RNA interference, skeletal muscle-specific Fbxo30 knockout mice and in vivo overexpression of Fbxo30, we will delineate an essential role of Fbxo30 in mediating weightlessness-induced muscle atrophy. By comparing the promoter sequences of Fbxo30 gene of human and mice, we found a conserved ATF4 binding site in the promoter region of Fbxo30, we will deeply explore the transcriptional regulatory mechanism that induces the expression of Fbxo30 in the process of weightlessness-induced muscle atrophy. Bioinformatic predictions show that there is a miRNA response element for miR-23 in the Fbxo30 3'UTR region. Given that miR-23 has been shown to target one of the atrophy-related genes, Atrogin-1, we will further reveal the interaction between Fbxo30 and Atrogin-1 in the process of weightlessness-induced muscle atrophy by working as competing endogenous RNA (ceRNA) reciprocally. This study will provide a new potential target for the development of effective protective measures or drugs for weightlessness-induced muscle atrophy.

失重性肌萎缩严重影响航天员在轨驻留时间及任务完成质量，但目前其分子机制尚未被完全揭示，妨碍了失重性肌萎缩有效防护措施的研制。我们应用转录组测序筛选到，泛素连接酶Fbxo30在失重性肌萎缩形成过程中显著上调，本研究借助于体内RNAi,骨骼肌特异性敲除动物及体内过表达等手段，将首先明确Fbxo30在失重性肌萎缩形成中的关键作用；通过比对人和小鼠Fbxo30基因启动子序列后显示，该基因启动子区域存在ATF4的保守结合位点，本研究将进一步探讨失重性肌萎缩形成过程中诱导Fbxo30表达的转录调控机制；生物信息学预测，Fbxo30 3’UTR区存在miR-23靶向位点，后者已被证明可以靶向肌萎缩基因Atrogin-1，我们将深入揭示Fbxo30与Atrogin-1在参与肌萎缩形成过程中互为竞争性内源RNA (ceRNA)的作用机制。该研究有望为研发失重性肌萎缩的有效防护措施或药物提供新的潜点。

失重性肌萎缩严重影响航天员在轨驻留时间及工作绩效，但现有的综合防护对抗措施不足以有效对抗失重性肌萎缩的发生。因此，深入揭示失重性肌萎缩发生的分子机制是开展肌萎缩有效防护对抗措施以保障未来载人深空探测任务成败的先决条件。本研究聚焦一种新近发现的泛素连接酶Fbxo30，利用公认的尾悬吊小鼠失重性肌萎缩模型发现：1）该基因在模拟失重性肌萎缩形成过程中显著上调，RNA干涉、骨骼肌特异性敲除Fbxo30转基因小鼠的结果表明，敲低或敲除该基因可以有效对抗失重性肌萎缩的发生，而体内过表达该基因可以诱导肌纤维萎缩，证实该基因的上调参与了失重性肌萎缩的发生；2）上游信号机制的研究结果证实，该基因转录调控区存在内质网应激下游重要转录因子ATF4的结合位点，内质网应激在失重性肌萎缩形成过程中也被显著激活，且ATF4因子的表达也与肌萎缩形成密切相关，利用牛磺酸脱氧胆酸钠（TUDCA）抑制内质网应激的激活后，也可以有效抑制Fbxo30和其它萎缩因子的表达，也明显对抗了失重性肌萎缩的形成和肌纤维类型的转变；体外实验进一步证实，骨骼肌中内质网应激通路的激活与另一重要代谢通路AMPK通路存在正反馈；3）在调控机制方面证实，ATF4通过结合在Fbxo30基因转录调控区的保守结合位点发挥了对后者的转录调控作用，而Fbxo30基因可以通过其3’UTR区域存在的miR-23结合位点，通过吸附miR-23，减少miR-23对另一重要萎缩基因Atrogin-1 3’UTR区域的结合作用，促进了Atrogin-1在蛋白水平的表达增加及肌萎缩的形成。本项目研究成果不仅为有针对性研发失重性肌萎缩的有效防护措施提供了新的潜在分子靶点，也为肌萎缩的防护对抗提供了有效的候选化合物TUDCA，作为已上市药物，该药物已经显示出较好的安全性和耐受性，具备推广应用至航天员或临床长期卧床病人以防护对抗其肌萎缩的潜在应用前景。