lpa-miR-nov-66调控羊驼黑色素细胞CDK5磷酸化作用的研究

The Effect of lpa-miR-nov-66 on CDK5 Phosphorylation in Alpaca Melanocyte

旨在研究lpa-miR-nov-66在调控羊驼黑色素细胞产生黑色素颗粒过程中对细胞周期依赖性激酶5(CDK5)磷酸化作用的影响。在体外培养的黑色素细胞中转染lpa-miR-nov-66后,采用实时定量PCR、免疫细胞化学、Western blotting、免疫共沉淀等方法检测功能性CDK5作用。结果显示:与阴性对照相比,黑色素细胞被转染lpa-miR-nov-66后,CDK5基因和蛋白表达量均下降,差异极显著(P<0.01);CDK5激活子是P35,CDK5磷酸化水平及其磷酸化底物——酪氨酸羟化酶(TH)的表达量均被上调,分别呈差异极显著(P<0.01)和差异显著(P<0.05)。结果表明:lpa-miR-nov-66过量表达可通过CDK5的激活子P35上调黑色素细胞CDK5发生磷酸化。该功能性CDK5激酶对底物TH发生磷酸化,是lpa-miR-nov-66调控黑色素生成分子机制的中间环节。

The objective of this research was to investigate the effect of lpa-miR-nov-66 on cyclin dependent kinase 5（CDK5）and its phosphoration on the pathway of melanogenesis in alpaca melanocytes.Following the transfection of alpaca melanocytes by lpa-miR-nov-66 in vitro,quantitative real-time PCR,immunocytochemistry,Western blotting and immunopricipitation were mainly used to determine the expression of the functional CDK5.The results showed that compared to the control,the over expression of lpa-miR-nov-66 in melanocytes resulted in decreased expression of CDK5 and increased expression of phosphoted CDK5 with significant difference（P〈0.01）;P35was the activator of CDK5.And it was found that CDK5-dependent phosphorylation of TH was up-regulated with the higher activity（P〈0.05）.The results suggested that lpamiR-nov-66 gave rise to up-regulation of phos-TH expression on the pathway of melanogenesis by functional CDK5 binding to P35,which was the intermediate link for the molecular mechanism of regulating melanogenesis.