MAP4K1促进皮肤黑色素瘤迁移和侵袭的机制研究

Study on mechanism of MAP4K1 in the promotion of migration and invasion of human cutaneous melanoma

目的探讨MAP4K1在黑色素瘤(cutaneous melanoma,CM)细胞和组织中表达及对恶性CM细胞A375迁移、侵袭的影响和机制。方法收集自2010年1月至2015年12月重庆市中医院皮肤科诊治的CM患者54例,采用GEPIA在线分析CM组织中MAP4K1的表达,实时定量聚合酶链法和免疫组织化学法检测CM组织中MAP4K1 m RNA和蛋白的表达水平;以MAP4K1基因表达最高的A375细胞为研究对象,采用脂质体转染法将MAP4K1抑制质粒和对照质粒转染至A375细胞;免疫蛋白印迹实验和实时定量聚合酶链法分别检测MAP4K1的基因敲除;分别通过Transwell实验检测MAP4K1敲除后对细胞侵袭的影响;荧光素酶报告实验检测MAP4K1对YAP/TAZ的调控;GEPIA验证MAP4K1与Hippo-YAP通路下游靶点的表达相关性;免疫蛋白印迹实验验证MAP4K1敲除后对Hippo-YAP信号通路的影响。结果与癌旁组织相比,MAP4K1 m RNA和蛋白在皮肤恶性CM细胞和组织中的表达上调(P<0.001);MAP4K1基因敲除A375细胞迁移和侵袭的能力明显受到抑制(P<0.001);MAP4K1敲除后YAP/TAZ的转录活性及下游的靶分子下调;MAP4K1基因敲除能抑制Hippo-YAP通路传导。结论MAP4K1在CM中表达上调,可能作为潜在的癌基因调控Hippo-YAP通路促进CM的发生发展。

Objective To explore the expression of MAP4 K1 in melanoma cell lines and tissues and the effect of MAP4 K1 on the migration and invasion of melanoma cell lines and molecular mechanism.Methods Fifty-four patients with melanoma admitted to the Department of Dermatology of Chongqing Traditional Chinese Medicine Hospital from January 2010 to December 2015 were selected.The expressionof MAP4 K1 in melanoma was analyzed by online database.The expression of MAP4 K1 m RNA and protein in melanoma was detected by qRT-PCR and immunohistochemistry.The MAP4 K1 gene knockout was detected by Western blot and RT-PCR.The effecton migration and invasion of A375 cells after MAP4 K1 gene knockout was detected by scratch assay and Transwell assay.The regulation of YAP/TAZ by MAP4 K1 was detected by dual luciferase reportergene assay.The correlation between MAP4 K1 and the expression of downstream target of Hippo-YAP pathway was proved by GEPIA online database.The effect on Hippo-YAP signaling pathway after MAP4 K1 gene knockout was verified by Westernblot.Results Compared with the adjacent tissues,the expression of MAP4 K1 m RNA and protein in cutaneous malignant melanoma cells and tissues was up-regulaged(P<0.001).The migration and invasionof A375 cells after MAP4 K1 gene knockout were significantly inhibited(P<0.001).The transcription activity of Yap/TAZ after MAP4 K1 gene knockout and downstream target molecules were down-regulated.The Hippo-YAP signaling transduction pathway was inhibited by MAP4 K1 gene knockout.Conclusion The expression of MAP4 K1 in melanoma is up-regulated.The MAP4 K1 can be served as potential oncogene to promote the development of melanoma by regulating Hippo-YAP pathway.