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中华结直肠疾病电子杂志 ›› 2023, Vol. 12 ›› Issue (02) : 117 -124. doi: 10.3877/cma.j.issn.2095-3224.2023.02.005

论著

m6A结合蛋白YTHDC2调节p38MAPK信号通路影响结直肠癌细胞凋亡
张师垚1, 徐岩岩1, 张琦1, 李春强1, 赵智成1, 刘刚1,()   
  1. 1. 300052 天津医科大学总医院普通外科
  • 收稿日期:2022-04-26 出版日期:2023-04-25
  • 通信作者: 刘刚
  • 基金资助:
    天津市131创新型人才第一层次项目(303077900808); 天津医科大学“十三五”学科建设项目(303071300302)

The m6A-binding protein YTHDC2 affects proliferation and apoptosis of colorectal cancer cells by regulating the p38MAPK signaling pathway

Shiyao Zhang1, Yanyan Xu1, Qi Zhang1, Chunqiang Li1, Zhicheng Zhao1, Gang Liu1,()   

  1. 1. Department of General Surgery, Tianjin Medical University General Hospital, Tianjin 300052, China
  • Received:2022-04-26 Published:2023-04-25
  • Corresponding author: Gang Liu
引用本文:

张师垚, 徐岩岩, 张琦, 李春强, 赵智成, 刘刚. m6A结合蛋白YTHDC2调节p38MAPK信号通路影响结直肠癌细胞凋亡[J/OL]. 中华结直肠疾病电子杂志, 2023, 12(02): 117-124.

Shiyao Zhang, Yanyan Xu, Qi Zhang, Chunqiang Li, Zhicheng Zhao, Gang Liu. The m6A-binding protein YTHDC2 affects proliferation and apoptosis of colorectal cancer cells by regulating the p38MAPK signaling pathway[J/OL]. Chinese Journal of Colorectal Diseases(Electronic Edition), 2023, 12(02): 117-124.

目的

探讨m6A结合蛋白YTHDC2在结直肠癌发展过程中的分子机制及其所参与的信号通路。

方法

通过The Human Protein Atlas和GEPIA网站分析肿瘤基因组图谱(TCGA)数据库中YTHDC2与结直肠癌的关系,探究相关的信号转导途径和生物学过程。体外培养结直肠癌HCT116和Caco2细胞系,对YTHDC2进行过表达或敲低后,采用RT-PCR和Western blot检测p38MAPK、p-p38MAPK及其下游凋亡相关蛋白在丝裂原活化蛋白激酶(MAPK)信号通路中的表达,流式细胞术检测细胞凋亡率。

结果

The Human Protein Atlas和GEPIA分析结果表明,肿瘤组织中YTHDC2表达水平较癌旁组织存在降低趋势,YTHDC2的表达增加与结直肠癌患者总体生存率提高呈正相关。基于cBioPortal和Xena数据库中结直肠癌相关的基因表达量和临床数据以及KEGG和GO数据库中的注释基因集,基因集富集分析(GSEA)提示,YTHDC2具有调控MAPK信号通路的作用。流式细胞术检测结果显示,敲除YTHDC2时细胞凋亡比率显著减少,过表达时则显著增加,Western blot检测结果显示,p38MAPK表达未见显著变化,p-p38MAPK在YTHDC2过表达时显著升高,敲除时显著降低。基因表达谱交互分析(GEPIA)结果提示,凋亡蛋白的表达与YTHDC2的表达呈正相关,与RT-PCR和Western blot检测结果相符合。

结论

YTHDC2激活p38MAPK信号途径中外源性死亡受体和内源性线粒体凋亡通路调控结直肠癌细胞的凋亡。

Objective

To investigate the molecular mechanism and signal pathway of m6A binding protein YTHDC2 in the development of colorectal cancer.

Methods

The relationship between YTHDC2 and colorectal cancer in the Cancer Genome Atlas (TCGA) database was analyzed through The Human Protein Atlas and GEPIA websites, and the related signal transduction pathways and biological processes were explored. Colorectal cancer cell lines HCT116 and Caco2 were cultured in vitro. After YTHDC2 was overexpressed or knocked down, the expression of p38MAPK, p-p38MAPK and their downstream apoptosis-related proteins in the mitogen-activated protein kinase (MAPK) signaling pathway was detected by RT-PCR and Western blot. The apoptosis rate was detected by flow cytometry.

Results

The Human Protein Atlas and GEPIA analysis results showed that the expression level of YTHDC2 in tumor tissues was lower than that in adjacent tissues, and the increase of YTHDC2 expression was positively correlated with the improvement of overall survival rate of patients with colorectal cancer. Based on the gene expression and clinical data related to colorectal cancer in cBioPortal and Xena databases and the annotated gene sets in KEGG and GO databases, gene set enrichment analysis (GSEA) suggested that YTHDC2 had a role in regulating the MAPK signaling pathway. The results of flow cytometry showed that the apoptosis rate was significantly reduced in YTHDC2 knockout group and increased in YTHDC2 overexpression group. Western blot showed that the expression of p38MAPK did not change significantly, while p-p38MAPK was significantly increased in YTHDC2 overexpression group and decreased in YTHDC2 knockout group. The results of gene expression profile interaction analysis (GEPIA) suggested that the expression of apoptotic proteins was positively correlated with the expression of YTHDC2, which was consistent with the results of RT-PCR and Western blot.

Conclusions

YTHDC2 activates exogenous death receptors and endogenous mitochondrial apoptosis pathways in the p38MAPK signaling pathway to regulate the apoptosis of colorectal cancer cells.

图1 YTHDC2表达生信分析结果。1A:从左至右依次为YTHDC2高表达和低表达时结肠癌、直肠癌、结直肠癌患者生存曲线分析;1B:YTHDC2在结肠癌和直肠癌组织中的表达量,其中红色代表肿瘤组织,灰色代表正常组织;1C:YTHDC2表达量高低与结直肠肿瘤分期的关系;1D:GSEA基因富集分析结果,按顺序分别为MAPK信号通路、固有凋亡信号通路、外源性凋亡信号通路的调控、细胞周期停滞;1E:外源性死亡受体激活通路相关凋亡蛋白与YTHDC2的相关性分析;1F:内源性线粒体凋亡相关通路相关凋亡蛋白与YTHDC2的相关性分析;1G:内源性内质网凋亡相关通路相关凋亡蛋白与YTHDC2的相关性分析
图2 结直肠癌细胞株凋亡、转移和侵袭的变化。2A:免疫荧光实验显示YTHDC2在Caco2细胞中的定位;2B:HCT116及Caco2细胞凋亡情况;2C:HCT116及Caco2细胞划痕实验结果
图3 HCT116及Caco2细胞中p38MAPK和p-p38MAPK蛋白表达情况
图4 YTHDC2对MAPK信号通路中凋亡相关蛋白的影响。4A:HCT116 细胞外源性死亡受体激活通路凋亡基因mRNA及蛋白表达量变化;4B:Caco2 细胞外源性死亡受体激活通路凋亡基因mRNA及蛋白表达量变化;4C:HCT116 细胞内源性线粒体凋亡相关通路凋亡基因mRNA及蛋白表达量变化;4D:Caco2 细胞内源性线粒体凋亡相关通路凋亡基因mRNA及蛋白表达量变化;4E:HCT116 细胞内源性内质网凋亡相关通路凋亡基因mRNA及蛋白表达量变化;4F:Caco2 细胞内源性内质网凋亡相关通路凋亡基因mRNA及蛋白表达量变化
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