KLF5调控的微小RNA对血管平滑肌细胞表型转化的作用及其分子机制研究

Hemodialysis access stenosis affects the mortality of chronic kidney disease patients. Phenotype modulation of vascular smooth muscle cells (VSMCs) results in cell proliferation and migration, which is the pathological basis of hemodialysis access stenosis. Our current study has shown that KLF5 is a key factor in stenosis through inducing phenotype modulation of VSMCs. However, the underlying mechanism is still not clear. Previous data have shown that KLF5 regulates miRNAs, and some miRNAs participate in the phenotype modulation of VSMCs and mesenchymal-epithelial transition (MET). Thus we hypothesize that KLF5-regulating miRNAs modulate the genes involving in MET, and result in phenotype modulation toward to de-differentiation in VSMCs. In addition, the phenotype modulation would promote VSMC proliferation and migration to cause hemodialysis access stenosis. The aim of this study is to examine the above hypothetical mechanism. In this study, we would elucidate the mechanism that KLF effects on VSMC toward to de-differentiation. We would identify KLF5-regulating miRNAs, and study the mechanism how KLF5 regulate these miRNAs. In addition, we would clarify the miRNAs targeting MET genes, and analyze that the miRNAs effect on MET. Finally, we would elucidate the mechanism that the KLF5-regulating miRNAs modulate MET genes to govern phenotype modulation of VSMCs, and VSMC proliferation and migration. These findings could shed light on treatments for hemodialysis access stenosis.

血液透析用动静脉瘘狭窄影响肾病患者的生存率。血管平滑肌细胞（VSMC）表型转化产生的增殖与迁移是血管狭窄的病理基础。课题组研究发现KLF5可诱导VSMC表型转化，但机制并不清楚。研究显示KLF5调控微小RNA；微小RNA参与VSMC表型转化并调控间质细胞-上皮细胞转化（MET）。因此我们推测，KLF5调控某些微小RNA的表达，这些微小RNA通过调控MET相关基因，诱导MET的发生，促进VSMC向未分化表型转化，造成VSMC增殖及迁移，最终导致血液透析用动静脉瘘狭窄。本研究拟明确KLF5作用VSMC向未分化表型转化的机制, 将针对KLF5调控的微小RNA进行鉴定，阐明KLF5对微小RNA的调控机制，明确微小RNA靶向MET的基因，分析微小RNA对MET的影响及以上对VSMC表型转化和细胞增殖迁移能力的作用，有望为血液透析用动静脉瘘狭窄治疗提供新靶点。

动静脉瘘管腔狭窄会显著影响规律血透患者的生存率，而这类患者接受肾脏移植后，移植肾动脉狭窄又显著影响移植肾的长期存活。血管平滑肌细胞（vSMC）表型转化产生的增殖与迁移是血管狭窄的病理基础。课题组通过前期研究发现KLF5可诱导VSMC表型转化，但具体机制不明，我们推测，KLF5调控某些微小RNA的表达，这些微小RNA通过调控MET相关基因，诱导MET的发生，促进VSMC向未分化表型转化，造成VSMC增殖及迁移，最终导致动脉血管的狭窄。课题组前期的miRNA 基因芯片检测提示KLF5过表达的vSMC中miR-30d、miR-365、miR-155等表达均显著上调，且均可能与MET相关。进一步的研究证实，KLF5可以正向调控miRNA-365的表达，抑制miRNA-365可以抑制vSMC的增殖以及迁移能力，过表达miRNA365则可以促进vSMC的增殖以及迁移能力，表明KLF5促进vSMC去分化过程是通过miRNA365介导的。这就为动脉受损之后vSMC的病理性增殖、新生内膜形成发现了一个新的可能的调控机制。同时我们进一步探讨了miRNA-365的靶向基因的表达情况。在KLF5过表达vSMC中，AMADTS-1和CyclinD1都有相应的上调和下调，而抑制或者过表达miRNA-365则可以逆转这一过程，这表明miRNA-365是通过对靶基因AMADTS-1和Cyclin D1来实现对vSMC去分化过程进行调控的。AMADTS-1和Cyclin D1已经被证实在动脉硬化以及细胞增殖方面存在确定的意义。最后，我们又探讨了MET相关的标志物在KLF5过表达vSMC中发生了相应的变化，结果显示表明vSMC的去分化过程与发生MET有关，同时通过抑制以及过表达miRNA-365可以逆转或促进MET的程度，这提示vSMC去分化过程中MET的发生与miRNA365有关系。因此KLF5可能是通过miRNA-365调控了MET导致了vSMC发生去分化改变。综上，本研究的主要意义在于，深入的探讨了KLF5在促进血管平滑肌细胞增殖、迁移以及去分化过程中具体的作用机制，基本阐明了miRNA-365在其中扮演了重要的角色，同时，我们也初步证实了MET是KLF5所诱导的血管平滑肌细胞去分化过程的主要机制。为更加深入的理解vSMC去分化的具体机制，并且进一步发现治疗靶点提供了帮助。