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. 2025 Feb 28;50(2):155–166. [Article in Chinese] doi: 10.11817/j.issn.1672-7347.2025.240385

EZH2通过EMT促进食管鳞状细胞癌的恶性生物学行为

EZH2 promotes malignant biological behavior in esophageal squamous cell carcinoma via EMT

JING Yuying 1,2,2, YANG Kaige 1,2, CHENG Yiting 1,2, HUANG Tianping 1,2, CHEN Sufang 1,2, CHEN Kai 1,2, HU Jianming 1,2,✉
Editors: 游 毅, 郭 征
PMCID: PMC13346017  PMID: 40523759

Abstract

Objective

Esophageal squamous cell carcinoma (ESCC) is characterized by complex pathogenesis and poor prognosis. In recent years, epithelial-mesenchymal transition (EMT) in tumor initiation and progression has attracted increasing attention. Enhancer of zeste homolog 2 (EZH2), which is aberrantly expressed in various tumors, may be closely related to the EMT process. This study aims to examine the expression and correlation of EZH2 and EMT markers in ESCC cells and tissues, evaluate the effects of EZH2 knockdown on ESCC cell proliferation, invasion, and migration, and explore how EZH2 contributes to the malignant biological behavior of ESCC.

Methods

Bioinformatics analyses were used to assess EZH2 expression levels in ESCC. Small interfering RNA was used to knock down EZH2 in ESCC cell lines EC109 and EC9706. Cell proliferation, invasion, and migration were evaluated using cell counting kit-8 (CCK-8), wound healing, and Transwell assays. Protein and mRNA expression levels of EZH2, E-cadherin (E-cad), and vimentin (Vim) were detected by Western blotting and real time fluorogenic quantitative PCR (RT-qPCR), respectively. Immunohistochemical (IHC) staining was performed on 70 ESCC tissue samples and 40 paired adjacent normal tissues collected from the First Affiliated Hospital of Shihezi University between 2010 and 2016 to assess the expression of EZH2, E-cad, and Vim, and to analyze their associations with clinicopathological feature and patient prognosis.

Results

Bioinformatics analysis showed that EZH2 was highly expressed in ESCC (P<0.001), and high EZH2 expression was associated with worse prognosis (P<0.001). CCK-8, wound healing, and Transwell assays demonstrated that EZH2 knockdown significantly suppressed the proliferation, invasion, and migration of ESCC cells (P<0.001). In addition, Vim expression was significantly reduced, while E-cad expression was significantly increased at both protein and mRNA levels in EZH2-silenced cells (all P<0.05). IHC staining analysis revealed higher expression of EZH2 and Vim and lower expression of E-cad in ESCC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that low expression of EZH2 and Vim and high expression of E-cad were associated with longer survival (all P<0.05).

Conclusion

EZH2 promotes malignant biological behavior in ESCC by mediating EMT. Elevated EZH2 expression is associated with poor prognosis in ESCC patients.

Keywords: esophageal squamous cell carcinoma, enhancer of zeste homolog 2, epithelial-mesenchymal transition, malignant biological behavior, prognosis

食管癌(esophageal cancer，EC)是一种全球范围内普遍存在的恶性肿瘤。全球癌症统计数据[1]显示：2020年中国新发EC 324 422例，死亡301 135例，分别占全球EC新发病例和死亡病例的53.70%和55.35%。按照病理组织学的分类标准，EC可分为食管鳞状细胞癌(esophageal squamous cell carcinoma，ESCC)和食管腺癌(esophageal adenocarcinoma，EAC)，其中ESCC约占80%[2]。尽管目前ESCC的诊断和治疗技术取得了一定的进展，但ESCC患者的 5年生存率仍不足30%[3]，其中容易发生转移是造成ESCC患者治疗不佳、预后差的主要原因[4]。因此，深入了解ESCC发病和转移的机制，寻找合适的治疗靶点，对改善ESCC患者的疗效和生存预后具有重要意义。

