ETV4对食管癌细胞生物学行为的调控作用及其分子机制

doi:10.13481/j.1671-587X.20260412

摘要/Abstract

摘要：

目的探讨ETS易位变异体4（ETV4）对食管癌细胞生物学行为的调控作用，并阐明其可能的分子机制。方法采用基因表达谱交互式分析2（GEPIA2）数据库分析食管癌组织和正常食管组织中ETV4 mRNA表达水平。从高通量基因表达综合数据库（GEO）获取食管癌转录组测序数据集GSE199967，采用R软件统计ETV4 mRNA表达水平。基于TCGA-ESCA数据集，利用UALCAN在线分析平台按临床分期、病理分级及组织学亚型分层评估ETV4的表达分布特征。将人食管癌KYSE-150细胞分为对照组和ETV4干扰组（shETV4组），分别转染对照慢病毒和靶向ETV4的短发夹RNA（shRNA）慢病毒，经嘌呤霉素筛选得到对照细胞及稳定干扰ETV4表达的细胞。采用实时定量PCR（RT-qPCR）法检测2组细胞中ETV4 mRNA表达水平，Werstern blstting法检测2组细胞中ETV4蛋白表达水平，细胞计数试剂盒8（CCK-8）法检测2组细胞增殖活性，Transwell小室实验检测2组迁移细胞数，划痕愈合实验检测2组细胞划痕愈合率。通过TF-Target Finder平台预测ETV4的靶基因，利用JASPAR数据库筛选MDM2结合蛋白（MTBP）启动子区ETV4的潜在结合位点，采用双荧光素酶报告基因实验验证ETV4对MTBP启动子的调控作用。建立稳定干扰MTBP表达的KYSE-150-shMTBP细胞，采用RT-qPCR法检测2组细胞中MTBP、细胞周期蛋白依赖性激酶抑制剂1A（CDKN1A）和细胞周期蛋白依赖性激酶抑制剂1B（CDKN1B）mRNA表达水平，流式细胞术检测不同细胞周期细胞百分率。结果生物信息学分析，与正常食管组织比较，食管癌组织中ETV4表达水平升高（P<0.05）。TCGA-ESCA队列中Ⅰ~Ⅳ期食管癌组织、G₁~G₃级分化肿瘤组织及腺癌、鳞状细胞癌亚型组织中ETV4 mRNA表达水平均高于正常组织（P<0.001）；而ETV4 mRNA表达水平在不同临床分期、病理分级及组织学亚型患者组间比较差异无统计学意义（P>0.05）。成功构建干扰ETV4表达的KYSE-150-shETV4细胞株，与对照组比较，shETV4组细胞中ETV4 mRNA和蛋白表达水平均降低（P<0.001）；CCK-8法检测，与对照组比较，shETV4组细胞增殖活性降低（P<0.001）；Transwell小室实验，与对照组比较，shETV4组迁移细胞数减少（P<0.01）；划痕愈合实验，与对照组比较，shETV4组细胞划痕愈合率降低（P<0.001）。成功构建干扰MTBP表达的KYSE-150-shMTBP细胞，与对照组比较，shMTBP组细胞中MTBP mRNA表达水平降低（P<0.001）。生物信息学分析和双荧光素酶报告基因实验验证MTBP为ETV4下游靶基因，其启动子区TFBS2是ETV4的关键结合位点。RT-qPCR法检测，与对照组比较，shMTBP组细胞中CDKN1A和CDKN1B mRNA表达水平升高（P<0.001）；流式细胞术检测，与对照组比较，shMTBP组G₀/G₁期细胞百分率升高（P<0.001），S期细胞百分率降低（P<0.001）。结论转录因子ETV4通过结合MTBP启动子激活其表达，调控细胞周期，促进食管癌细胞增殖与迁移，其机制与ETV4/MTBP轴有关。

