Yes相关蛋白(YAP)通过激活PI3K/AKT通路促进皮肤鳞状细胞癌细胞侵袭和迁移

Yes-associated protein(YAP)promotes invasion and migration of cutaneous squamous cell carcinoma cells by activating the PI3K/AKT pathway

目的探讨Yes相关蛋白(YAP)在皮肤鳞状细胞癌(cSCC)中表达及与cSCC侵袭和迁移中的作用。方法通过免疫组织化学染色法检测cSCC、鲍温病(BD)、癌旁正常皮肤组织中YAP的表达水平,并分析与临床病理参数之间的关系;利用慢病毒转染构建YAP基因敲低的A431稳定细胞株,利用四甲基罗丹明标记的鬼笔环肽检测A431细胞微丝分布和数量,Transwell TM实验检测细胞侵袭能力,划痕实验检测A431细胞的迁移能力;免疫荧光细胞化学染色法观察敲低YAP后上皮间质转化(EMT)相关标志物上皮钙黏素(E-cadherin)、锌指转录因子Snail的表达;Western blot法检测E-cadherin、Snail、β-catenin、磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(AKT)、磷酸化的蛋白激酶B(p-AKT)、核糖体蛋白S6(S6)、磷酸化S6(p-S6)、4E结合蛋白1(4EBP1)、磷酸化的4EBP1(p-4EBP1)的表达。结果YAP在cSCC和BD中表达显著高于癌旁正常皮肤组织;cSCC中YAP高表达与肿瘤大小、分化程度、侵袭程度密切相关,与患者的性别、年龄、发病部位、形态类型、是否神经脉管侵犯不相关;敲低A431细胞中YAP后,肿瘤细胞的侵袭、迁移能力降低,细胞微丝变细、伪足变少;E-cadherin表达增加,Snail和β-catenin蛋白表达降低,p-AKT、p-S6及p-4EBP1蛋白表达降低。结论YAP在cSCC中高表达,YAP激活PI3K/AKT信号通路促进cSCC的侵袭、迁移及EMT过程。

Objective To investigate the expression of Yes-associated protein(YAP)in cutaneous squamous cell carcinoma(cSCC)and its effects on invasion and migration.Methods Immunohistochemical staining was used to detect the expression of YAP in cSCC,Bowen disease(BD),and adjacent normal tissues,and analyzed the correlation between YAP expression and clinicopathological characteristics of cSCC.A stable cell line in A431 cells with YAP gene silencing was established through lentiviral infection.Tetramethylrhodamine isothiocyanate(TRITC)-phalloidin staining was performed to analyze the distribution and number of microfilaments in A431 cells.Transwell TM chamber assay was performed to detect the invasion ability of cells,and scratch healing assay was used to determine the migration ability.Immunofluorescence cytochemistry was used to detected the expression of EMT-related markers,including epithelial-cadherin(E-cadherin),zinc-finger transcription factors Snail in A431 cells with YAP silencing.Western blot analysis was employed to detect the expression of E-cadherin,snail,β-catenin,phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),phosphorylaedAKT(p-AKT),ribosomal proteinS6(S6),phosphorylatedS6(p-S6),4E-binding protein 1(4EBP1),and phosphorylated 4EBP1(p-4EBP1).Results The expression of YAP was significantly higher in BD and cSCC tissues compared to adjacent tissues.The strong positive rate of YAP in cSCC tissues was associated with tumor size,differentiation and the level of invasion.However,there was no correlation between YAP expression and gender,age,tumor location,morphological type,or nerve and vascular invasion.After silencing the expression of YAP in A431 cells,the migration and invasion ability of tumor cells were significantly reduced,and cell microfilaments became thinner with reduced pseudopodia.The expression of E-cadherin was increased,while the expression of snail,β-catenin,p-AKT,p-S6 and p-4EBP1were decreased.Conclusion YAP is highly expressed in cSCC tissues,and promotes the cell migration and invasion of cSCC cells by activating the PI3K/AKT signaling pathway and EMT.