Zeste基因增强子同源物2(enhancer of zeste homolog 2，EZH2)属于多梳群基因(polycomb group genes，PcGs)，是一类重要的表观遗传调控因子。EZH2的突变或表达异常通常与癌症的发生和发展密切相关[5-6]。研究[7]表明EZH2在ESCC组织中高表达，且与ESCC的浸润和转移密切相关，但其具体机制尚未阐明。上皮-间充质转化(epithelial-mesenchymal transition，EMT)在胚胎发育、组织重塑、创伤修复等生理过程中扮演关键的角色，也是肿瘤侵袭和转移的重要生物学过程[8]。通常EMT的发生受到转录因子、表观遗传调控因子、细胞外基质和细胞因子等的影响[9]。已有研究[10]报道EZH2与宫颈癌、肝癌等恶性肿瘤EMT过程的调控之间存在关联，但EZH2对EMT过程的具体调控作用还有待进一步的探明。因此，本研究从细胞水平和人体组织水平探讨EZH2与EMT之间的调控关系及其在ESCC浸润、转移中的作用。

1. 材料与方法

1.1. 伦理声明

本研究已获得石河子大学第一附属医院伦理委员会的批准(审批号：KJ2023-073-01)，并且经过患者本人及其家属的签字同意。

1.2. 组织及细胞来源

收集石河子大学第一附属医院2010至2016年间的70例ESCC组织及其中40例配对癌旁组织的石蜡标本。所有ESCC患者均经2名病理医师确诊为ESCC，并且在手术前未接受放化疗或免疫治疗。

ESCC细胞系EC109购自中国医学科学院，EC9706购自上海复祥生物科技有限公司。

1.3. 主要试剂

EZH2的小干扰RNA(small interfering RNA，siRNA)EZH2-Homo-1743和EZH2-Homo-1952、siRNA阴性对照(siRNA negative control，siNC)均购自上海吉玛制药技术有限公司；细胞计数试剂盒-8(cell counting kit-8，CCK-8)购自北京达科为生物技术有限公司；Transwell小室购自康宁公司(美国)；EZH2一抗购自Cell Signaling Technology公司(美国)；上皮钙黏素(E-cadherin，E-cad)、波形蛋白(vimentin，Vim)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase，GAPDH)一抗均购自武汉三鹰生物技术有限公司；兔二抗、鼠二抗均购自北京中杉金桥生物技术有限公司；增强化学发光(enhanced chemiluminescence，ECL)显影液购自Thermo Fisher Scientific公司(美国)；逆转录试剂盒及逆转录荧光定量试剂盒均购自北京康为世纪生物科技有限公司。

1.4. 生物信息学分析

利用癌症基因组图谱(The Cancer Genome Atlas，TCGA)[11-12]计算EZH2的每百万转录本数(transcripts per million，TPM)，并对TPM进行对数变换。考虑到TPM可能为0，为避免对0取对数时出现无意义的情况，在TPM基础上加1，最后得到log2(TPM+1)，以分析EZH2在多种癌症中的表达情况。

下载183例ESCC组织和13例癌旁组织的mRNA数据，并采用Limma R软件包进行基因表达谱的标准化处理，以评估EZH2在EC中的表达水平。从基因表达综合(Gene Expression Omnibus，GEO)数据库中下载GSE53625和GSE161533数据集，其中GSE53625包含179对ESCC组织样本(癌和癌旁组织配对)的测序数据，GSE161533包括28对ESCC组织及其癌旁组织样本，比较EZH2在ESCC组织和癌旁组织中的表达情况，并对EZH2高表达、低表达组进行生存分析。

1.5. 细胞转染

将EC109、EC9706细胞接种于含10%胎牛血清的RPMI 1640培养基中，置于37 ℃的CO2培养箱中培养。将处于生长对数期的EC109、EC9706细胞分为siNC组(转染siNC)、siEZH2#1组(转染EZH2-Homo-1743)、siEZH2#2组(转染EZH2-Homo-1952)，6 h后更换为完全培养基，继续转染24 h后，提取RNA；转染48 h后，提取蛋白质。

