金丝桃苷通过ERK通路调控miR-34a抑制糖尿病肾病系膜增生的分子机理研究

Diabetic nephropathy (DN) is the major diabetic complication characterized by mesangial proliferation. Previous studies by our groups showed that miR-34a regulates mesangial cell proliferation by targeting GAS1. It has recently been found that hyperoside (Hyp) ameliorated mesangial matrix expansion in the glomeruli of db/db mice. Hyp inhibited the mesangial cell proliferation and the expression of miR-34a. The inhibition of ERK pathway activation was also observed. The result of bioinformatic analysis showed that there are four putative ERK mediating binding sites for CREB and c-Ets in miR-34a promoter region. These results suggest that Hyp inhibits mesangial proliferation of DN by regulating miR-34a through the ERK pathway. This study aims to explore the relationship between mesangial proliferation inhibited by Hyp and miR-34a and ERK pathway in DN mice. The expression of miR-34a, GAS1 and the cell phenotype will be investigated in vitro by activation or inhibition of ERK pathway with Hyp treatment. Furthermore, the function of promoter will be confirmed by luciferase report gene assay, EMSA, ChIP associated with the construction of plasmids containing highly expressed CREB, c-Ets, siRNA, and luciferase reporter genes. Our study will elucidate the molecular mechanism by which Hyp regulates miR-34a through the ERK pathway, and will probably benefit the research of Hyp for DN treatment.

糖尿病肾病（DN）的病理特征之一为系膜增生。前期工作表明miR-34a靶向GAS1促进系膜细胞增殖。近期发现金丝桃苷（Hyp）能减少DN小鼠系膜基质扩张，对系膜细胞增殖和miR-34a的表达均有抑制作用，同时抑制ERK活化；生物信息学分析发现miR-34a启动子区存在4个由ERK所调控转录因子CREB、c-Ets的推定结合位点。推测：Hyp通过ERK通路调控miR-34a抑制DN系膜增生。本项目拟在小鼠模型上观察Hyp抑制DN系膜增生与miR-34a和ERK通路的关系；体外培养系膜细胞，通过抑制或激活ERK通路，观察细胞表型、miR-34a和GAS1表达，验证Hyp的干预作用机制；采用报告基因、ChIP等实验验证CREB、c-Ets对miR-34a的转录调控，明确ERK通路对miR-34a的调控机制。研究结果将阐明Hyp抑制DN系膜增生的确切分子机制，并为其用于DN的治疗提供理论基础。

糖尿病肾病（DN）的病理特征之一为系膜增生。前期工作表明miR-34a靶向GAS1促进系膜细胞增殖。近期发现金丝桃苷能减少DN小鼠系膜基质扩张，对系膜细胞增殖和miR-34a的表达均有抑制作用，同时抑制ERK活化。为进一步明确金丝桃苷对DN系膜增生的保护作用机制，本研究在前期工作基础上，建立DN小鼠模型，进一步观察金丝桃苷对DN小鼠血糖、肾功能、肾小球病理形态的影响；应用定量PCR、Western Blot、免疫组化方法检测肾组织内细胞外基质蛋白表达，证实金丝桃苷对DN小鼠的保护作用；在细胞水平观察高糖条件下金丝桃苷对系膜细胞表型、ERK通路活化、CREB磷酸化和miR-34a表达的影响，并分别干扰miR-34a及ERK通路，研究金丝桃苷抑制DN系膜增生的作用机制；构建CREB高表达质粒、shRNA质粒及miR-34a启动子荧光素酶报告基因质粒，采用荧光素酶报告基因和ChIP实验确定CREB对miR-34a的转录调控。结果表明，金丝桃苷处理的DN小鼠血糖浓度及肾脏重量显著减低，肾功能相关生化指标和尿蛋白与肌酐比值明显得到明显改善；同时，金丝桃苷能显著降低DN小鼠肾组织中FN、COLIV和TIMP-1蛋白的表达，显著升高MMP-9和MMP-2蛋白表达，体内实验证实了金丝桃苷对DN小鼠具有保护作用，能够减少系膜基质扩张、基底膜增厚及肾小球硬化。体外实验中，金丝桃苷能抑制高糖诱导的系膜细胞细胞增殖，呈时间和剂量依赖性。采用agomir-34a上调miR-34a后，金丝桃苷抑制细胞增殖的作用减弱。ERK特异性抑制剂U0126处理细胞，高糖诱导的细胞增殖、ERK活化、CREB磷酸化和miR-34a表达均受到抑制。我们通过生物信息学对miR-34a启动子区进行分析，发现了CREB结合位点，ChIP实验和DNA测序证实了CREB介导的miR-34a转录调控依赖于CREB与miR-34a启动子区域特点位点的直接结合。荧光素酶报告基因实验确定了miR-34a启动子区转录因子CREB的结合位点是TGAGGCCA、TGACCCCA和TGACCTAA。研究结果揭示了ERK/CREB/miR-34a信号通路在金丝桃苷抑制DN系增生中的作用及ERK调控miR-34a的确切分子机制，为DN临床防治策略的开发提供了新思路。