Lnc-GRM1-1-8在HCV相关肝纤维化中的作用及机制研究

HCV-related hepatic fibrosis is the critical pathway leading to end-stage liver diseases. Early diagnosis and reversion of liver fibrosis progression is the vital problem to be solved. Long non-coding RNA (lncRNA) play a pivotal modulated role in the progression of liver fibrosis. Our pre-experiment data showed that was significantly upregulated in plasma and liver tissue of patients with HCV-related hepatic fibrosis. However, the role and molecular mechanism of lnc-GRM1-1-8 in HCV-related hepatic fibrosis was still unclear. In this study, we plan to carry out the following investigations: ① to examine the expression of lnc-GRM1-1-8 in plasma and liver tissue of patients with HCV-related hepatic fibrosis, and to analyze the correlation of plasma lnc-GRM1-1-8 level and the stage of liver fibrosis; ② overexpression and knockdown of lnc-GRM1-1-8 in hepatic stellate cell line, and CCl4-induced mouse model of fibrosis, then to observe the modulated effect on phenotype of HSC, such as activation, proliferation, apoptosis of HSC and the production of collagen, and the progression of liver fibrosis; ③to verify the biding sites between lnc-GRM1-1-8, miR-29 and its target genes, and to clarify the role and molecular signal pathway of lnc-GRM1-1-8 as ceRNA to modulate miR-29 and its target genes on the progression of HCV-related liver fibrosis. This study might provide the theoretical basis for novel biomarker and new target therapy for HCV-related liver fibrosis.

HCV相关肝纤维化是导致HCV相关终末期肝病的重要阶段，早期诊断及逆转肝纤维化进展亟待解决。我们前期研究发现，lnc-GRM1-1-8在HCV相关肝纤维化中表达显著升高，可能参与调控肝纤维化进程，但其在该病中的作用及分子机制尚未清楚。本课题拟进行系统研究：①临床水平：检测HCV相关肝纤维化患者血浆及肝组织lnc-GRM1-1-8表达，分析lnc-GRM1-1-8表达与肝纤维化分期相关性；②细胞水平及肝纤维化动物模型：在肝星状细胞系及CCl4小鼠肝纤维化模型中过表达及沉默lnc-GRM1-1-8表达，明确其对肝星状细胞表型及肝纤维化进程的调控作用；③分子层面：验证lnc-GRM1-1-8与miR-29及其靶基因结合位点，并在体内外模型中验证lnc-GRM1-1-8作为ceRNA调控进而调节肝纤维化进程的分子机制及及关键信号通路；为该疾病的诊断标志及基因治疗靶点的确立提供重要理论依据。

目的：HCV相关肝纤维化是导致HCV相关终末期肝病的重要阶段，早期诊断及逆转肝纤维化进展亟待解决。我们前期研究发现，lnc-GRM1-1-8在HCV相关肝纤维化中表达显著升高，可能参与调控肝纤维化进程，但其在该病中的作用及分子机制尚未清楚。.方法：本课题拟通过系统研究:1临床水平:检测HCV相关肝纤维化患者血浆及肝组织lnc-GRM1-1-8表达，分析lnc-GRM1-1-8表达与 肝纤维化分期相关性;2细胞水平及肝纤维化动物模型:在肝星状细胞系及CCl4小鼠肝纤维化模型中过表达及沉默lnc-GRM1-1-8表达，明确其对肝星状细胞表型及肝纤维化进程的调控作用;3分子层面:验证lnc-GRM1-1-8与miR-29及其靶基因结合位点，并在体内外模型中验证lnc-GRM1-1-8作为ceRNA调控进而调节肝纤维化进程的分子机制及及关键信号通路。.结果：HCV相关肝纤维化患者血浆及肝组织lnc-GRM1-1-8表达水平显著上调，与肝纤维化分期呈正相关，对肝纤维化分期诊断效能为0.604，优于FIB-4的0.593(P =0.000)。双荧光素酶报告基因检测显示lnc-GRM1 -1-8与miR-29a-3p具有结合位点并且miR-29a-3p与COL1A1及PDGFRb有结合位点。在 LX-2 细胞中过表达lnc-GRM1-1-8，miR-29 表达下降，下游靶基因 COL1A1，PDGFRb蛋白表达显著升高；在 LX-2 细胞中敲减 lnc-GRM1-1-8 后miR-29表达显著升高，下游靶基因COL1A1、PDGFRb蛋白表达显著下降。在CCL4 诱导的C57BL/6J小鼠肝纤维化模型中，AAV-pcDNA3.1-GRM1组肝脏胶原组织及α-SMA表达较AAV-pcDNA3.1组显著增加。.结论：lnc-GRM1-1-8通过竞争性结合miR-29，促进肝星状细胞活化及肝纤维化进展，是HCV相关肝纤维早期诊断生物学标志及潜在的基因治疗靶点。