1.6. 蛋白质印迹法

用放射免疫沉淀法缓冲液(radio immunoprecipitation assay，RIPA)和苯甲基磺酰氟(phenylmethylsulfonyl fluoride，PMSF)按100꞉1配比的裂解液提取转染后各组(siNC、siEZH2#1、siEZH2#2)ESCC细胞中的总蛋白质，上样20 μg蛋白质，经电泳、电转、封闭后，将聚偏二氟乙烯(polyvinylidene fluoride，PVDF)用EZH2(稀释比1꞉800)、E-cad(稀释比1꞉5 000)、Vim(稀释比1꞉4 000)、GAPDH(稀释比1꞉5 000)一抗在4 ℃冰箱中孵育过夜，与二抗(稀释比1꞉10 000)在40 r/min、室温条件下孵育2 h后使用ECL显影液显影。

1.7. 实时荧光定量PCR

使用TRIzol裂解液提取各组ESCC细胞中总的RNA，使用逆转录试剂盒将RNA逆转录为互补脱氧核糖核酸(complementary DNA，cDNA)，使用逆转录荧光定量试剂盒进行定量PCR，以GADPH为内参对照。配置20 μL反应体系，反应程序设定为：预变性95 ℃ 30 s，变性95 ℃ 5 s，60 ℃ 30 s，共40个循环。采用2-ΔΔCt法计算EZH2、E-cad、Vim的mRNA相对表达量，引物序列见表1。

表1.

引物序列

Table 1 Primer sequences

Gene name Primer sequences(5'-3')
EZH2 Forward: CATCGATTTCTTTCCCTGTGAA
Reverse: TCTTGGAGCTTATTAAAGGCATTC
E-cad Forward: GCCATCGCTTACACCATCCTCAG
Reverse: CTCTCTCGGTCCAGCCCAGTG
Vim Forward: CCTTCGTGAATACCAAGACCTGCTC
Reverse: AATCCTGCTCTCCTCGCCTTCC
GAPDH Forward: GTCTCCTCTGACTTCAACAGCG
Reverse: ACCACCCTGTTGCTGTAGCCAA
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EZH2: Enhancer of zeste homolog 2; E-cad: E-cadherin; Vim: Vimentin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

1.8. CCK-8实验

将转染后各组EC109、EC9706细胞以3 000个/孔的密度接种于96孔板中，孵育6 h后，弃去各孔培养基，每孔加入100 μL完全培养基、10 μL CCK-8试剂。孵育2 h后，分别在第0、24、48、72 h时测定各孔450 nm波长处的吸光度值。

1.9. 细胞划痕实验

将转染后各组EC109、EC9706细胞以1×106个/孔的密度接种于6孔板中，当细胞融合率达到单层100%时，用100 μL无酶枪头垂直于孔板划线，每孔平行划取3条竖线。在第0、24、48 h时用显微镜(4×)拍照取图。通过ImageJ软件测量划痕宽度，并计算细胞迁移率=(初始划痕宽度-最终划痕宽度)/初始划痕宽度×100%。

1.10. Transwell侵袭及迁移实验

Transwell侵袭实验：将基质胶与无血清培养基按1꞉8的比例混合，吸取50 μL稀释后的基质胶，垂直加入Transwell小室。将小室放置在37 ℃、5%CO2培养箱中孵育2 h固化。弃去多余液体后，每孔加入200 μL无血清培养基，检查是否漏液，于37 ℃、5% CO2培养箱中继续孵育30 min使得基底膜水化。将转染后各组EC109、EC9706细胞以2.5×105个/孔的密度接种于无血清培养基的上室，下室加入含20%血清的完全培养基，培养48 h后，使用磷酸盐缓冲液(phosphate buffered saline，PBS)清洗3遍，放置于4%多聚甲醛溶液中固定，使用结晶紫染色后，置于显微镜(10×)下观察、拍照，采用ImageJ软件计数。

