Nuclear EGFR cooperates with YY1 to drive CEMIP expression and enhance hepatocellular carcinoma invasion

TU Li; LIU Yang; YU Ying; OUYANG Shiqi; WANG Zengli; TANG Xiaolong

doi:10.16016/j.2097-0927.202505079

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Nuclear EGFR cooperates with YY1 to drive CEMIP expression and enhance hepatocellular carcinoma invasion

Monographic Reports | 更新时间：2025-09-11

- Nuclear EGFR cooperates with YY1 to drive CEMIP expression and enhance hepatocellular carcinoma invasion
- Journal of Army Medical University Vol. 47, Issue 17, Pages: 1994-2011(2025)
- 作者机构：
  
  安徽理工大学医学院生物化学与分子生物学教研室，安徽淮南
- 作者简介：
  
  TANG Xiaolong， E-mail： txljd2006@126.com
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(82071862);the Medical Special Cultivation Project of Anhui University of Science and Technology(YZ2023H1A005)
- DOI：10.16016/j.2097-0927.202505079
  CLC： R394.3;R730.23;R735.7
- Received：30 May 2025，
  
  Revised：2025-07-20，
  
  Accepted：28 July 2025，
  
  Published：15 September 2025
- 稿件说明：
移动端阅览
涂丽，刘洋，于滢，等.核表皮生长因子受体与转录因子YY1协同驱动CEMIP表达促进肝细胞癌侵袭［J］.陆军军医大学学报，2025，47（17）：1994-2011.DOI：10.16016/j.2097-0927.202505079.

TU L，LIU Y，YU Y，et al.Nuclear EGFR cooperates with YY1 to drive CEMIP expression and enhance hepatocellular carcinoma invasion［J］.J Army Med Univ，2025，47（17）：1994-2011.DOI：10.16016/j.2097-0927.202505079.
涂丽，刘洋，于滢，等.核表皮生长因子受体与转录因子YY1协同驱动CEMIP表达促进肝细胞癌侵袭［J］.陆军军医大学学报，2025，47（17）：1994-2011.DOI：10.16016/j.2097-0927.202505079. DOI：

TU L，LIU Y，YU Y，et al.Nuclear EGFR cooperates with YY1 to drive CEMIP expression and enhance hepatocellular carcinoma invasion［J］.J Army Med Univ，2025，47（17）：1994-2011.DOI：10.16016/j.2097-0927.202505079. DOI：

摘要

目的

揭示核表皮生长因子受体（nuclear epidermal growth factor receptor， nEGFR）通过与转录因子YY1形成复合物协同调控细胞迁移诱导蛋白（cell migration-inducing protein， CEMIP）基因表达的分子机制，并探究nEGFR-YY1-CEMIP信号轴在肝细胞癌（hepatocellular carcinoma， HCC）侵袭中的生物学功能。

方法

将血清饥饿24 h的HCC细胞用100 ng/mL EGF进行处理，设置对照组和不同时间点的EGF处理组，通过Western blot及间接免疫荧光实验（immunofluorescence， IF）检测EGFR的核内表达和定位变化。为明确nEGFR与YY1的相互作用，设置对照组及EGF处理组，采用IF观察核内共定位情况，并通过免疫共沉淀（co-immunoprecipitation， Co-IP）验证二者是否存在相互作用。通过高通量测序技术（RNA sequencing， RNA-seq）检测细胞基因组转录水平差异，结合GO功能富集分析和JASPAR数据库预测，最终锁定候选靶基因CEMIP，为验证调控机制构建YY1的 siRNA后，联合EGF处理，分为：Control组、EGF组、siYY1组、siYY1+EGF组，通过Western blot和RT-qPCR检测CEMIP蛋白及mRNA水平。为明确nEGFR/YY1结合CEMIP启动子的分子机制，设置对照组和EGF处理组，采用染色质免疫沉淀-定量PCR（chromatin immunoprecipitation-qPCR， ChIP-qPCR）实验检测CEMIP启动子区的富集情况。通过转染野生型EGFR（EGFR-WT）、核定位缺陷突变体（EGFR-dNLS）、YY1过表达质粒（YY1-OE）及功能缺失突变体（YY1-DN），利用荧光素酶报告基因实验检测启动子活性变化。通过划痕愈合实验、Transwell实验检测细胞的迁移侵袭能力，采用ELISA实验检测透明质酸（hyaluronic acid， HA）的含量，并通过Western blot实验检测基质金属蛋白酶（MMP2/9）的含量，评估nEGFR/YY1-CEMIP轴对HCC细胞迁移侵袭的调控作用。通过分析国家生物技术信息中心（National Center for Biotechnology Information， NCBI）公共数据库中HCC患者的CEMIP表达谱，验证其与肿瘤转移风险的潜在关联。

结果

Western blot和IF检测显示EGF处理可显著诱导EGFR的核易位，并在30 min达到峰值（

0.001）。Co-IP和IF证明了nEGFR和YY1存在相互作用，且在细胞核内共定位。RNA-seq分析显示，CEMIP表现为差异显著基因。GO富集分析显示CEMIP主要富集于细胞促侵袭等生物学功能。JASPAR预测发现CEMIP启动子区存在YY1的潜在结合区域。Western blot和RT-qPCR实验进一步证实EGF处理可上调CEMIP蛋白和mRNA的水平，而敲低YY1则显著抑制CEMIP的表达，外源性补充EGF能够恢复YY1缺陷细胞的CEMIP表达（

