基于miRNA调控关系的维医异常黏液质型阳痿的机制研究

Uyghur Traditional Medicine have exceptionally good theory of system for defining the symtoms of impotence and treatment. According to its categorization, the abnormal balgam type of impotence is one of the most common type among the erectile dysfunction patients. It is well known that the Uyghur Traditional Medicine have certain advantages for treating this type of disease, and the Yimusake (in form of tablets) is one of the recognized classic uyghur medicines for this purpose. Our research team have succeed on establishing the animal model of the abnormal balgam type of impotence. The penile tissue miRNA expression analysis was performed using Solexa sequencing for initial screen, has also found the difference of microRNA expression spectrum between model rats and normal rats. 17 differentially expressed miRNAs which related to the development of the disease were selected as candidate miRNAs. For the validation of the results numbers of the subjects and samples will be increased to 20 rats, and for the verification of the miRNA expression by qRT-PCR. The trend of the expression of 2 candidate miRNAs is in accordance with the that of miRNA sequencing. The candidate target gene of these two miRNAs was determined according to miRNA analysis databases.The regulatory field of these candidate genes focus on fibrosis, energy metabolism, material metabolism process.The preliminary results of the study also indicate that the fibrosis, dysfunction of energy metabolism, material metabolism are the reason of the disease.Therefore, this study aims to validate the rest of the 15 candidate miRNA, establish biomarker system of this disease. The verification of the target genes in penis tissue by RT-PCR and Western blot. Construct the dual-flouresence luciferase reporter gene of candidate target gene to validate the relationship between miRNA expression and target gene transcription. Pull down, co-IP, immunofluorescence, western blot, RT-PCR was used to analyze the role of the target gene in the designated signal pathway. Providing a new basic information about the mechanism involved in the pathogenesis of the abnormal balgam type of impotence and the targets for the drug intervention, providing the scientific proof to explain Uygur medicine’s concept of “combination of disease and syndrome”, “correspondence between prescription and syndrome”.

维医在阳痿的诊断和治疗上具有优势，异常黏液质型阳痿是其主要证型，伊木萨克片为对证药物。前期利用miRNA测序分析技术，在维医异常黏液质型ED病证结合大鼠的阴茎组织筛选出17个候选差异表达miRNA，经qRT-PCR技术在阴茎组织确认的2个与测序结果一致的miRNA，其预测靶基因集中于纤维化、物质和能量代谢信号通路，与我团队已有结果一致，提示miRNA与该病证的发生发展相关。本研究欲进一步筛选和确认与测序结果一致的miRNA，建立异常黏液质证候与ED病证相关的miRNA标志物体系；在阴茎组织qPT-PCR和western blot技术筛查miRNA预测靶基因的基础上，构建荧光报告基因载体在原代培养的阴茎海绵体平滑肌细胞上确认miRNA靶基因；免疫荧光、pull down、co-IP等技术明确miRNA及其靶基因在信号通路中作用，从转录后水平探讨该病证发病机制及对证方药作用机理和潜在靶点。

阳痿也称阴茎勃起功能障碍(erectile dysfunction，ED)是最常见的男性性功能疾病，严重影响患者心理健康与家庭和睦。本研究复制异常黏液质证侯和阳痿病证结合大鼠模型，APO勃起实验和交配实验从中筛选出ED大鼠，中药伊木萨克片干预3周后，提取阴茎组织总RNA进行miRNA Illumina高通量测序，筛选出与ED发生发展相关差异表达microRNA（miRNA）共121个(P<0.05)，其中以log2 Ratio的绝对值>0.8为条件，找到ED相关miRNA候选标志物9个；筛选出伊木萨克片干预靶点相关差异表达miRNA 137个(P<0.05)，其中以log2 Ratio的绝对值>0.8，找到伊木萨克片干预相关miRNA候选靶点标志物16个。经qRT-PCR验证，rno-miR-140-3p，rno-miR-100-5p，rno-miR-125a-5p，rno-miR-125b-5p，rno-miR-127-3p，rno-miR-204-5p，rno-miR-486，rno-miR-222-3p的表达与测序结果趋势一致，可作为ED发生及药物干预靶点相关的候选标志物。对上述已验证miRNA的预测靶基因进行GO分析和KEGG分析显示，这些miRNA可能通过调节其相应靶基因网络，参与组织纤维化的形成，及糖、脂代谢的调控。构建人PTEN 基因 3’端非翻译区野生型及突变型双荧光素酶报告载体，同时应用PI3K/AKT信号通路抑制剂LY294002，发现miR-486通过抑制PTEN的表达，激活PI3K/AKT信号通路，促进人HUMEC内细胞间质转化，加速纤维化进程。本研究有助于深刻揭示ED发生的现代医学机制，也为伊木萨克片的的推广使用提供了理论基础。