关键词: 食管肿瘤, 转录因子, ETS易位变异体4, 细胞周期蛋白依赖性激酶抑制剂1A, 细胞周期

Abstract:

Objective To discuss the regulatory role of ETS translocation variant 4 （ETV4） in the biological behavior of esophageal cancer cells， and to clarify its possible molecular mechanism. Methods The expression levels of ETV4 mRNA in esophageal cancer tissue and normal esophageal tissue were analyzed using the Gene Expression Profiling Interactive Analysis 2 （GEPIA2） database. The esophageal cancer transcriptome dataset GSE199967 was obtained from the Gene Expression Omnibus （GEO） database， and the ETV4 mRNA expression level was analyzed using R software. Based on the TCGA-ESCA dataset， the expression distribution characteristics of ETV4 were systematically evaluated according to clinical stage， pathological grade， and histological subtype using the UALCAN online platform. The human esophageal cancer KYSE-150 cells were divided into control group and ETV4 interference group （shETV4 group）； the cells were transfected with control lentivirus or ETV4-targeted short hairpin RNA （shRNA） lentivirus， and after puromycin selection， the control cells and cells with stable ETV4 interference were established. Real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the ETV4 mRNA expression level in the cells in two groups； Western blotting method was used to detect the ETV4 protein expression levels in the cells in two groups； cell counting kit-8 （CCK-8） method was used to detect the proliferation activities of the cells in the two groups； Transwell chamber assay was used to count the numbers of migration cells in two groups； wound healing assay was used to detect the wound healing rates of the cells in two groups. The target genes of ETV4 were predicted using the TF-Target Finder platform； potential binding sites of ETV4 in the promoter region of MDM2 binding protein （MTBP） were screened using the JASPAR database； dual-luciferase reporter assay was used to verify the regulatory effect of ETV4 on the MTBP promoter. The KYSE-150-shMTBP cells with stable MTBP interference was established； RT-qPCR method was used to detect the expression levels of MTBP， cyclin-dependent kinase inhibitor 1A （CDKN1A）， and cyclin-dependent kinase inhibitor 1B （CDKN1B） mRNA in the cells in two groups； flow cytometry was used to detect the percentages of the cells at different cell cycles. Results The bioinformatics analysis results showed that compared with normal esophageal tissue， the expression level of ETV4 mRNA in esophageal cancer tissues was increased （P<0.05）. In the TCGA-ESCA cohort， the expression levels of ETV4 mRNA in esophageal cancer tissues at clinical stages Ⅰ-Ⅳ， in tumor tissues with pathological grades G₁-G₃， and in adenocarcinoma and squamous cell carcinoma subtypes were all higher than those in normal tissue （P<0.001）； however， there was no statistically significant difference in ETV4 mRNA expression levels among the subgroups stratified by clinical stage， pathological grade， and histological subtype （P>0.05）. The KYSE-150-shETV4 cells with stable ETV4 interference was successfully constructed. Compared with control group， the ETV4 mRNA and protein expression levels in the cells in shETV4 group were decreased （P<0.001）. The CCK-8 assay results showed that compared with control group， the proliferation activity of the cells in shETV4 group was decreased （P<0.001）. The Transwell chamber assay results showed that compared with control group， the number of migration cells in shETV4 group was decreased （P<0.01）. The wound healing assay results showed that compared with control group， the wound healing rate of the cells in shETV4 group was decreased （P<0.001）. The KYSE-150-shMTBP cells with stable MTBP interference was successfully constructed. Compared with control group， the MTBP mRNA expression level in the cells in shMTBP group was decreased （P<0.001）. The bioinformatics analysis and dual-luciferase reporter assay results confirmed that MTBP was a downstream target gene of ETV4， and TFBS2 in its promoter region was the key binding site for ETV4. The RT-qPCR results showed that compared with control group， the expression levels of CDKN1A and CDKN1B mRNA in the cells in shMTBP group were increased （P<0.001）. The flow cytometry results showed that compared with control group， the percentage of cells in shMTBP group at G₀/G₁ phase was increased （P<0.001）， and the percentage of the cells at S phase was decreased （P<0.001）. Conclusion The transcription factor ETV4 activates the expression of MTBP by binding to its promoter， regulates the cell cycle， and promotes the proliferation and migration of esophageal cancer cells； its mechanism is related to the ETV4/MTBP axis.

