p53-SP1复合体诱导miR-29b转录激活在抑制青光眼术后滤过道瘢痕形成中的作用及机制

Excessive scarring leading to failure of the filtering bleb continues to be a major problem after glaucoma filtration surgery. Our previous study obtained specific miRNA expression profiles of scar tissue in the filtration channel. The profile showed miR-29b significantly lower expression in scar tissue. The increased expression of miR-29b can be suppressed scar formation after glaucoma surgery. Recently, we found that the expression of miR-29b was positively correlated with p53. The reasons for the expression of miR-29b decline are not well understood. So, we are ready to clone the promoter region of miR-29b and mutant p53 binding sites; we are ready to studied the mechanism of scar formation in the filtration channel which p53 transcriptional regulate miR-29b by luciferase reporter gene technology, EMSA, ChIP experiments; we are ready to study the interaction between p53 and Sp1 by Co-IP and double immunofluorescent labeling; we are ready to clear whether p53 regulation of miR-29b expression is dependent on Sp1 by siRNA technology. We studied the effect and mechanism of p53-SP1 complex induced transcriptional activation integratedly, which miR-29b inhibits scar formation after glaucoma filtering surgery. We hope to provide new ideas for the prevention of scarring after glaucoma filtration surgery.

青光眼术后滤过道瘢痕形成是手术失败的主要原因。我们前期研究获得了滤过道瘢痕组织特异性miRNA表达谱，并显示miR-29b在瘢痕组织中呈现显著的低表达，其表达水平上调能抑制滤过道瘢痕形成。但是，miR-29b表达下降的原因未明。近期我们发现，miR-29b的表达与p53的表达呈正相关。对此我们的问题是：p53如何激活miR-29b转录表达并抑制青光眼术后滤过道瘢痕形成？为此我们拟克隆miR-29b启动子区并对p53结合部位突变，通过荧光素酶报告基因技术、EMSA、ChIP等实验研究滤过道瘢痕形成中p53对miR-29b的转录调控；通过Co-IP及免疫荧光双标等探讨p53和SP1之间的相互作用；通过siRNA技术了解p53对miR-29b的激活是否依赖于SP1等。整合研究p53-SP1复合体诱导miR-29b转录激活抑制青光眼术后滤过道瘢痕形成中的机制，为临床青光眼术后瘢痕的防治提供新思路。

青光眼术后滤过道瘢痕形成是滤过性手术失败的主要原因。本项目在前期研究基础上开展了miR-29b与青光眼术后滤过道瘢痕形成发病机制相关性研究。课题组完成高通量转录组测序，得到一系列miR-29b表达调控的基因，搜寻涉及青光眼术后滤过道瘢痕形成中细胞周期、凋亡、转导、分化发育等相关差异基因的表达。通过原代培养人Tenon’s囊成纤维细胞、制造兔青光眼术后滤过道瘢痕形成动物模型，利用免疫组化、PCR、western-blotting、siRNA等技术分析研究p53、SP1和miR-29b之间的相互作用；通过生物信息学预测，分析miR-29b 启动子上游转录因子与p53和SP1可能的结合位点；通过CO-IP、ChIP、EMSA等基因功能研究，明确p53、SP1与miR-29b的相关性以及基因表达、调控等机制。本研究发现青光眼术后滤过道瘢痕形成中存在“mRNA-miRNA”调控模式。在青光眼术后滤过道瘢痕组织中检测到p53和SP1基因，且与miR-29b成负向表达关系。miR-29b启动子上游转录因子不存在与p53结合位点，存在SP1结合位点。p53和SP1形成蛋白复合体，经SP1位点介导，转录抑制miR-29b表达，进而调控胶原基因COL1A1表达增强等瘢痕化临床表型。为深入理解青光眼术后滤过道瘢痕形成发病机制奠定了理论基础，为药物靶点研发、疾病预防与诊疗提供新的视角。