Transwell迁移实验：将转染后各组EC109、EC9706细胞以2.5×105个/孔的密度接种无血清培养基的上室，下室加入含20%血清的完全培养基，培养24 h后，用PBS清洗3遍，放置于4%多聚甲醛溶液中固定，用结晶紫染色后，置于显微镜(10×)下观察、拍照，用ImageJ软件计数。

1.11. 免疫组织化学染色

ESCC组织及癌旁组织切片放入60 ℃烘箱中烤30 min，经过二甲苯和乙醇脱水、乙二胺四乙酸(ethylenediaminetetra-acetic acid，EDTA)高温修复、过氧化氢孵育10 min后，加入EZH2(稀释比1꞉50)、E-cad(稀释比1꞉10 000)、Vim(稀释比1꞉12 000)一抗，放置在4 ℃冰箱中孵育过夜后，使用PBS清洗3遍，再加入二抗孵育30 min，加入二氨基联苯胺(diaminobenzidine，DAB)显色6 min，苏木精染色 3 min、盐酸-乙醇分色5 s，自来水返蓝5 min后使用乙醇脱水，封片。在显微镜(400×)下选取5个不同视野，细胞核、细胞质或细胞膜出现黄色或棕黄色为阳性。染色强度评分：无着色为0分，弱阳性为1分，中等阳性为2分，强阳性为3分。阳性细胞比例评分：无阳性肿瘤细胞为0分，1%~25%为1分，26%~50%为2分，51%~75%为3分，76%~100%为4分。最终分值=染色强度评分×阳性细胞比例评分，其中<4分计为阴性，≥4分计为阳性。

1.12. 统计学处理

采用SPSS26.0软件对数据进行统计学处理，以 P<0.05为差异有统计学意义。计量资料以均数±标准差表示。2组间的比较采用t检验，2组以上的比较采用方差分析。使用Cox比例风险模型来评估数据库中EZH2高、低表达组与生存期之间的关系。采用卡方检验分析ESCC组织中EZH2的表达阳性率及不同临床特征ESCC患者癌组织中EZH2、E-cad、Vim的表达水平。采用Spearman相关性系数分析E-cad、Vim在ESCC组织中的表达水平与EZH2之间的关系。采用Kaplan-Meier法分析EZH2、E-cad、Vim与ESCC患者生存期之间的关系。

2. 结 果

2.1. EZH2 在ESCC中高表达且与预后不良相关

通过TCGA的泛癌分析发现EZH2在大部分类型的肿瘤组织中均有表达，且其mRNA水平高于癌旁组织(图1A)。进一步分析TCGA数据库中EC患者的数据发现EZH2在ESCC组织中的表达水平显著高于癌旁组织(P<0.001，图1B)。在GEO数据库GSE53625和GSE161533数据集中得到同样的结果 (P<0.001，图1C)。Cox比例风险模型生存分析结果显示在ESCC患者中，EZH2高表达组相比低表达组的预后更差(P<0.001，图1D)。

图1.

