解剖学报

解剖学报 ›› 2020, Vol. 51 ›› Issue (6): 912-918.doi: 10.16098/j.issn.0529-1356.2020.06.016

• 肿瘤生物学 • 上一篇    下一篇

蛋白激酶CβⅡ诱导上皮-间质转化及血管新生促进肝癌发展

刘敏1,2 曾云1,2 祝珊珊3 巫佳翠1 高洪泉1 伊雪1,2*   

  1. 1.厦门医学院基础医学部; 2.机能与临床转化福建省高等学校重点实验室; 3.厦门医学院科学实验中心，福建 厦门 361023
  • 收稿日期:2019-11-12 修回日期:2020-04-16 出版日期:2020-12-06 发布日期:2020-12-06
  • 通讯作者: 伊雪 E-mail:1772650252@qq.com
  • 基金资助:
    厦门医学院博士启动基金;福建省大学生创新训练项目;厦门医学院科技类课题

Protein kinase CβⅡ promoting the development of hepatocellular  carcinoma via inducing epithelial-mesenchymal transition and angiogenesis

LIU Min1, 2  ZENG Yun1,2  ZHU Shan-shan3  WU Jia-cui1  GAO Hong-quan1  YI Xue1,2*   

  1. 1.Department of Basic Medicine, Xiamen Medical College; 2.Key Laboratory of Functional and Clinical Translational Medicine in Fujian Province; 3.Centre of Science Laboratory, Xiamen Medical College, Fujian Xiamen 361023, China
  • Received:2019-11-12 Revised:2020-04-16 Online:2020-12-06 Published:2020-12-06
  • Contact: YI Xue E-mail:1772650252@qq.com

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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、HepG2、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。

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

Abstract:

Objective  To explore the function and mechanism of protein kinase CβⅡ(PKCβⅡ) in the progression of hepatocellular carcinoma(HCC).   Methods  The expression of PKCβⅡ in hepatic cell L02 and HCC cell lines (SK-hep1, HepG2, BEL-7404, 7721, Hep3B and huh7)was examined by Western blotting. Stable HCC cells over-expressing PKCβⅡ 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 PKCβⅡ 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.

Key words: Protein kinase CβⅡ, Hepatocellular carcinoma, Epithelial-mesenchymal transition, Angiogenesis, Western blotting, Nude mouse

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