miR-155、miR-192调控Rho A/Rho GTPase信号通路对原发性开角型青光眼的干预

Glaucoma is a common blinding disease worldwide and is frequently associated with elevated intraocular pressure (IOP) caused by increased resistance to aqueous humor outflow through the trabecular meshwork (TM). Upregulation of CTGF and extracellular matrix (ECM) deposits in TM is main molecular change of primary open angle glaucoma (POAG). Research confirmed that both miR155 and miR192 can regulate increase of expression of ECM which leads to renal fibrosis. Our result confirmed that miR192 inhibitor can decrease expression of ECM in TM cells. Our previous result also indicated that expression of miR155 increase significantly in TM cell transfected with Ad-CTGF. We suppose that miR155 play important role in the regulation of ECM expression in TM of POAG. To confirm the hypothesis, we will comparison between miR155 and miR192 on expression of CTGF, neuromedin U, ECM and morphological change of TM and Schlemm' canal which was based on Ad transfection, cultured TM cells with three dimension microfluidic technique, anterior segmental perfusion of tracer based on fluid dynamics by using rat primary TM cells and CTGF conducted POAG model through molecular, cell and whole model level. We also explore the target gene of miR155 on the RhoA/Rho GTPase signaling pathway which is downscream of CTGF. The completion of these specific aims should provide new insights into the molecular mechanism involving in the regulation relevant to the maintenance of normal levels of intraocular pressure in the eye and the pathogenesis of the TM in the glaucoma. This information should be useful in identifying novel targets for therapeutic approaches aimed at delaying or preventing the abnormal elevation of intraocular pressure, which constitutes the best-characterized risk factor of delevoping glaucoma.

结缔组织生长因子（CTGF）上调和小梁网细胞外间质沉积是原发性开角型青光眼主要发病机制；miR155和miR192是调控细胞外间质表达的重要基因。我们证实miR192抑制物可减少小梁网细胞外间质表达，发现CTGF诱导小梁网细胞中miR155表达明显增加，但其机制有待探讨。我们提出假说miR155可能调控小梁网细胞外间质表达，导致小梁网组织结构和功能变化。为验证这一假说，将采用腺病毒转染、微流体三维细胞培养、基于流体力学的眼前节灌注等手段，从分子、细胞以及动物整体水平等方面，对比研究miR-155、miR-192对小梁网细胞CTGF、Neuromedin U、细胞外间质蛋白表达的影响，及对小梁网、Schlemm管等结构的影响，探讨miR155对信号通路RhoA/Rho GTPase的调控。本研究将从miR-155这个新视点为揭示原发性开角型青光眼的调控机制奠定基础，为治疗青光眼提供新的思路。

小梁网组织尤其是近小管组织的阻力增加在原发性开角型青光眼的发病机制中有主要作用。细胞外基质成份如纤维粘连蛋白、胶原IV等在小梁网组织的沉积增加房水循环阻力。我们观察miR-155、miR-192-5p 转染后对小梁网组织细胞外基质表达的影响。我们的结果证实CTGF转染后小梁网组织中表达增加。采用western blot纤维粘连蛋白、胶原、层粘连蛋白等的表达。我们的结果表明纤维粘连蛋白、胶原I、胶原IV、层粘连蛋白、SPARC等的表达，miR-155 模拟物＋CTGF组、miR-192-5p 抑制物组＋CTGF和对照组有显著差异，miR-155抑制物＋CTGF组、miR-192-5p模拟物＋CTGF组和对照组没有显著差异。.我们的结果证实，转染Adv-CTGF可升高大鼠眼压，与对照大鼠相比增加了35.2%。对照组和Adv-CTGF组大鼠平均眼压分别为12.5±22.1mmHg和25.6±3.5mmHg, miR-155mimic 和miR-192inhibitor对眼压可以明显降低，分别为18.5±1.5mmHg、19.5±2.1mmHg。Adv-CTGF组和对照组的房水流出系数的改变如图所示。在对照组中，房水流出系数较基线增加了25.2±9.5%。在Adv-CTGF组中，房水流出系数较基线显著下降52.5±12.3%, miR-155mimic 和miR-192inhibitor组的房水流出系数较基线显著下降35.1±5.2%、29.5±3.5%。.用共聚焦显微镜发现了对照组眼中小梁网内示踪剂的分布。相反，在Adv-CTGF眼中小梁网内未发现示踪剂。MiR155mimic组（75.2±5.2%）、miR-192 inhibitor组（79.3±8.5%）和Adv-CTGF组（1.5±2.1%）有显著差异，和PEFL比对照组（95.3±15.2%）无显著差异。因此，MiR155mimic和miR-192inhibitor转染对房水流出模式有显著影响。观察到了眼压与PEFL之间的负相关，PEFL下降时，眼压升高。实验结果证实，MiR155mimic和miR-192inhibitor转染可以增加PEFL，增加房水流出系数，降低眼内压。