图1

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TCGA数据库检测ESCC组织中 EZH2 的表达

Figure 1 TCGA database detects EZH2 expression in ESCC tissues

A: Expression of EZH2 in each tumor in TCGA database; B: Expression of EZH2 in ESCC and CAN tissues and pairing difference analysis in TCGA database; C: Expression of EZH2 in ESCC and CAN tissues in GSE53625 and GSE161533 datasets; D: Survival analysis of EZH2 high and low expression groups. ***P<0.001. TCGA: The Cancer Genome Atlas; EZH2: Enhancer of zeste homolog 2; ESCC: Esophageal squamous cell carcinoma; CAN: Cancer adjacent normal; TPM: Transcripts per million; ACC: Adrenocortical carcinoma; BLCA: Bladder urothelial carcinoma; BRCA: Breast invasive carcinoma; CESC: Cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL: Cholangiocarcinoma; COAD: Colon adenocarcinoma; DLBC: Lymphoid neoplasm diffuse large B-cell lymphoma; ESCA: Esophageal carcinoma; GBM: Glioblastoma multiforme; HNSC: Head and neck squamous cell carcinoma; KICH: Kidney chromophobe; KIRC: Kidney renal clear cell carcinoma; KIRP: Kidney renal papillary cell carcinoma; LGG: Lower-grade gliomas; OV: Ovarian serous cystadenocarcinoma; MESO: Mesothelioma; LIHC: Liver hepatocellular carcinoma; LUAD: Lung adenocarcinoma; LUSC: Lung squamous cell carcinoma; PAAD: Pancreatic adenocarcinoma; PRAD: Prostate adenocarcinoma; PCPG: Pheochromocytoma & paraganglioma; READ: Rectum adenocarcinoma; SARC: Sarcoma; SKCM: Skin cutaneous melanoma; LAML: Acute myeloid leukemia; TGCT: Testicular germ cell tumor; THCA: Thyroid carcinoma; THYM: Thymoma; STAD: Stomach adenocarcinoma; UCEC: Uterine corpus endometrioid carcinoma; UCS: Uterine carcinosarcoma; UVM: Uveal melanoma.

2.2. 敲减 EZH2 能抑制ESCC细胞的增殖、侵袭及迁移能力

CCK-8实验结果表明：敲减EZH2基因后，2种ESCC细胞的增殖能力均减弱(均P<0.001，图2A)。Transwell实验结果表明：敲减EZH2基因后，2种ESCC细胞的侵袭及迁移能力均减弱(均P<0.001， 图2B)。细胞划痕实验结果表明：敲减EZH2基因后，2种ESCC细胞的迁移率均降低(均P<0.001，图2C)。

图2.

图2

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敲减 EZH2 表达可抑制ESCC细胞的增殖、侵袭及迁移能力

Figure 2 Knockdown of EZH2 inhibits the proliferation, invasion, and migration of ESCC

A: Proliferation abilities of EC109 and EC9706 cells in siNC, siEZH2#1, and siEZH2#2 groups were assessed using CCK-8 assay. B: Invasion and migration abilities of EC109 and EC9706 cells in each group were determined using Transwell assay. C: Migration ability of EC109 and EC9706 cells in each group was determined using cell scratch assay. ***P<0.001. siNC: Cells transfected with small interfering RNA negative control; siEZH2#1: Cells transfected with small interfering EZH2-Homo-1743; siEZH2#2: Cells transfected with small interfering EZH2-Homo-1952; CCK-8: Cell counting kit-8.

2.3. 敲减 EZH2 能抑制ESCC细胞的EMT

实时荧光定量PCR和蛋白质印迹法结果(图3)显示：敲减EZH2基因后，2种ESCC细胞中上皮标志物E-cad的蛋白质和mRNA表达水平均显著上调(均 P<0.05)，而间充质标志物Vim的蛋白质和mRNA表达水平均显著下调(均P<0.05)。

图3.

图3

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敲减 EZH2 可抑制ESCC中EZH2、E-cad、Vim的蛋白质和mRNA表达

Figure 3 Knockdown of EZH2 inhibits protein and mRNA expression of EZH2, E-cad, and Vim in ESCC

A: Western blotting was used to detect the protein expressions of E-cad, EZH2, and Vim after knockdown of EZH2 in EC109 and EC9706 cells. B: Real time fluorogenic quantitative PCR was used to detect the mRNA expressions of EZH2, E-cad, and Vim after knockdown of EZH2 in EC109 and EC9706 cells. *P<0.05, **P<0.01, ***P<0.001. E-cad: E-cadherin; Vim: Vimentin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

