Pax6 PAI亚结构域在黑色素细胞中对MITF、TYR、TYRP1和TYRP2的影响

Influences of Pax6 PAI Subdomain on MITF, TYR, TYRP1 and TYRP2 in Melanocytes

【目的】高度保守的PAX转录因子家族在黑色素细胞的分化和黑色素的生成中起重要的作用,其家族共有的PD结构域是其与下游基因结合的主要位点,而PD结构域氨基端的PAI亚结构域在其与下游基因的结合过程中发挥重要的作用。研究表明Pax6在视网膜上皮黑色素细胞的分化中发挥至关重要的作用,本试验借助研究Pax6 PAI亚结构域的功能来对PAX转录因子家族共有的PD结构域和PAI亚结构域进行研究。【方法】首先通过Psipred对Pax6 PD结构域的结构进行分析,使用NCBI对Pax6 PD结构域与下游基因的结合位点进行分析,使用Jaspar对MITF、TYR、TYRP1和TYRP2启动子中Pax6 PD结构域可能的作用位点进行预测。使用普通PCR克隆Pax6PAI亚结构域,将其连入T载体,酶切后连入慢病毒载体,并送公司测序确认。将构建好的PAI亚结构域过表达载体通过细胞转染导入到培养的小鼠黑色素细胞中,使其过量表达。收集细胞,分别通过观察绿色荧光蛋白检测转染效率,使用RT-PCR和Western blot来检测MITF、TYR、TYRP1和TYRP2在m RNA和蛋白水平的变化,同时检测黑色素细胞中黑色素生成量的变化。【结果】通过NCBI分析可知,Pax6 PD结构域与下游基因的作用位点主要集中在氨基端的PAI亚结构域。通过Jaspar预测分析,得知,MITF启动子-695处存在Pax6 PD结构域的结合位点,TYR启动子-873和-1133处存在Pax6 PD结构域的结合位点,TYRP1启动子-629处存在Pax6 PD结构域的结合位点,TYRP2启动子-655处存在Pax6 PD结构域的结合位点。在黑色素细胞中过表达Pax6 PAI亚结构域后,与空载组相比,试验组MITF m RNA升高2.05倍(P<0.01),蛋白质升高1.7倍(P<0.01);TYR m RNA升高2.09倍,蛋白质升高2倍(P<0.05);TYRP1 m RNA升高2.93倍(P<0.05),蛋白质升高1.9倍(P<0.01);TYRP2 m RNA升高3.62倍(P<0.01),蛋白质升高1.37倍。同时试验组的黑色素含量是空载组黑色素含量的1.33倍(P<0.001)。【结论】在小鼠黑色素细胞中,过表达Pax6 PAI亚结构域可以促进MITF、TYR、TYRP1和TYRP2的表达,进而使黑色素细胞黑色素的生成量增加。

【Objective】 Highly conserved PAX family has important effects on the differentiation of melanocytes and production of melanin. It has mainly binding sites with target gene in PD domain that is included by all of PAX family, on the other hand, the PAI subdomain, which is located in amino terminal of PD domain, has most important effects on PD domain which bound with target gene. Many reports show that Pax6 has the most important effects on the differentiation of retinal pigment epithelial cells. This experiment studies the function of PD domain and PAI subdomain of PAX family by analysing the function of Pax6 PAI subdomain.【Method】The structure of Pax6 PD domain was analyzed by Psipred. The target gene binding sites of Pax6 PD domain was analyzed by NCBI. The binding sites of Pax6 PD domain to the promoter of MITF, TYR, TYRP1, and TYRP2 were analyzed by Jasper. The coding sequences of Pax6 PAI subdomain was amplified by PCR and the Pax6 PAI subdomain was cloned into the T-Vector, meanwhile, confirmed by sequencing. The fragment was then subcloned into a mammalian expression vector, resulting in a construction that contained a promoter driving the expression of green fluorescent protein（GFP）. The plasmid vector was confirmed by sequencing. Then, the mouse melanocytes were transfected with the vector using Liposome 2000. Three methods were used in the result test, they were quantitative real-time PCR, western blot and melanin content measurement. 【Result】The target gene binding sites of Pax6 PD domain was mainly distributed in PAI subdomain which is located in amino terminal of PD domain. There was a binding site of Pax6 PD domain at-695 base of MITF promoter; Two binding sites of Pax6 PD domain at-873 base and-1133 base of TYR promoter; One binding site of Pax6 PD domain at-629 base of TYRP1 promoter; And one binding site of Pax6 PD domain at-655 base of TYRP2 promoter. The RT-PCR and western blot results showed that the four target genes and melanin content were significantly increased. MITF m RNA was significantly increased by 2.05 times（P0.01）, TYR m RNA was increased by 2.09 times, TYRP1 m RNA was increased by 2.93 times（P0.05）, TYRP2 m RNA was increased by 3.62 times（P0.01）. Compared with the control group, MITF protein was significantly increased by 1.7 times（P0.01）, TYR protein was increased to 2 times（P0.05）, TYRP1 protein was increased by 1.9 times（P0.01）, TYRP2 protein was increased to 1.37 times. Meanwhile, the melanin content was significantly increased by 1.33 times（P0.001）.【Conclusion】Results of the study demonstrated that the Pax6 PAI subdomain still promoted the expression of MITF, TYR, TYRP1, and TYRP2, while increased the production of melanin of melanocytes.