甲羟戊酸激酶基因在汗孔角化症中的作用机制研究

MVK is the causal gene of Disseminated superficial actinic porokeratosis (DSAP), which found by the whole exome sequencing, bioinformatics technology, and repoorted causal gene loci. Previous studies found that MVK gene expression play an important role in KCs differentiation induced by chloride, and could prevent UVA-induced apoptosis of KCs. The result point out MVK mutation may cause the dysfunction of KCs, such as differentiation and apoptosis, but the exact mechanism of the involvement of MVK in the pathogenesis of DSAP remains to be clarified.This study: we will construct the eukaryotic expression recombinant of MVK gene and shRNA vector targeting against MVK, then the recombninants will be transfected into HaCat cells and primary keratinocytes(KCs) to set up cell models with over or low MVK expression. The cells will be exposed to UV or added the mevalonate pathway intermediate metabolite, to analyze the affect of the intermediate metabolite on the proliferation, apoptosis and diffirentiation of KCs, and MVK expression and the intermediate metabolite on Small G protein esterification and relevant ways.Then explore the role of MVK in the pathogenesis of the DSAP. The mechanism research of DSAP caused by MVK mutation has not been reported, this research has original innovation. This study will lay foundation for revealing the pathogenesis, clinical diagnosis and treatment of DSAP.

MVK是我们用全基因组外显子测序和生物信息学技术，结合基因定位，筛选发现的播散性浅表性光化性汗孔角化症（Disseminated superficial actinic porokeratosis, DSAP)的致病基因。前期研究发现：MVK基因表达在角质形成细胞（KCs）氯化钙诱导的分化中发挥重要作用，且能阻止KCs的UVA诱导的凋亡，提示MVK突变可能导致KCs分化、凋亡等生理功能异常，但具体发病机制不明。本研究拟：通过构建野生型MVK和shRNA表达载体，转染HaCat细胞和原代KCs，建立MVK表达调控细胞模型，结合紫外线、甲羟戊酸途径中间代谢产物处理细胞模型，观察其中间产物对角质形成细胞增殖、分化和凋亡的影响，和对小G蛋白酯化及其相关途径的影响，并评判其可能的分子机制。MVK基因突变致DSAP的机制研究尚未见报道，本研究具有源头创新性，可为揭示DSAP发病机制、临床诊治提供帮助。

前期我们通过全基因组外显子测序技术，结合生物信息学技术及初步的功能学研究发现了播散性浅表性光化性汗孔角化症（Disseminated superficial actinic porokeratosis, DSAP)的致病基MVK（甲羟戊酸激酶基因）。2015年1月获得该项目的资助（甲羟戊酸激酶基因在汗孔角化症中的作用机制研究）以来，首先在14个不同类型的汗孔角化症患者中进行了甲羟戊酸激酶途径4个基因（MVD，PMVK，FDPS，MVK）的突变筛查，同时按照计划完成了通过慢病毒载体转染HaCat细胞建立MVK低表达和高表达细胞模型，结合紫外线、甲羟戊酸途径中间代谢产物焦磷酸法尼酯（FPP）和牻牛儿基牻牛儿基焦磷酸（GGPP）处理细胞模型，观察甲羟戊酸途径中间产物对角质形成细胞的增殖、分化及凋亡的影响。且对总异戊二烯化水平及小G蛋白H-ras，RhoE、LaminA、RhoA、Rac1、cdc42、RhoB 的异戊二烯化修饰水平进行了检测及FPP和GGPP反加补救分析。研究发现：1.在11个不同类型的汗孔角化症患者中发现了MVK/MVD基因突变；2. 甲羟戊酸激酶途径中间产物FPP和 GGPP对MVK下调引起的增值、分化、凋亡相关指标有选择性的补救作用；3. MVK表达与总异戊二烯化水平有关，GGPP和FPP对MVK下调引起的异戊二烯化水平下降有补救作用.4.MVK基因的表达影响小G蛋白H-ras，RhoE，LaminA，RhoA，Rac1，cdc42， RhoB的异戊二烯化水平，FPP和GGPP有选择性的补救作用(FPP: H-ras，RhoE，LaminA，RhoB,GGPP: RhoA，Rac1，cdc42， RhoB)。本研究结果有助于今后汗孔角化症的分子诊断，遗传咨询及产前诊断的开展，为新药开发提供理论依据。