表皮松解性掌跖角化症KRT9R163W突变表达对细胞骨架结构影响研究

Effect of KRT9R163W mutation on cytoskeleton structure in epidermolysis palmoplantar keratoderma

目的利用siRNA靶向沉默永生化人角质形成细胞(HaCaT细胞)KRT9R163W表达,共聚焦显微镜观察转染前后细胞骨架结构变化,探讨KRT9R163W突变在表皮松解性掌跖角化症的发病机制中的作用.方法①构建KRT9野生型及KRT9R163W突变型质粒转染入HaCaT细胞过表达.②设计针对KRT9R163W基因的特异性野生型及突变型siRNA,利用脂质体转染HaCaT细胞,并设立阴性对照组、阳性对照组及空白对照组.③通过荧光定量聚合酶链反应(qPCR)及Western印迹检测HaCaT细胞KRT9基因mRNA及蛋白水平表达.④共聚焦显微镜观察siRNA转染前后细胞骨架结构变化.结果①KRT9R163WsiRNA转染HaCaT细胞48 h后,细胞的KRT9基因mRNA表达水平明显降低(0.242±0.051,P<0.05);转染72 h后,靶向siRNA对蛋白表达有统计学差异(0.289±0.036,P<0.01).②KRT9R163W高表达时,细胞骨架结构为错综复杂的网状结构,且部分细胞骨架结构消失.③siRNA特异性沉默KRT9R163W基因后,细胞骨架结构恢复正常的束状排列.结论KRT9R163W高表达可影响细胞的正常细胞结构,通过siRNA技术进一步验证KRT9R163W为表皮松解性掌跖角化症的致病突变基因,为表皮松解性掌跖角化症发病机制研究及靶点治疗提供新的理论基础.

Objective To investigate the effect of keratin 9(KRT9) gene on the pathogenesis of epidermolysis palmoplantar keratoderma.Methods(1)KRT9 wild-type and KRT9R163W mutant plasmids were constructed and transfected into HaCaT cells, resulted in overexpression.(2)Specific siRNA directed to KRT9 wild-type and KRT9R163W mutant were designed and transfected into HaCaT cells by liposome,meanwhile the negative control group, positive control group and the blank control group were also established.(3)Gene silencing effect was evaluated by fluorescence quantitative polymerase chain reaction(qPCR) and Western blot method.(4)The changes of cytoskeleton structure before and after siRNA transfection were observed by confocal microscope. Results(1)After transfection of KRT9R163W mutant plasmid into HaCaT cells for 48 hours, the expression level of KRT9 gene mRNA decreased significantly(0.242±0.051, P<0.05);72 hours after transfection, there was significant difference in protein expression(0.289±0.036, P<0.01).(2)In the condition of KRT9R163W overexpression, the cytoskeleton structure of the HaCaT cells presented a complex network structure, and some cytoskeleton structures disappeared.(3)After KRT9R163W gene specifically silenced by siRNA, the cytoskeleton structure returned to normal bundle arrangement. Conclusion KRT9R163W overexpression may affect the normal skeleton structure of keratinocytes, which may be a pathogenic mutant result in the occurrence of epidermolysis palmoplantar keratoderma, above findings may provide a new theoretical basis for the study of the pathogenesis and target therapy of the disease.