HSC中特异性调节炎症与纤维化串话通路miR-192-Rictor/mTORC2-TLR4的研究

Even quiescent hepatic stellate cells (HSCs) express high levels of TLR4 and are highly responsive to LPS treatment, suggesting that HSCs is not passively involved in inflammation, but positively act as sentinels that promote inflammatory and thus fibrogenic responses after increased hepatic exposure to TLR4 agonists such as LPS. In previous work, we found that miR-192 may directly target Rictor, implicated in mTORC2 signaling pathway. In HSCs which was activated by LPS-TLR4 signals, the downregulation of miR-192 was observed significantly, and restoration of miR-192 in activated-HSCs may lead to alterations of both mTORC2 and TLR4 signaling spontaneously. Hence, we present a hypothesis that miR-192-Rictor/mTORC2 participates in the modulation of TLR4 signaling pathway, and plays a pivotal role in the cascade effects of inflammation and fibrogenesis. In this project, we will further investigate the role of miR-192 in the cascade effects of inflammation and fibrogenesis in terms of biological function of HSCs, verify the signaling pathway of miR-192-Rictor/mTORC2-TLR4 in HSCs, and utilize our established technique e.g. artificial intron-derived microRNAs (Id-miRNA) expression system to realize the activated-HSC-cell-specific gene silencing. Our project would guarantee further investments to develop miR-192 as an effective molecule for intervening therapy of liver fibrosis.

新近研究提示，肝星状细胞（HSC）并非如已往认为的那样，只是肝纤维化的被动参与者，它通过TLR4通路主动参与炎症，进而调控炎症与肝纤维化的级联反应。前期工作发现，miR-192通过靶向Rictor参与mTORC2通路调控，预实验结果显示在LPS-TLR4激活的HSC中miR-192表达下调，过表达miR-192可引起mTORC2和TLR4信号改变。据此我们提出科学假设：miR-192-Rictor/mTORC2参与TLR4信号调控，在炎症和肝纤维化的级联反应中发挥重要作用。本课题拟在前期工作基础上，进一步探讨miR-192在HSC炎症和纤维化级联反应中的作用，重点验证miR-192-Rictor/mTORC2-TLR4信号通路，并应用已建立的内含子miRNA表达技术，实现对HSC特异性的miR-192表达，以期为小分子物质miR-192成为有效而特异的纤维化干预靶点提供理论依据和应用前景。

肝纤维化是各种理化因素引发肝脏组织持续的慢性炎症，进而导致肝内纤维沉积破坏原有的肝脏生理结构。肝纤维化各类并发症以及继发的肝癌的风险严重损害了患病者的生活质量。肝星状细胞是肝内的一类间质细胞，在慢性的肝脏炎症时分泌大量胶原纤维导致肝脏组织纤维沉积。肝星状细胞活化被认为是肝纤维化发展的中心环节。目前临床上针对肝纤维化的治疗尚且只能针对病因治疗，仍然需要探究肝星状细胞活化的分子机制开发新的治疗靶点。miRNA分子是一类长约22个核苷酸的非编码小RNA，能在转录后水平广泛调节基因的表达。本研究采用四氯化碳腹腔注射构建大鼠的肝纤维化模型，发现miR-192作为肝组织内源性表达丰富的miRNA分子之一，在肝脏纤维化中显著下调。TGF-β1激活的肝星状细胞内miR-192表达下调，而恢复miR-192的表达可以抑制TGF-β1对肝星状细胞的活化作用。生物信息学方法及荧光素酶报告实验证实mTORC2通路的关键组分Rictor分子，作为miR-192下游靶点介导了miR-192抑制肝星状细胞活化作用。进一步的分子机制研究突破来自我们同期展开的miR-192-Rictor对肝癌细胞抗肿瘤作用的研究。实验发现miR-192通过调节Rictor能在具有野生型p53的肝癌细胞内发挥显著的抗肿瘤作用，而在突变型的p53的肝癌细胞中仅发挥微弱的抗肿瘤作用。免疫荧光及免疫共沉淀证实了Rictor能同野生型p53结合。荧光定量PCR试验检测p53的下游分子Bax、p21等分子证实Rictor能通过结合p53分子而抑制其转录活性。同样的在肝星状细胞中，miR-192能通过靶向调节Rictor促进p53功能进而影响肝星状细胞活化。本研究探究了肝纤维化发展过程中miR-192-Rictor-p53通路的失调，为肝纤维化及肝癌治疗提供了一个潜在的靶点。