Association of Plasma EPB41L3 Gene Expression and Methylation with the Risk of Developing Colorectal Cancer
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摘要:
目的 探讨血浆EPB41L3基因表达水平及甲基化状态与结直肠癌患者患病风险的关联性，分析其作为结直肠癌早期筛查及风险评估生物标志物的潜在价值。 方法 选取2022年5月至2025年10月期间于江苏省人民医院宿迁医院确诊的210例结直肠癌患者作为病例组，并以同期健康体检者207例作为对照组。采集外周血血浆样本，运用qRT-PCR技术测定EPB41L3基因mRNA的表达量；采用重亚硫酸盐转化联合甲基化特异性荧光定量PCR法（MethyLight）对EPB41L3基因启动子区甲基化状态进行定性检测，通过比较样本与甲基化阳性对照的Ct值判定甲基化阳性或阴性；采用免疫印迹技术对血浆样本中的EPB41L3蛋白表达量进行测定；分析病例组与对照组在EPB41L3基因表达水平及启动子区甲基化状态上的差异；通过多因素Logistic回归模型分析EPB41L3基因表达异常及甲基化状态与结直肠癌患病风险的独立关联；分析不同临床特征对结直肠癌患者EPB41L3基因表达及启动子区甲基化状态的差异及其影响，评估相关单项及联合指标对结直肠癌的诊断效能。 结果 病例组中EPB41L3基因甲基化阳性率显著高于对照组，血浆中EPB41L3基因mRNA的相对表达量以及EPB41L3蛋白的相对表达量均显著降低（均P < 0.05）。EPB41L3基因mRNA高表达和EPB41L3蛋白高表达均与结直肠癌发病风险降低显著相关（P < 0.05）；EPB41L3基因甲基化阳性与结直肠癌患病风险增高显著相关（P < 0.05）。EPB41L3基因mRNA和蛋白表达水平在临床分期晚（Ⅲ～Ⅳ期）、低分化及有淋巴结转移的肿瘤患者中显著降低，其甲基化阳性率在相应组别中显著升高（均P < 0.05）。EPB41L3基因甲基化、mRNA表达和蛋白表达三者联合检测对结直肠癌及早期结直肠癌（Ⅰ～Ⅱ 期）显示良好的诊断价值，AUC分别为0.913和0.894，表现出良好的灵敏度和特异度。 结论 血浆EPB41L3基因的高甲基化状态及其表达下调与结直肠癌的发病风险增加及不良临床病理特征密切相关。将这三者进行联合检测，在结直肠癌（包含早期结直肠癌）的诊断方面有着重要的潜在辅助作用。 Abstract:Objective To investigate the correlation between plasma EPB41L3 gene expression level and methylation status with colorectal cancer （CRC）, and to evaluate its potential as a biomarker for early screening and risk assessment of CRC. Methods A total of 210 colorectal cancer patients diagnosed between May 2022 and October 2025 at Suqian Hospital affiliated with Jiangsu Province People’s Hospital were enrolled as the case group, with 207 healthy individuals from the same period as the control group. Peripheral blood plasma samples were collected. The expression level of EPB41L3 gene mRNA was measured using quantitative real-time PCR （qRT-PCR）. The methylation status of the EPB41L3 gene promoter region was qualitatively detected using bisulfite conversion combined with methylation-specific fluorescent quantitative PCR （MethyLight）. Methylation positivity or negativity was determined by comparing the Ct values of samples with methylation-positive controls. Plasma EPB41L3 protein expression was measured using Western blotting. Differences in EPB41L3 expression and promoter methylation between the case and control groups were compared. A multivariate logistic regression model was used to analyze the independent association of EPB41L3 expression and methylation with CRC risk. Differences in EPB41L3 gene expression and promoter region methylation status across different clinical characteristics and their effects were analyzed, and the diagnostic efficacy of individual and combined indicators for colorectal cancer was evaluated. Results The positivity rate of EPB41L3 methylation was