基于磷酸化修饰组学分析WNK2调控卵巢癌细胞骨架形成

李凤杰; 贾永琴; 梁振; 胡彬; 贺思敏; 王延洲; 杨泽宇; 梁志清

doi:10.16016/j.2097-0927.202110107

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基于磷酸化修饰组学分析WNK2调控卵巢癌细胞骨架形成

基础医学 | 更新时间：2026-01-09

- 基于磷酸化修饰组学分析WNK2调控卵巢癌细胞骨架形成
- Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing
- 陆军军医大学学报 2022年44卷第6期页码：513-521
- 作者机构：
  
  1.400038 重庆，陆军军医大学(第三军医大学)第一附属医院妇产科
  2.410031 长沙，中南大学生命科学学院分子生物学研究中心
  3.410028 长沙，中南大学湘雅公共卫生学院
  4.400013 重庆，重庆市人民医院甲状腺乳腺外科
- 作者简介：
  
  [梁志清，E-mail：zhi_qingliang@163.com LIANG Zhiqing, E-mail: zhi_qingliang@163.com
- 基金信息：
  
  国家重点研发计划项目(2019YFC1005202)
- DOI：10.16016/j.2097-0927.202110107
  中图分类号： R345; R394.3; R737.31
- 收稿：2021-10-19，
  
  修回：2021-12-08，
  
  纸质出版：2022-03-30
- 稿件说明：
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李凤杰, 贾永琴, 梁振, 等. 基于磷酸化修饰组学分析WNK2调控卵巢癌细胞骨架形成[J]. 陆军军医大学学报, 2022,44(6):513-521.

Fengjie LI, Yongqin JIA, Zhen LIANG, et al. Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing[J]. Journal of Army Medical University, 2022, 44(6): 513-521.
李凤杰, 贾永琴, 梁振, 等. 基于磷酸化修饰组学分析WNK2调控卵巢癌细胞骨架形成[J]. 陆军军医大学学报, 2022,44(6):513-521. DOI： 10.16016/j.2097-0927.202110107.

Fengjie LI, Yongqin JIA, Zhen LIANG, et al. Analysis of WNK2 regulating cytoskeleton formation in ovarian cancer cells based on phosphorylation modification sequencing[J]. Journal of Army Medical University, 2022, 44(6): 513-521. DOI： 10.16016/j.2097-0927.202110107.

摘要

目的

通过蛋白质磷酸化修饰组学质谱测序，分析蛋白激酶WNK2在卵巢癌细胞中调控的生物过程。

方法

用WNK2干扰慢病毒感染SKOV3细胞系，药筛获得稳转细胞株，提取总蛋白进行蛋白质磷酸化修饰组学质谱测序，根据生物信息学分析干扰WNK2前后SKOV3细胞中蛋白质磷酸化水平的变化，筛选磷酸化差异蛋白，KEGG及GO富集分析获取差异蛋白在卵巢癌细胞中调控的生物学过程并进行实验验证。

结果

通过生物信息学分析磷酸化修饰组学质谱数据，结果显示干扰WNK2后，SKOV3细胞中共有745个蛋白磷酸化水平显著下降，其中包含1 078个磷酸化修饰位点发生变化，328个蛋白磷酸化水平显著上升，共415个磷酸化修饰位点发生变化。将磷酸化水平发生变化的差异蛋白进行KEGG、GO富集分析，发现WNK2主要参与了细胞骨架形成、信号传导、细胞组成、激酶活性调控等生物过程。干扰或过表达WNK2后，利用鬼笔环肽进行细胞骨架染色，结果显示，干扰WNK2后，细胞骨架断裂，排列紊乱；过表达WNK2后，细胞骨架重排增强。

结论

WNK2通过调控vimentin等蛋白的磷酸化水平参与卵巢癌细胞骨架形成过程。

Abstract

Objective

To analyze the biological processes regulated by protein kinase WNK2 in ovarian cancer cells based on protein phosphorylation modified mass spectrometry.

Methods

Ovarian cancer cell line SKOV3 was transfected with lentiviral vector to knockdown WNK2 and the obtained cells were further screened by drug screening. Then the total protein was extracted for protein phosphorylation modification mass spectrometry analysis. Bioinformatics analysis was adopted to detect the phosphorylation changes before and after WNK2 knockdown

and differential proteins were screened out. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were employed to explore the functions of differentially phosphorylated proteins

and experiments were performed to verify the biological processes regulated by WNK2.

Results

Phosphorylation modified mass spectrometry indicated that WNK2 knockdown significantly decreased the phosphorylation of 745 proteins with 1 078 altered protein sites

and increased the phosphorylation of 328 proteins with 415 altered protein sites. KEGG and GO enrichment analyses showed WNK2 influences biological processes such as cytoskeleton organization

signal transduction

cell composition

and regulation of kinase activity. Phalloidin staining indicated after WNK2 knockdown resulted in broken and disordered cytoskeleton

while its overexpression promoted cytoskeleton remodeling.

Conclusion

WNK2 is involved in the cytoskeleton formation in ovarian cancer cells by regulating the phosphorylation of vimentin and other proteins.

关键词

Keywords

references

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