摘要
目的探讨蛋白激酶CβⅡ(PKCβⅡ)在肝细胞癌(HCC)发展中的作用机制。方法免疫印迹法观察PKCβⅡ在肝细胞系L02和肝癌细胞系SK-hep1、Hep G2、BEL-7404、7721、Hep3B和huh7中的表达,构建稳定高表达PKCβⅡ的细胞系,倒置相差显微镜下观察细胞形态变化,免疫荧光观察E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)表达的变化;通过免疫印迹法、实时定量聚合酶链反应(Real-time PCR)和放线菌酮(CHX)追踪实验,观察PKCβⅡ调控E-cadherin、N-cadherin和Snail表达的分子机制;小室迁移和侵袭实验(transwell assay)以及裸鼠尾静脉注射观察PKCβⅡ对肝癌细胞转移的影响;成管实验观察PKCβⅡ对人脐静脉内皮细胞(HUVECs)血管形成能力的影响,酶联免疫吸附法(ELISA)观察PKCβⅡ对肝癌细胞上清中血管内皮生长因子A(VEGFA)含量的影响。结果 PKCβⅡ在肝癌细胞系中的表达高于肝细胞系L02,PKCβⅡ促进肝癌细胞形态从鹅卵石样上皮细胞向梭行间质样细胞的转变,通过mRNA水平下调E-cadherin(P <0. 05)和上调N-cadherin(P <0. 01)的蛋白表达,通过翻译水平上调Snail蛋白表达,PKCβⅡ还促进了肝癌细胞的迁移、侵袭(P <0. 01)以及VEGFA的分泌(P <0. 01)和血管新生(P <0. 01)。结论蛋白激酶CβⅡ诱导上皮-间质转化及血管新生在肝癌的发展中有重要作用。
Objective To explore the function and mechanism of protein kinase Cβ Ⅱ( PKCβ Ⅱ) in the progression of hepatocellular carcinoma( HCC). Methods The expression of PKCβⅡ in hepatic cell L02 and HCC cell lines( SK-hep1,Hep G2,BEL-7404,7721,Hep3 B and huh7) was examined by Western blotting. Stable HCC cells overexpressing PKCβ Ⅱ were constructed, the morphological change was observed under inverted phase microscope,immunofluorescence was applied to examine the expression of E-cadherin and N-cadherin. Western blotting,Real-time PCR and cycloheximide( CHX) chase assay were used to examine the molecular mechanism of PKCβ Ⅱ in modulating the expression of E-cadherin,N-cadherin and Snail. The effect of PKCβⅡ on the metastasis of HCC was examined by using transwell assay and nude mice tail vein injection. Tube formation assay was used to detect the impact of PKCβⅡ on the tube formation by human umbilical vein endothelial cells( HUVECs),enzyme linked immunosorbent assay( ELISA) was used to detect the impact of PKCβⅡ on the content of vascular endothelical growth factor( VEGF) A in the supernatant of HCC cells. Results The expression of PKCβⅡ in HCC cells was higher than that in hepatic cell L02,PKCβⅡ promoted the morphologic phenotype changed from cobblestone-like epithelial cells to spindle-shaped mesenchymal-like cells,with the expression of E-cadherin down-regulated( P < 0. 05) and N-cadherin up-regulated( P < 0. 01) at mRNA level,as well as Snail up-regulated at translation level. Meanwhile,PKCβⅡ improved migratory and invasive abilities( P < 0. 01) of HCC cells as well as VEGFA secretion( P < 0. 01) and angiogenesis( P < 0. 01). Conclusion Protein kinase CβⅡ promotes the development of hepatocellular carcinoma via inducing epithelial-mesenchymal transition and angiogenesis.
作者
刘敏
曾云
祝珊珊
巫佳翠
高洪泉
伊雪
LIU Min;ZENG Yun;ZHU Shan-shan;WU Jia-cui;GAO Hong-quan;YI Xue(Department of Basic Medicine,Xiamen Medical College;Key Laboratory of Functional and Clinical Translational Medicine in Fujian Province;Centre of Science Laboratory,Xiamen Medical College,Fujian Xiamen 361023,China)
出处
《解剖学报》
CAS
CSCD
北大核心
2020年第6期912-918,共7页
Acta Anatomica Sinica
基金
厦门医学院博士启动基金(K2016-30)
福建省大学生创新训练项目(201812631006)
厦门医学院校内课题(K2016-35)。
关键词
蛋白激酶CβⅡ
肝细胞癌
上皮-间质转化
血管新生
免疫印迹法
裸鼠
Protein kinase CβⅡ
Hepatocellular carcinoma
Epithelial-mesenchymal transition
Angiogenesis
Western blotting
Nude mouse