2.4. ESCC组织中EZH2、E-cad和Vim的蛋白质表达情况及其相关性

免疫组织化学染色结果(图4A)显示：EZH2主要表达在ESCC细胞的细胞核中，E-cad主要表达在ESCC细胞的细胞膜上，而Vim主要表达在ESCC细胞的细胞质中；与癌旁组织相比，ESCC组织中EZH2和Vim的表达水平较高，而E-cad的表达水平较低；ESCC组织中EZH2的表达阳性率为60.0%(42/70)，明显高于癌旁组织的32.5%(13/40)，差异有统计学意义(P=0.000 6)。Spearman相关性分析结果(图4B)显示：E-cad的表达与EZH2呈负相关(r=-0.262，P=0.029)，而Vim的表达与EZH2呈正相关(r=0.438，P=0.000 12)。

图4.

图4

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ESCC组织中EZH2、Vim、E-cad的蛋白质表达及相关性分析

Figure 4 Protein expression and correlation analysis of EZH2, Vim, and E-cad in ESCC tissues

A: Immunohistochemical staining showing the protein expression of EZH2, E-cad, and Vim in ESCC and CAN tissues; B: Spearman correlation analysis of the correlation between EZH2 and E-cad or Vim in ESCC.

2.5. 不同临床特征ESCC患者癌组织中EZH2、

E-cad、Vim的表达情况

分析不同临床特征的ESCC患者癌组织中EZH2、E-cad、Vim的表达水平(表2)发现：浸润深度≥3 cm的ESCC患者相比于浸润深度<3 cm的患者，其EZH2表达阳性率更高(P=0.042)；肿瘤淋巴结转移的ESCC患者相比于无转移的患者，其癌组织中Vim表达阳性率更高(P=0.011)；肿瘤淋巴管浸润的ESCC患者相比于无浸润的患者，其癌组织中Vim表达阳性率更高(P=0.007)；临床分期更高(III~IV期)的ESCC患者相比于临床分期更低(I~II期)的患者，其癌组织中EZH2表达阳性率更高(P=0.008)。提示EZH2高表达和EMT与ESCC的浸润、转移、临床分期密切相关。

表2.

不同临床特征ESCC患者癌组织中EZH2、E-cad、Vim的表达情况

Table 2 Expressions of EZH2, E-cad, and Vim in cancer tissues of ESCC patients with different clinical characteristics

Clinical characteristics n E-cad/[n(%)] P Vim/[n(%)] P EZH2/[n(%)] P
Negative Positive Negative Positive Negative Positive
Age/Years
<60 43 22(51.2) 21(48.8) 0.076 23(53.5) 20(46.5) 0.160 15(34.9) 28(65.1) 0.270
≥60 27 8(29.6) 19(70.4) 19(70.4) 8(29.6) 13(48.1) 14(51.9)
Gender
Male 48 22(45.8) 26(54.2) 0.457 30(62.5) 18(37.5) 0.528 18(37.5) 30(62.5) 0.528
Female 22 8(36.4) 14(63.6) 12(54.5) 10(45.5) 10(45.5) 12(54.5)
Differentiation
Low 22 8(36.4) 14(63.6) 0.129 14(63.6) 8(36.4) 0.877 11(50.0) 11(50.0) 0.065
Moderate 37 16(43.2) 21(56.8) 22(59.5) 15(40.5) 16(43.2) 21(56.8)
High 11 8(72.7) 3(27.3) 6(54.5) 5(45.5) 1(9.1) 10(90.9)
Infiltration depth/cm
<3 25 13(52.0) 12(48.0) 0.515 15(60.0) 10(40.0) 0.856 14(56.0) 11(44.0) 0.042
≥3 45 27(60.0) 18(40.0) 27(60.0) 18(40.0) 14(31.1) 31(68.9)
Lymph node metastasis
No 38 14(36.8) 24(63.2) 0.268 28(73.7) 10(26.3) 0.011 17(44.7) 21(55.3) 0.378
Yes 32 16(50.0) 16(50.0) 14(43.8) 18(56.2) 11(34.4) 21(65.6)
Lymphatic vessel invasion
No 20 6(30.0) 14(70.0) 0.169 17(85.0) 3(15.0) 0.007 9(45.0) 11(55.0) 0.589
Yes 50 24(48.0) 26(52.0) 25(50.0) 25(50.0) 19(38.0) 31(62.0)
Clinical stage
I~II stage 39 13(33.3) 26(66.7) 0.071 26(66.7) 13(33.3) 0.203 21(53.8) 18(46.2) 0.008
III~IV stage 31 17(54.8) 14(45.2) 16(51.6) 15(48.4) 7(22.6) 24(77.4)
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ESCC: Esophageal squamous cell carcinoma.