significantly higher in the case group than in the control group, while the relative expression levels of EPB41L3 mRNA and protein were significantly lower in the case group （all P < 0.05）. High expression of EPB41L3 gene mRNA and high expression of EPB41L3 protein were both significantly associated with decreased colorectal cancer risk （P < 0.05）. Positive methylation of EPB41L3 gene was significantly associated with increased colorectal cancer risk （P < 0.05）. EPB41L3 mRNA and protein expression levels were significantly lower in patients with advanced clinical stage （III–IV）, poor differentiation, and lymph node metastasis, while the methylation positivity rate was significantly higher in the corresponding groups （all P < 0.05）. Combined detection of EPB41L3 methylation, mRNA expression, and protein expression showed good diagnostic value for CRC and early-stage CRC （stage I–II） with AUC values of 0.913 and 0.894, respectively, showing good sensitivity and specificity. Conclusion Hypermethylation and downregulation of EPB41L3 in plasma are closely associated with an increased risk of CRC and adverse clinicopathological features. Combined detection of these three markers has important potential auxiliary value in the diagnosis of colorectal cancer, including early-stage colorectal cancer. -
Key words:
- Colorectal cancer /
- EPB41L3 gene /
- Methylation /
- Disease risk
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图 1 两组典型病例图片（HE染色）
A：病例组，镜下可见淋巴结结构被密集的肿瘤细胞巢破坏，肿瘤细胞呈浸润性生长，与周围正常淋巴组织界限不清，符合结直肠癌淋巴结转移的典型病理特征；B：对照组，黏膜上皮排列规则，隐窝结构完整，细胞形态一致，无明显异型性及浸润性生长
Figure 1. Typical cases of the two groups （HE staining）
图 2 病例组与对照组EPB41L3基因mRNA表达水平比较
*P < 0.05。
Figure 2. Comparison of EPB41L3 gene mRNA expression levels between the case group and the control group
图 3 结直肠癌病例组与健康对照组血浆EPB41L3蛋白表达水平的Western blot检测结果
A：两组各12例代表性样本的蛋白条带图；B：两组全部样本蛋白相对表达量的定量柱状图；病例组与对照组相比，*P < 0.05。
Figure 3. Western blot analysis of plasma EPB41L3 protein expression levels in colorectal cancer case group and healthy control group
图 4 EPB41L3基因相关指标对结直肠癌的诊断效能评估
Figure 4. Diagnostic performance evaluation of EPB41L3 gene-related indicators for colorectal cancer
图 5 EPB41L3 基因相关指标对结直肠癌（早期：Ⅰ～Ⅱ 期）的诊断效能评估
Figure 5. Assessment of the diagnostic performance of EPB41L3-associated biomarkers in early-stage （stage I–II） colorectal cancer
表 1 qRT-PCR引物序列信息
Table 1. qRT-PCR primer sequences
基因名称 引物类型 引物序列（5′→3′） 扩增片段长度（bp） 退火温度（℃） EPB41L3 上游 AGCCTCAACGACCAGATCCA 156 60 EPB41L3 下游 TGTAGCCGACTCCGTCTTC 156 60 GAPDH 上游 GTCTCCTCTGACTTCAACAGCG 138 60 GAPDH 下游 ACCACCCTGTTGCTGTAGCCAA 138 60 
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表 2 MethyLight法引物序列信息
Table 2. Primer sequence for MethylLight method
