运用Hes1 decoy ODN 干预Hes1和miR-23b/27b/24-1基因簇之间的调节环进而逆转肝纤维化的研究

The imbalance of the TGF-β and BMP-7 signaling pathways is a critical step in promoting hepatic stellate cell sustained activation during the process of damage-inflammation-repairation of liver. Our study has found Gremlin1 contributes to impairment of the balance of TGF-β and BMP-7-mediated signal transduction, in turn, accelerating hepatic fibrogenesis. Our study has also found miR-23b/27b/24-1 cluster may synergistically silence Gremlin1 and LOX genes resulting in correcting the imbalance of the TGF-β and BMP-7 signaling pathways.to inhibit the activation of hepatic stellate cells.. Hes1 as a transcription factor promotes the activation of hepatic stellate cells, while Hes1 decoy ODN down-regulates the promoter activities of α-smooth muscle actin and Collagen Iα2 in hepatic stellate cell-T6 cells. Further research has found that there are binding sites both for miR-23b and for miR-27b in 3′ untranslated region of Hes1, which means that the two microRNAs can down-regulate Hes1 potentially. At the same time, it contains three Hes1 binding sites in the promoter of miR-23b/27b/24-1 cluster. Preliminary experiments have also verified there is the down-regulation effect of Hes1 on the promoter. There seems to be a regulative circuit between Hes1 and miR-23b/27b/24-1 cluster.. Therefore, we put forward a scientific hypothesis that there is a regulative circuit between Hes1 and miR-23b/27b/24-1 cluster, and the circuit plays an important positive role in the process of the activation of hepatic stellate cells. It may be an effective strategy using Hes1 decoy ODN to interfere with the circuit in therapy of hepatic fibrosis.. This project aims to confirm a regulative circuit between Hes1 and miR-23b/27b/24-1 cluster using the techniques of molecular biology. In order to reverse the activation of hepatic stellate cells and hepatic fibrosis in vivo and in vitro, we intend to use Hes1 decoy ODN strategy to interfere with the regulative circuit. To reach this goal, biodegradable CPP-P(NIPAM-co-B15C5Am)-b-PMMA polymeric micelles, which have potassium-ion-responsive characteristics being modified on the surface by hepatic stellate cells targeted cell penetrating peptides will be prepared to deliver Hes1 decoy ODN to hepatic stellate cells.

HSC活化引起肝纤维化，导致基因簇miR-23b/27b/24-1的表达降低；该基因簇通过直接靶向沉默Gremlin1 和 LOX，抑制HSC活化；而转录因子Hes1促进HSC活化，在基因簇miR-23b/27b/24-1的启动子上，存在3个Hes1的结合位点，预实验证实，Hes1能下调该基因簇的启动子活性，提示Hes1与基因簇的表达改变有关联。另一方面，Hes1的3′非编码区也存在miR-23b/27b的结合位点，具有被二者负调控的潜能。我们推测，Hes1与基因簇miR-23b/27b/24-1之间可能存在相互作用的调节环；HSC活化时，该调节环可能起着关键性作用。本项目旨在确认Hes1与基因簇miR-23b/27b/24-1之间的调节环；用Hes1 decoy ODN策略干预该调节环，采用HSC特异性的细胞穿膜肽修饰的钾离子响应型纳米胶束递送，通过体内外实验确认其逆转肝纤维化的效果。

肝纤维化是由多种病因引起肝细胞损伤和炎症反应，进而演变为慢性进行性肝病，最终发展为肝硬化甚至原发性肝癌的病理过程。肝纤维化的发生机制极为复杂，迄今仍缺乏有效的治疗方法。近年来的研究显示肝纤维化可逆转。肝星状细胞（HSC）活化引起肝纤维化，导致基因簇miR-23b/27b/24-1的表达降低；该基因簇通过直接靶向沉默Gremlin1 和 LOX，抑制HSC活化；而转录因子Hes1促进HSC活化，在基因簇miR-23b/27b/24-1的启动子上，存在3个Hes1的结合位点，提示Hes1能下调该基因簇的启动子活性。另一方面，Hes1的3′非编码区也存在miR-23b/27b的结合位点，具有被二者负调控的潜能。据此我们提出，Hes1与基因簇miR-23b/27b/24-1之间可能存在相互作用的调节环，该调节环在HSC活化时可能起着关键作用，干预该调节环可能为抑制肝星状细胞活化，甚至逆转肝纤维化提供新的思路；在该项目中，我们通过构建miR-23b/27b/24-1基因簇启动子的双荧光素酶报告基因质粒证实了Hes1对该基因簇的调节；同时构建Hes1-3’UTR及其突变体的荧光素酶报告质粒，验证了miR-23b和miR-27b对Hes1的直接靶向下调潜能。将真核表达质粒pCDH-miR-23b/27b/24-1转染至HSC-T6细胞中，证实了该基因簇能有效下调Hes1的蛋白表达。运用适配体AC4WJF将Hes1 decoy ODN递送到HSC-T6细胞及TGF-β刺激活化的HSC-T6细胞中，证实了decoy ODN干预了Hes1与miR-23b/27b/24-1基因簇之间的调节环后能有效下调肝纤维化相关基因的表达。此外，干预该调节环后能有效抑制原代HSC细胞的活化。该课题将为适配体AC4WJF体内递送Hes1 decoy ODN应用于肝纤维化和肝硬化的治疗奠定坚实的理论基础。