2.6. E-cad低表达和Vim、EZH2高表达与ESCC患者的较差预后相关

Kaplan-Meier生存分析结果(图5)显示：E-cad高表达患者的预后相比于E-cad低表达患者更好(P=0.031 7)，而EZH2(P=0.043 4)、Vim(P=0.040 8)高表达患者的预后相比于低表达的患者更差。提示EZH2高表达和EMT与ESCC患者的较差预后相关。

图5.

图5

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Kaplan-Meier生存曲线分析 EZH2 、 E-cad 、 Vim 对ESCC患者预后的影响

Figure 5 Kaplan-Meier survival analysis was used to analyze the effects of EZH2, E-cad, and Vim on the prognosis of ESCC patients

3. 讨 论

ESCC是一种常见的消化道恶性肿瘤，主要发生在东亚地区，其中中国的发病率和病死率均较高[13-15]。ESCC发病隐匿，病情进展快，一经发现多为晚期，导致其5年生存率只有30%[16]。因此，深入探究ESCC的发病机制对于提升治疗效果和患者生存率至关重要。

EZH2参与多种生物学过程，如细胞周期、转录调控、代谢和免疫调节[17]。EZH2的过表达或功能异常可能导致抑癌基因的沉默或致癌基因的激活，从而促进肿瘤的发生和发展[18]。已有研究[19]表明：EZH2在多种癌症中表达水平上调，包括前列腺癌、乳腺癌和卵巢癌等，与肿瘤的增殖、转移、侵袭、分级、耐药、不良预后等密切相关。既往研究[20]表明：EZH2在ESCC中的表达水平显著升高，并与ESCC的侵袭和转移密切相关。EZH2可能是调控ESCC发展的关键分子。本研究的结果与这些发现一致，也观察到ESCC组织中EZH2的表达水平高于癌旁组织，且与ESCC的浸润深度、临床分期及不良预后相关。本研究通过CCK-8实验、细胞划痕实验、Transwell侵袭与迁移实验，进一步证实了敲减EZH2能够显著抑制ESCC细胞的增殖、侵袭和迁移能力。这些发现为未来的治疗策略提供了潜在的分子靶点。

EMT是胚胎发育和器官形成的关键生物学过程，可促使极化的上皮细胞失去其上皮特性并获取间充质特征。在生理状态下，EMT过程是可逆的，但在病理状态下，EMT的异常激活能够赋予上皮源性癌细胞迁移和侵袭的能力[21]。这一去分化过程通常涉及多种转录因子的调控网络。现有研究[22-23]表明：EZH2在EMT中的作用与多个转录因子密切相关，包括SNAIL、TWIST和ZEB2等，这些转录因子在EMT中扮演核心角色，且EZH2能够通过调节转录因子的活性或表达水平来影响EMT的进程。然而，EZH2在ESCC中调控EMT的具体机制仍待进一步阐明。本研究发现EZH2的敲减能够显著增高E-cad的表达水平，同时降低Vim的表达水平，并且E-cad的表达与EZH2呈负相关，而Vim的表达与EZH2呈正相关。这些结果表明，EZH2可能通过促进ESCC中的EMT过程，从而增强ESCC细胞的增殖、侵袭和迁移能力。此外，Kaplan-Meier生存分析显示，EZH2高表达的患者具有较差的预后。基于这些发现，EZH2可能通过促进EMT过程影响ESCC的恶性生物学行为及患者的临床预后。