0.05）。ChIP-qPCR显示nEGFR/YY1复合物特异性富集于CEMIP启动子区域且EGF刺激可以显著增强其结合强度（

0.001）。荧光素酶报告基因实验证实nEGFR/YY1可以增强CEMIP的启动子活性（

0.01），而EGFR-dNLS或YY1-DN则无此效应。功能表型实验证明nEGFR/YY1-CEMIP轴通过促进HA分解和上调MMP2/9表达，显著增强HCC细胞的迁移侵袭能力（

0.05）。基于NCBI数据库分析显示，CEMIP在HCC肿瘤组织中的表达水平显著高于癌旁正常组织（

0.001），且CEMIP高表达的HCC患者具有更高的转移风险（

0.001）。

结论

nEGFR通过与YY1形成转录因子复合物协同激活CEMIP表达促进HCC侵袭。

Abstract

Objective‍　‍To reveal the molecular mechanism by which nuclear epidermal growth factor receptor (nEGFR) synergistically regulates the expression of cell migration-inducing protein (CEMIP) by forming a complex with the transcription factor Yin Yang 1 (YY1)

and to investigate the biological functions of the nEGFR-YY1-CEMIP signaling axis in invasion of hepatocellular carcinoma (HCC). Methods‍　‍After HCC cells were serum-starved for 24 h

the cells were treated with 100 ng/mL EGF. Thus

the cells were divided into a control group and EGF-treated groups at different time points. Nuclear expression and localization changes of EGFR were detected by Western blotting and immunofluorescence (IF). To investigate the interaction between nEGFR and YY1

their nuclear colocalization and interaction were examined by IF and co-immunoprecipitation (Co-IP)

respectively. Transcriptional profiling was performed using RNA sequencing (RNA-seq) to identify differentially expressed genes at the genome-wide level. Combined with Gene Ontology (GO) fun

ctional enrichment analysis and transcription factor binding profiles

via

using the JASPAR database

CEMIP was identified as a candidate target gene. To validate the regulatory mechanism

the following experimental groups were established

Control

EGF

siYY1

and siYY1+EGF. The expression of CEMIP at protein and mRNA levels was detected by Western blotting and RT-qPCR. To elucidate the molecular mechanism of nEGFR/YY1 binding to the CEMIP promoter

the control and EGF-treated groups were established. Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) was performed to assess the enrichment of nEGFR/YY1 at the CEMIP promoter region. Luciferase reporter assay was conducted following transfection with either wild-type EGFR (EGFR-WT)

nuclear localization-deficient mutant (EGFR-dNLS)

YY1 overexpression plasmid (YY1-OE)

or dominant-negative YY1 mutant (YY1-DN) to evaluate changes in promoter activity. Subsequently

cell migration and invasion capabilities were evaluated using scratch wound healing assay and Transwell assay

while hyaluronic acid (HA) level was quantified by ELISA. The expression of matrix metalloproteinases (MMP2/9) was analyzed

via

Western blotting to assess the regulatory role of the nEGFR/YY1-CEMIP axis in the migration and invasion of HCC cells. By analyzing the CEMIP expression profiles in HCC patients from National Center for Biotechnology Information (NCBI) public databases

its potential association with tumor metastasis risk was validated. Results‍　‍Western blotting and IF demonstrated that EGF treatment significantly induced nuclear translocation of EGFR

peaking at 30 min (

0.001). Co-IP and IF assays indicated both physical interaction and nuclear co-localization between nEGFR and YY1. RNA-seq analysis identified CEMIP as a significantly differentially expressed gene. GO enrichment analysis revealed that CEMIP was significantly enriched in biological processes related to cell invasion promotion. JASPAR prediction

identified conserved YY1 potential binding region within the CEMIP promoter region. Western blot and RT-qPCR analyses confirmed that EGF treatment up-regulated CEMIP at both protein and mRNA levels

0.05). Notably

YY1 knockdown significantly suppressed CEMIP expression

while exogenous EGF supplementation restored CEMIP level in YY1-deficient cells (

0.05). ChIP-qPCR analysis demonstrated specific enrichment of the nEGFR/YY1 complex at the CEMIP promoter region

with EGF stimulation significantly enhancing its binding affinity (

0.001). Luciferase reporter assay confirmed that nEGFR/YY1 robustly enhanced CEMIP promoter activity (

0.01)

while either the EGFR-dNLS or the YY1-DN substantially attenuated this transcriptional activation. Functional phenotyping showed that the nEGFR/YY1-CEMIP axis significantly enhanced the migration and invasion of HCC cells by promoting HA catabolism and up-regulating MMP2/9 expression (

0.05). Analysis of NCBI datasets revealed that CEMIP expression was significantly up-regulated in HCC tumor tissues than adjacent normal tissues (

0.001). Moreover

HCC patients with elevated CEMIP expression exhibited higher risk of metastasis (

0.001). Conclusion‍　‍nEGFR promotes HCC invasion by forming a transcriptional complex with YY1 to cooperatively activate CEMIP expression.

关键词

Keywords

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Related Author

LI Jianwei

WANG Xingru

LIU Yang

WANG Yuran

YU Ying

TU Li

WANG Zengli

OUYANG Shiqi

Related Institution

No.953，Xigaze， Tibet Autonomous Region

Institute of Hepatobiliary Surgery of PLA，First Affiliated Hospital， Army Medical University （Third Military Medical University）

Grade Majoring in Clinical Medicine， School of Medicine， Anhui University of Science and Technology

Class

Department of Biochemistry and Molecular Biology

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