基因名称 引物类型 引物序列（5′→3′） 扩增片段长度（bp） 退火温度（℃） 靶向区域（GRCh38） EPB41L3 上游 GGTTTTTTGGGTTGTTTAGTTTTTC 110 60 启动子区（−350至−240） EPB41L3 下游 CAAAAACTAAAAACCCCAAACCTC 110 60 启动子区（−350至−240） ACTB（内参） 上游 TGGTGATGGAGGAGGTTTAGTAAGT 133 60 − ACTB（内参） 下游 AACCAATAAAACCTACTCCCTCCCTTAA 133 60 −
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表 3 研究对象基线资料比较[（$\bar x \pm s $）/n（%）]
Table 3. Comparison of baseline characteristics[（$\bar x \pm s $）/n（%）]
基线资料 病例组
（n = 210）对照组
（n = 207）t/χ2 P 年龄（岁） 62.43 ± 10.87 61.89 ± 9.76 0.534 0.594 性别 0.108 0.742 男 118（56.19） 113（54.59） 女 92（43.81） 94（45.41） 吸烟史 0.252 0.616 是 78（37.14） 72（34.78） 否 132（62.86） 135（65.22） 饮酒史 0.013 0.909 是 65（30.95） 63（30.43） 否 145（69.05） 144（69.57） BMI（kg/m2） 24.16 ± 3.42 23.87 ± 3.05 0.913 0.362 肿瘤家族史 16.549 < 0.001* 是 46（21.90） 16（7.73） 否 164（78.10） 191（92.27） *P < 0.05。
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表 4 两组EPB41L3基因甲基化状态比较[n（%）]
Table 4. Comparison of EPB41L3 gene methylation status between the two groups[n（%）]
CpG位点 病例组
（n = 210）对照组
（n = 207）χ2 P EPB41L3基因
甲基化142.026 < 0.001* 阳性 152（72.38） 30（14.49） 阴性 58（27.62） 177（85.51） *P < 0.05。
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表 5 EPB41L3基因表达及甲基化状态与结直肠癌患病风险的多因素Logistic回归分析
Table 5. Multivariate Logistic regression analysis of EPB41L3 gene expression and methylation status associated with colorectal cancer risk
变量 β SE Wald χ2 P OR 95%CI 模型1 EPB41L3基因mRNA表达 −1.78 0.24 54.08 < 0.001* 0.17 0.11～0.27 EPB41L3蛋白表达 −1.23 0.22 31.27 < 0.001* 0.29 0.19～0.45 EPB41L3基因甲基化阳性 2.69 0.25 113.76 < 0.001* 14.76 8.99～24.23 模型2 < 0.001* EPB41L3基因mRNA表达 −1.55 0.26 35.52 < 0.001* 0.21 0.13～0.36 EPB41L3蛋白表达 −1.14 0.24 22.56 < 0.001* 0.32 0.20～0.51 EPB41L3基因甲基化阳性 2.42 0.27 80.36 < 0.001* 11.25 6.61～19.14 模型1：未校正混杂因素；模型2：校正年龄、性别、吸烟史、饮酒史、BMI、肿瘤家族史等；*P < 0.05。
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表 6 EPB41L3基因相关指标对结直肠癌的诊断效能评估
Table 6. Diagnostic performance evaluation of EPB41L3 gene-related indicators for colorectal cancer
检测指标 AUC（95%CI） P 灵敏度（%） 特异度（%） 阳性预测值（%） 阴性预测值（%） EPB41L3基因mRNA表达 0.771（0.716～0.826） < 0.001* 75.64 76.21 76.35 75.50 EPB41L3蛋白表达 0.785（0.734～0.836） < 0.001* 77.12 78.45 78.60 76.97 EPB41L3基因甲基化阳性 0.792（0.759～0.871） < 0.001* 78.26 81.50 79.65 80.11 甲基化+mRNA表达 0.850（0.808～0.892） < 0.001* 82.31 84.17 84.29 82.18 甲基化+蛋白表达 0.855（0.814～0.896） < 0.001* 83.59 85.26 85.39 83.45 甲基化+mRNA表达+蛋白表达 0.913（0.838～0.988） < 0.001* 90.13 91.42 90.55 91.01 *P < 0.05。
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表 7 EPB41L3基因表达与结直肠癌临床病理特征的关系[（$\bar x \pm s $）/n（%）]
Table 7. Correlation of EPB41L3 expression with clinicopathological characteristics in colorectal cancer[（$\bar x \pm s $）/n（%）]
