TRPM1/miR-211协同调控紫外线照射诱导表皮黑素小体转移与黑素细胞稳态

Microphthalmia-associated transcription factor (MITF) is generally considered as the master regulator for melanocyte (MC) survival and melanogenesis. A novel target gene, the transient receptor potential cation channel subfamily M member 1 (TRPM1), has been recently identified in normal human epidermal MCs. TRPM1 gene codes two transcripts: TRPM1 channel protein in its exons and miR-211 in its sixth intron, and both miR-211 and TRPM1 channel protein are regulated by MITF. We propose a dual role for TRPM1 gene where the activity of TRPM1 channel protein is involved to ultraviolet radiation (UVR)-induced melanosome transfer, while the expression of miR-211 is linked to melanocyte homeostasis in the epidermal melanin unit. In this study, we will perform whole-cell patch clamp, Fluo-3 calcium labelling, and 14C-thiouracil incorporation to measure transient calcium influx and melanosome transfer in the melanocytes upon exposure to UVR. Meanwhile, we also investigate the upregulation of miR-211 by transient transfection of pCMV-p53 plasmid affects the matrix metallopeptidase 9 (MMP9) activity and the migration of p53-transfected cells using gelatin zymography and Transwell assay, respectively. The proposed studies are of considerable significances to understand the “igniting” event in UVR-induced melanosome transfer and to explore the new generation of skin color modifier and a novel strategy to stimulate rapid repigmentation in vitiligo lesions.

一个能被小眼相关转录因子（MITF，普遍认为是黑素生成主调控因子）调控的新靶基因，瞬时受体电位阳离子通道M亚家族1（TRPM1）基因，在人表皮黑素细胞（MC）被识别。该基因的外显子编码TRPM1通道蛋白，而它的第6个内含子还能编码加工成微小RNA-211（miR-211），推测二者可能协同参与调节着黑素小体转移和表皮MC的稳态。本课题拟用膜片钳和钙荧光探针等技术检测当MC暴露紫外线照射（UVR）时细胞膜TRPM1介导的瞬时钙离子内流以及用放射性核素标记技术测定黑素小体转移速率的变化；同时，用转染P53基因方法间接调节MC的miR-211表达，观察其对MC分泌基质金属蛋白酶9（MMP9）活性以及细胞移行能力的影响。本课题实施对认识UVR “触发（igniting）”黑素小体转移的分子事件，寻找新型皮肤色素调节剂和刺激白癜风皮损快速复色的药物有着重大意义。

【摘要】 目的 TRPM1基因的外显子编码生成一种钙离子通道TRPM1（瞬时受体电位阳离子通道M亚家族1）蛋白，该基因的第6内含子还能编码生成一个微小RNA211（miR-211），且二者的表达水平均受小眼相关转录因子（MITF）的调节。然而，TRPM1与miR-211是否同时参与黑素细胞（MC）对紫外光照射做出的光应答（UV photoresponse）至今仍缺乏认识。本课题旨在研究：① 是否TRPM1介导的瞬时钙离子内流影响黑素小体自MC向角质形成细胞（KC）转移；② 是否miR-211水平改变参与对MMP9介导MC移行调节。方法 ① 建立原代人表皮MC和KC单纯培养和共培养，用siRNA技术沉默TRPM1基因表达，用流式细胞仪和双重免疫荧光染色测定在TRPM1基因沉默MC与KC共培养体系中黑素小体转移变化；② 用Fluo-4钙离子探针标记结合双光子荧光显微镜观察在UVA和UVB照射下，记录MC钙离子内流变化。用蛋白印迹和qRT-PCR技术测定视蛋白3（OPN3）和OPN5表达水平在UVA/UVB照射前后的改变；③ 用miRNA模拟物或重组慢病毒质粒转染技术使人表皮MC上调miR-211或过表达P53，用蛋白印迹和qRT-PCR测定p53-TRPM1/miR-211-MMP9轴相关分子的表达水平，用Transwell小室法测定MC在IV型胶原表面的移行；④ 观察了抗坏血酸及其衍生物对培养人表皮MC酪氨酸酶活性的影响，用吖啶橙和嗜酸性荧光探针测定细胞内pH变化；⑤ 磨皮术诱导皮肤发生治疗性创伤，在豚鼠背部色沉斑模型上观察色沉斑边缘黑素颗粒扩展。结果 ① UVA和UVB照射可上调TRPM1蛋白表达，激发2个不同时相的钙内流，连续刺激黑素小体发生转移；② UVB照射后，人表皮MC的p53-TRPM1/miR-211-MMP9轴活化，调节MMP9介导的MC移行；③ MC胞内酸化或诱导皮肤治疗性创伤，可改变MC的黑素生成活性及体内MC的移行。结论 TRPM1和miR-211对人MC的UV诱导光应答具有新的调控作用，认识这一机制有助于理解皮肤的光保护能力，找到黄褐斑和白癜风等疾病新的治疗药物与方法。