阻抑长链非编码RNA-HOTAIR以诱导肌成纤维细胞重分化对肝纤维化的防治意义

Myofibroblast (MFB), which plays the critical part in hepatic fibrogenesis, is derived from mesenchymal cells by phenotypic de-differentiation. Although various procedures, such as inhibiting the generation of MFB and eradicating the existing ones, have been employed, the difficulties in MFB redifferentiation still make it a hard task to prevent and reverse the liver fibrosis. Recently, one of the long non-coding RNAs, HOTAIR, is verified to highly express in MFB. Its effects on chromatin remodeling induce the gene depression in HOX loci, and then the de-differentiation phenotype. We, therefore, attempt to restore the chromatin structure by HOTAIR knockdown. The activation of redifferentiation genes, being obtained as a result, will help to abolish the dedifferentiation-related functions (excessive proliferation, extracellular matrix production/secretion, motility and chemotaxis) and acquire the redifferentiation -related functions (lipid metabolism, bile secreton). In vivo down-regulation of HOTAIR using lentiviral vectors provide another insight into its role on MFB redifferentiation, and fibrosis interference.

肝脏间质细胞失去分化表型，转化为肌成纤维细胞(myofibroblast, MFB)是肝纤维化过程的关键事件。既往的干预措施局限于抑制MFB生成，加速其清除，无法诱导MFB重新向成熟表型分化，因而难以实现阻断乃至逆转肝纤维化的目标。我们近期发现，长链非编码RNA（long non-coding RNA, lnRNA）-HOTAIR在MFB中特异性高表达，并通过同源异型盒基因座的染色质重塑，导致分化相关基因的表达缺失。为此，本研究拟以siRNA实现HOTAIR的基因沉默，从而逆转染色质重塑，恢复促分化基因活性，在此基础上消除MFB的过度增殖、细胞外基质合成/分泌、迁移、趋化等去分化表型，恢复脂质代谢、胆汁分泌等成熟细胞功能。此外借助慢病毒载体进行体内HOTAIR"敲减"，观察诱导MFB重分化对肝纤维化的防治效应。

本课题通过筛选MFB相关的长链非编码RNA（lncRNA），发现Vof16出现特异性高表达，并与MFB的去分化表型密切相关。为揭示lncRNA-Vof16在维持MFB表型及诱导肝纤维化过程中的作用，课题组采用pull-down、RIP联合RNA建库及二代测序（RIP-sequencing）检测了Vof16与MFB相关蛋白的交互作用。证实Vof16主要作用于HNRPG、K1C1、K2C1、K2C6A、RL19、ROA1、RS3A、TRA2B、ZCHC7等蛋白，且在RNA转录后剪切环节发挥重要的调控作用。以lncRNA基因沉默为基础，可见Vof16表达下调能够恢复MFB的凋亡敏感性，提高HNF1α、RARα、RXRα mRNA水平，从而诱导MFB重分化，表型向静止的肝星状细胞（hepatic stellate cell, HSC）转化。构建携带α-SMA启动子、Vof16靶向性shRNA序列的重组慢病毒，由此进行Vof16表达的在体干预，则可上调MFB凋亡率，显著减少MFB表型的细胞数量；进而抑制纤维组织形成，改善四氯化碳（CCl4）诱导的大鼠实验性肝纤维化。上述成果揭示了lncRNA-Vof16在MFB表型分化中的调控效应及其确切机制，在MFB重分化的基础上阐明了靶向干预Vof16对纤维化进程的影响，从而为发现肝纤维化防治的新途径提供了理论依据与可能靶点。