靶向调控Claudin-4基因表达介导DNA损伤修复增强膀胱癌细胞化疗敏感性的研究

艾克帕尔·阿布拉江; 崔涛; 杜恒

doi:10.11659/jjssx.07E022080

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靶向调控Claudin-4基因表达介导DNA损伤修复增强膀胱癌细胞化疗敏感性的研究

基础研究 | 更新时间：2023-04-24

- 靶向调控Claudin-4基因表达介导DNA损伤修复增强膀胱癌细胞化疗敏感性的研究
- Targeted regulation of Claudin-4 gene expression to mediate DNA damage repair and enhance chemosensitivity of bladder cancer cells
- 局解手术学杂志 2023年32卷第4期页码：279-284
- 作者机构：
  
  新疆医科大学第二附属医院泌尿外科，新疆乌鲁木齐 830000
- 作者简介：
  
  杜恒，E-mail：536559255@qq.com
- 基金信息：
  
  新疆维吾尔自治区自然科学基金(2021D01C364)
- DOI：10.11659/jjssx.07E022080
  中图分类号： R737.14
- 纸质出版日期：2023-04-25，
  
  收稿日期：2022-07-21，
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艾克帕尔·阿布拉江 ,崔涛,杜恒.靶向调控Claudin-4基因表达介导DNA损伤修复增强膀胱癌细胞化疗敏感性的研究[J].局解手术学杂志,2023,32(04):279-284.

Aikepaer·Abulajiang ,CUI Tao,DU Heng.Targeted regulation of Claudin-4 gene expression to mediate DNA damage repair and enhance chemosensitivity of bladder cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2023,32(04):279-284.
艾克帕尔·阿布拉江 ,崔涛,杜恒.靶向调控Claudin-4基因表达介导DNA损伤修复增强膀胱癌细胞化疗敏感性的研究[J].局解手术学杂志,2023,32(04):279-284. DOI： 10.11659/jjssx.07E022080.

Aikepaer·Abulajiang ,CUI Tao,DU Heng.Targeted regulation of Claudin-4 gene expression to mediate DNA damage repair and enhance chemosensitivity of bladder cancer cells[J].JOURNAL OF REGIONAL ANATOMY AND OPERATIVE SURGERY,2023,32(04):279-284. DOI： 10.11659/jjssx.07E022080.

摘要

目的

探究靶向下调紧密连接蛋白4（Claudin-4）基因表达对膀胱癌细胞DNA损伤修复及其化疗敏感性的影响。

方法

采用si-Claudin-4及阴性对照(si-NC)转染人膀胱癌细胞系EJ下调Claudin-4的表达，采用qRT-PCR和Western blot检测细胞Claudin-4的表达，以确定转染效果。不同浓度顺铂（DDP）处理转染后的EJ细胞，CCK-8法检测细胞存活率。将EJ细胞随机分为对照组、si-Claudin-4组、DDP组、DDP+si-Claudin-4组。经转染处理与DDP处理后，CCK-8法检测各组细胞存活率；平板克隆形成实验检测各组细胞克隆形成能力；细胞免疫荧光染色观察各组细胞内γ-H2AX焦点生成情况；单细胞凝胶电泳法检测各组细胞DNA损伤情况；Western blot检测各组细胞内γ-H2AX、Ku70、Ku80以及DNA-PKcs的蛋白表达水平。

结果

细胞转染后，si-Claudin-4组细胞内Claudin-4 mRNA和蛋白相对表达量均显著低于对照组和si-NC组（

0.05）。经不同浓度DDP处理后，si-Claudin-4组EJ细胞存活率显著低于对照组和si-NC组（

0.05）。与对照组比较，si-Claudin-4组和DDP组细胞存活率显著下降，细胞克隆形成数目显著减少，细胞内γ-H2AX焦点形成显著增加，细胞出现典型拖尾形状，彗星尾长和尾部DNA比例均显著增加，γ-H2AX蛋白相对表达量显著上调，Ku70、Ku80及DNA-PKcs的蛋白相对表达量显著下调，差异均具有统计学意义（

0.05）；与DDP组比较，DDP+si-Claudin-4组细胞存活率显著下降，细胞克隆形成数目显著减少，细胞内γ-H2AX焦点形成显著增加，细胞拖尾现象更加明显，细胞彗星尾长和尾部DNA比例均进一步增加，同时，γ-H2AX蛋白相对表达量显著上调，且Ku70、Ku80、DNA-PKcs的蛋白相对表达量均显著下调，差异均具有统计学意义（

0.05）。

结论

在膀胱癌细胞系EJ中靶向下调Claudin-4基因表达，能够增强肿瘤细胞对DDP的敏感性，该作用可能是通过抑制DNA损伤修复能力实现的。

Abstract

Objective

To investigate the effect of targeted down-regulation of Claudin-4 gene expression on DNA damage repair and chemosensitivity of bladder cancer cells.

Methods

Human bladder cancer cell line EJ was transfected with si-Claudin-4 and negative control（si-NC） to down-regulate the expression of Claudin-4.The expression of Claudin-4 in cells was detected by qRT-PCR and Western blot to determine the transfection effect.The transfected EJ cells were treated with different concentrations of cisplatin （DDP），and the survival rate of cells was detected by CCK-8 method.The EJ cells were randomly divided into control group，si-Claudin-4 group，DDP group，DDP+si-Claudin-4 group.After transfection and DDP treatment，the CCK-8 method was used to detect the survival rate of cells in each group；Plate colone formation assay was used to detect the colony formation ability of cells in each group；Immunofluorescence staining was used to observe the γ-H2AX foci formation in each group；Single-cell gel electrophoresis was used to detect DNA damage of cells in each group；Western blot was used to detect the protein expression levels of γ-H2AX，Ku70，Ku80 and DNA-PKcs of cells in each group.

Results

After transfection，the relative expression of Claudin-4 mRNA and protein in si-Claudin-4 group were significantly lower than those in control group and si-NC group （

0.05）.The survival rate of EJ cells which treated with different concentrations of DDP in si-Claudin-4 group was significantly lower than those in control group and si-NC group （

0.05）.Compared with control group，the survival rates of cells in si-Claudin-4 group and DDP group were significantly decreased，the numbers of cell clones were significantly reduced，the intracellular γ-H2AX foci formations were significantly increased，and the cells showed typical trailing shape，the comet tail length and tail DNA ratio were significantly increased，the relative expression of γ-H2AX protein were significantly up-regulated，and the relative protein expression of Ku70，Ku80 and DNA-PKcs were significantly down-regulated，the differences were statistically significant （

0.05）.Compared with DDP group，the survial rate of cells and the number of cell clones were decresed，the intracellular γ-H2AX foci formation was increased，the cell tailing is more obvious，the comet tail length and tail DNA ratio were increased，the relative expression of γ-H2AX protein was significantly up-regulated，the relative protein expression of Ku70，Ku80 and DNA-PKcs were significantly down-regulated in DDP+si-Claudin-4 group，and the differences were statistically significant （

0.05）.

Conclusion

Targeted down-regulation of Claudin-4 gene expression in bladder cancer cell line EJ can enhance the sensitivity of tumor cells to DDP，which may be achieved by inhibiting the ability of DNA damage repair.

关键词

膀胱癌紧密连接蛋白4顺铂化疗敏感性DNA损伤修复

Keywords

bladder cancerClaudin-4cisplatinchemosensitivityDNA damage repair

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