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

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。

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.