Key words: Esophageal neoplasm, Transcription factor, ETS translocation variant 4, Cyclin-dependent kinase inhibitor 1A, Cell cycle

中图分类号:

R735.1

麻丽霞,汪悦,郝相端,郭颖. ETV4对食管癌细胞生物学行为的调控作用及其分子机制[J]. 吉林大学学报(医学版), 2026, 52(4): 999-1010.

Lixia MA,Yue WANG,Xiangduan HAO,Ying GUO. Regulatory effect of ETV4 on biological behavior of esophageal cancer cells and its molecular mechanism[J]. Journal of Jilin University(Medicine Edition), 2026, 52(4): 999-1010.

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Primer name	Primer sequence（5'-3'）
ShETV4-F	CCGGCCCTGTGTACATATAAATGAACTCGAGTTCATTTATATGTACACAGGGTTTTTG
ShETV4-R	AATTCAAAAACCCTGTGTACATATAAATGAACTCGAGTTCATTTATATGTACACAGGG
ShMTBP-F	CCGGGGAGAGTGTTCTAGCTATTATCTCGAGATAATAGCTAGAACACTCTCCTTTTTG
ShMTBP-R	AATTCAAAAAGGAGAGTGTTCTAGCTATTATCTCGAGATAATAGCTAGAACACTCTCC
Scrambled shRNA-F	CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG
Scrambled shRNA-R	AATTCAAAAACAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTG
ETV4-F (BamHⅠ)	AAAGGATCCGGATCCGATGGAGCGGAGGATGAA
ETV4-R (EcoRⅠ)	CCCGAATTCCTAGTAAGAGTAGCCACC
MTBP pro-F (KpnⅠ)	AAAGGTACCGGTGGCTGCATTTTCCCATC
MTBP pro-R (MluⅠ)	CCCACGCGTCGTCGGTTTTCCGCAGTTTT
TFBS1 mut-F	AAAATTTTTATTTTACTTTGGTGTATTTATCTCATCA
TFBS1 mut-R	TGATGAGATAAATACACCAAGTAAAATAAAAATTTT
MTBP TFBS2-R MluⅠ	AAAACGCGTTATGACGCATTTTCGGTTCCCTTCGT
TFBS3 mut-F	AACACAGTAATTATTCCTTGGTGGTTTCCTGTTAGC
TFBS3 mut-R	GCTAACAGGAAACCACCAAGGAATAATTACTGTGTT
TFBS4 mut-F	CCTATACTCAATATTCACCAAGTCCTACTGGCTCCAC
TFBS4 mut-R	GTGGAGCCAGTAGGACTTGGTGAATATTGAGTATAGG

Gene name	Primer sequence（5'-3'）
qETV4	F：CAGTGCCTTTACTCCAGTGCC
qETV4	R：CTCAGGAAATTCCGTTGCTCT
qMTBP	F：TCGTCGGGTGAGGGTACTG
qMTBP	R：AACCACTTCTTGGAACCAGGT
qCCND1	F：GCTGCGAAGTGGAAACCATC
qCCND1	R：CCTCCTTCTGCACACATTTGAA
qCDKN1A	F：TGTCCGTCAGAACCCATGC
qCDKN1A	R：AAAGTCGAAGTTCCATCGCTC
qCDKN1B	F：TAATTGGGGCTCCGGCTAACT
qCDKN1B	R：TGCAGGTCGCTTCCTTATTCC
qGAPDH	F：ACAACTTTGGTATCGTGGAAGG
qGAPDH	R：GCCATCACGCCACAGTTTC