EZH2与EMT均被认为是肿瘤发生和发展的重要因素，部分学者[24]提出EZH2通过调控EMT相关基因(如E-cad、Vim等)对肿瘤细胞的特性和功能产生影响。本研究运用生物信息学技术对ESCC中的EZH2表达水平进行深入分析，并在体外实验中验证了EZH2对ESCC细胞的增殖、侵袭、迁移等多方面的影响。此外，本研究进一步揭示了EZH2和EMT之间的正向调控关系，并通过ESCC临床样本评估了EZH2与ESCC的不同临床特征、EMT关键分子之间的关联性，同时对患者的生存预后进行了深入分析。

然而，本研究也存在局限性。首先，尽管已有研究[25-26]表明EZH2能够促进癌细胞的恶性转移，并调控EMT关键分子的表达，与本研究的结果一致，但本研究的结果是基于70例ESCC组织和40例癌旁组织得出，样本量相对较小，可能存在一定的偏差，因此后续研究需要扩大样本规模，以提高研究的代表性和可靠性。其次，本研究未深入探讨EZH2在ESCC中的具体分子机制，仅在细胞实验中对EZH2进行敲减处理后，检测EMT及恶性生物学行为的变化，未来应考虑进行EZH2的过表达及回复实验，以便更深入地阐明EZH2如何通过影响EMT进程参与ESCC的发生和发展。此外，EZH2同时具有组蛋白甲基化酶的作用，能够催化组蛋白H3第27位赖氨酸残基(histone H3 lysine 27，H3K27)的甲基化以调控抑癌基因的表达[27]，可能也是参与ESCC发生和发展的潜在机制。对于EZH2与EMT之间的具体分子机制，如其上下游信号通路的相互作用与反馈调节等，也需要更深入地探索，以全面揭示EZH2与EMT在ESCC发病机制中的确切作用和调控机制。

综上所述，本研究揭示了EZH2在ESCC中高表达，并且通过调控EMT进程增强ESCC细胞的增殖、侵袭和迁移能力。针对EZH2引发的EMT可能成为改善ESCC患者预后的有效策略。这些发现为深入理解ESCC的发生和发展机制及开发潜在的治疗靶点提供了重要的理论基础，同时也为未来的研究指明了方向。

基金资助

国家自然科学基金(82460597)；消化道早癌医师共同成长计划科研项目(GTCZ-2023-XJ-02)；兵团指导性科技计划项目(2022ZD003，2023AB058)；石河子大学基础研究培育计划项目(MSPY202407)；石河子大学自主资助科研创新项目(ZZZC2023029)；石河子大学大学生研究训练计划项目(SRP2024459)。This work was supported by the National Natural Science Foundation (82460597), the Gastrointestinal Early Cancer Physician Common Growth Plan Research Project (GTCZ-2023-XJ-02), the Guiding Scientific and Technological Research Project in the Xinjiang Production and Construction Corps (2022ZD003 and 2023AB058), the Basic Research Project of Shihezi University (MSPY202407), the Shihezi University Independently Funded Research and Innovation Project (ZZZC2023029), and the Shihezi University Undergraduate Research Training Program (SRP2024459), China.开放获取(Open access)：本文遵循知识共享许可协议，允许第三方用户按照署名-非商业性使用-禁止演绎4.0(CC BY-NC-ND 4.0)的方式，在任何媒介以任何形式复制、传播本作品(https://creativecommons.org/licenses/by-nc-nd/4.0/)。

利益冲突声明

作者声称无任何利益冲突。

作者贡献

井玉莹 研究设计，数据分析，论文撰写与修改；杨凯歌、胡建明 研究设计，论文指导与修改；程仪婷，黄田平 数据采集；陈素芳，陈凯 研究设计，论文修改。所有作者阅读并同意最终的文本。

Footnotes

http://dx.chinadoi.cn/

原文网址

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202502155.pdf

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