miR-146通过调控megalin参与胆囊胆固醇结石形成的机制研究

Gallstone disease (GSD) is one of the most common biliary tract diseases worldwide with various prevalence across populations, and the incidence of gallstones in China showed a trend of rising. It is important to understand the pathogenesis of gallstone disease so that effectively prevent the formation of gallstones. MicroRNAs (miRNA) plays an important role in many diseases, but in formation of gallbladder stone, its roles were unclear. We have made the high throughput sequencing analysis of miRNA for this disease, the results showed miR146b expression are markedly changed, which related to the changes of gallbladder bile.However, its participation in the gallbladder stone formation process, throughwhat channels involved is unclear.We found miR-146b were targeted to megalin.Therefore,we speculated that some pathological factors changed the miR-146 levels, which regulating megalin, result in alteration of gallbladder function.including bile secretion, cholesterol saturation degree, etc., and it may be part of the involved in the gallbladder stone formation.In this study we try to prepared experimental gallstone animal models, and detected the levels and distribution of miR-146b by quantitative PCR and fluorescence probe hybridization technique, detected the composition of gallbladder bile and cholesterol saturatio by physiological and biochemical analysis, and understood the relationship between miR-146b and megalin or the activity of its related factors. The study will help to understand the molecular mechanism of gallstone formation, and maybe provides a new train of thought to prevent the formation of gallstones.

阐明胆囊结石的发病机制预防胆囊结石的形成是当前研究的热点问题。microRNA在很多疾病的分子机制中都具有重要作用，课题组前期的研究发现miR-146及其潜在靶基因megalin在胆囊结石患者、非结石患者和熊去氧胆酸治疗患者中呈现差异性表达，且二者呈现负相关。已有的研究表明Megalin能够影响胆囊胆汁成分的变化，与胆囊结石的形成和治疗具有一定相关性。本项目尝试探索miR-146是否能够通过靶向调控megalin基因，进而改变胆囊胆汁分泌、胆固醇饱和程度等，参与到胆囊结石的形成或治疗。本项目首先在体外细胞水平验证miR-146同megalin基因的靶向关系，随后利用体外动物模型，通过生理生化分析，了解miR-146的改变对胆囊胆汁成分和胆固醇饱和状态的影响，并通过阻断试验探讨megalin的关键用。本研究有助于探讨胆石成因，寻找改变影响胆囊结石的可控因素，为防治胆囊结石提供临床依据。

项目背景：胆汁中胆固醇、钙离子及粘蛋白等促成石成份浓度增高是结石形成的重要诱因，但具体机制不清。我们在前期研究的基础上，提出miR-146是否能够通过靶向调控megalin基因，进而影响胆囊胆汁分泌、胆固醇饱和程度等，参与到胆囊结石的形成或治疗。.方法：在本项目中，课题组首先在细胞中验证了miR-146同megalin的靶向关系，随后在以高胆固醇饮食构建C57BL/6小鼠胆囊胆固醇结石模型中，采用RT-PCR、Western blot和免疫组化检测正常饮食组以及致石组胆囊粘膜megalin及相关通路基因的表达差异。采用FISH技术检测胆囊组织中miR-146的表达；了解miR-146a同这些指标的相互作用情况。我们以鹅脱氧胆酸（CDCA）为对照，检测了miR-146a对于胆囊结石、胆囊胆汁分泌细胞以及成分变化的影响。.结果：（1）miR-146能够靶向调节megalin及FXR/megalin/cubilin信号通路，在胆囊组织中也具有负相关性。（2）抑制miR-146能够促进总胆红素和胆汁酸的排泄，降低小鼠胆囊结石的发生率。熊去氧胆酸治疗组中miR-146的表达以及megalin、cublin、LXR也明显降低。（3）FXR激动剂GW4064能够抑制胆囊结石的形成，但上调miR-146后，能够增加成石率；拮抗胆囊胆汁分泌和胆固醇吸收；并改变megalin/cubilin以及下游相关通路ApoE、FXR、CTGF、Dab2等因子的变化。.结论及科学意义：胆囊结石形成过程中胆囊功能异常是多条信号通路及调控因子表达异常的结果。本项目的结果显示miR-146调控的megalin可能对胆囊上皮细胞的活性和功能具有抑制作用。通过本项目的系统研究，能够加深对胆囊结石发病机理的理解，为预防和治疗胆囊胆固醇结石提供理论依据和实验基础。