临床特征 n mRNA 相对表达量 蛋白相对表达量 EPB41L3基因甲基化阳性（%） 年龄（岁） < 65 116 0.37 ± 0.06 0.29 ± 0.07 82（70.69） ≥65 94 0.38 ± 0.11 0.28 ± 0.08 70（74.47） t/χ2 − 0.838 0.965 0.371 P − 0.403 0.335 0.543 性别 男 118 0.34 ± 0.05 0.26 ± 0.06 87（73.73） 女 92 0.35 ± 0.08 0.27 ± 0.09 65（70.65） t/χ2 − 1.109 0.963 0.090 P − 0.269 0.336 0.764 肿瘤家族史 是 46 0.31 ± 0.09 0.27 ± 0.07 33（71.74） 否 164 0.34 ± 0.11 0.29 ± 0.08 119（72.56） t/χ2 − 1.696 1.538 0.012 P − 0.091 0.126 0.912 肿瘤分期 Ⅰ～Ⅱ期 107 0.31 ± 0.08 0.27 ± 0.08 67（62.62） Ⅲ～Ⅳ期 103 0.24 ± 0.06 0.20 ± 0.06 85（82.52） t/χ2 − 7.152 7.152 10.404 P − < 0.001* < 0.001* < 0.001* 分化程度 高、中分化 132 0.33 ± 0.11 0.29 ± 0.09 81（61.36） 低分化 78 0.26 ± 0.08 0.22 ± 0.05 71（91.03） t/χ2 − 4.904 6.314 21.578 P − < 0.001* < 0.001* < 0.001* 淋巴结转移 t/χ2 83 0.24 ± 0.05 0.21 ± 0.07 69（83.13） P 127 0.36 ± 0.10 0.27 ± 0.08 83（65.35） t/χ2 − 10.130 5.578 7.936 P − < 0.001* < 0.001* 0.005* *P < 0.05。
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表 8 EPB41L3 基因相关指标对早期结直肠癌（Ⅰ～Ⅱ 期）的诊断效能评估
Table 8. Diagnostic performance evaluation of EPB41L3 gene-related indicators for early-stage （Ⅰ–Ⅱ） colorectal cancer
检测指标 AUC（95%CI） P 灵敏度（%） 特异度（%） 阳性预测值（%） 阴性预测值（%） EPB41L3基因mRNA表达 0.732（0.684～0.840） < 0.001* 72.10 74.02 73.52 74.67 EPB41L3蛋白表达 0.753（0.693～0.856） < 0.001* 74.67 75.06 74.51 76.27 EPB41L3基因甲基化阳性 0.761（0.670～0.832） < 0.001* 77.24 80.57 78.95 79.88 甲基化+mRNA表达 0.842（0.720～0.934） < 0.001* 79.43 81.34 80.48 80.30 甲基化+蛋白表达 0.848（0.734～0.942） < 0.001* 80.48 79.37 80.47 80.39 甲基化+mRNA表达+蛋白表达 0.894（0.779～0.953） < 0.001* 88.29 90.72 89.75 89.26 *P < 0.05。
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[1] Wu S, Zhang Y, Lin Z, et al. Global burden of colorectal cancer in 2022 and projections to 2050: Incidence and mortality estimates from GLOBOCAN[J]. BMC Cancer, 2025, 25(1): 1770. doi: 10.1186/s12885-025-15138-0 [2] Yin Y, Zhang X. Analysis of trends in the burden of colorectal cancer in China and globally from 1990 to 2021 with projections for the next 15 years: A cross-sectional study based on the GBD database[J]. Front Public Health, 2025, 13: 1518536. doi: 10.3389/fpubh.2025.1518536 [3] Alsaialy D, Kulhanova I, Lustigova M. Barriers to colorectal cancer screening: Insights and opportunities for enhanced participation[J]. Eur J Public Health, 2025, 35(Supplement_4): ckaf161.510. doi: 10.1093/eurpub/ckaf161.510 [4] Zarrinkamar M, Ebrahimi M, Zahedi M, et al. Prevalence, determinants, and barriers to follow-up colonoscopy after a positive fecal immunochemical test in Qaemshahr, Iran: A population-based cross-sectional study from 2022 to 2023[J]. Prev Med Rep, 2026, 61: 103331. doi: 10.1016/j.pmedr.2025.103331 [5] Lakatos E, Gunasri V, Zapata L, et al. Epigenetically driven and early immune evasion in colorectal cancer evolution[J]. Nat Genet, 2025, 57(12): 3039-3049. doi: 10.1038/s41588-025-02349-1 [6] Qian S, Song H, Huang L, et al. DNA, RNA, and histone methylation regulation enzymes and their crosstalk in colorectal carcinogenesis and progression: A review of molecular mechanisms, clinical implications, and future perspectives[J]. Cell Mol Biol Lett, 2025, 30(1): 142. doi: 10.1186/s11658-025-00823-6 [7] 旦真甲, 曹钦兴, 严力, 等. DNA甲基化生物标志物在结直肠癌早期诊断与治疗中的应用进展[J]. 实用医院临床杂志, 2024, 21(4): 176-180. [8] Hajebi Khaniki S, Shokoohi F. Data-driven identification of early cancer-associated genes via penalized trans-dimensional hidden Markov models[J]. Biomolecules, 2025, 15(2): 294. doi: 10.3390/biom15020294 [9] Werren E A, Rodriguez Bey G, Majethia P, et al. Biallelic EPB41L3 variants underlie a developmental disorder with seizures and myelination defects[J]. Brain, 2024, 147(12): 4033-4042. doi: 10.1093/brain/awae299 [10] 张永涛. miR-181b-5P和EPB41L3蛋白在喉癌中的表达及临床意义[J]. 实用癌症杂志, 2025, 40(4): 548-550. [11] Molano M, Machalek D A, Phillips S, et al. DNA methylation at individual CpG-sites of EPB41L3, HTERT and FAM19A4 are useful for detection of cervical high-grade squamous intraepithelial lesions (HSIL) or worse: Analysis of individual CpG-sites outperforms averaging[J]. Tumour Virus Res, 2024, 18: 200288. doi: 10.1016/j.tvr.2024.200288 [12] 国家卫生健康委员会医政司, 中华医学会肿瘤学分会. 国家卫健委中国结直肠癌诊疗规范(2023版)[J]. 中国实用外科杂志, 2023, 43(6): 602-630. [13] Bustin S A, Ruijter J M, van den Hoff M J B, et al. MIQE 2.0: Revision of the minimum information for publication of quantitative real-time PCR experiments guidelines[J]. Clin Chem, 2025, 71(6): 634-651. doi: 10.1093/clinchem/hvaf043 [14] He C, Huang Q, Zhong S, et al. Screening and identifying of biomarkers in early colorectal cancer and adenoma based on genome-wide methylation profiles[J]. World J Surg Oncol, 2023, 21(1): 312. doi: 10.1186/s12957-023-03189-1 [15] 贾子琪, 林耿斌, 袁文静, 等. 基于MRI影像组学评估局部进展期直肠癌新辅助放化疗反应: 一项两中心多设备研究[J]. 中国临床医学影像杂志, 2025, 36(3): 194-199. doi: 10.12117/jccmi.2025.03.009 [16] 谢媛媛, 许禹, 邓亚男. 不可手术治疗的局部晚期直肠癌患者根治性放化疗后血行转移的影响因素分析[J]. 实用癌症杂志, 2025, 40(3): 467-471, 488. doi: 10.3969/j.issn.1001-5930.2025.03.031 [17] Jiang W, Yang K, Xiao C, et al. Multimodal tumor microenvironment signature of colorectal cancer for prediction prognosis and chemotherapy benefit[J]. npj Precis Oncol, 2025, 9: 270. doi: 10.1038/s41698-025-01069-3 [18] Li Q, Geng S, Luo H, et al. Signaling pathways involved in colorectal cancer: Pathogenesis and targeted therapy[J]. Signal Transduct Target Ther, 2024, 9: 266. doi: 10.1038/s41392-024-01953-7 [19] Reynolds I S, O’Connell E, Fichtner M, et al. An insight into the driver mutations and molecular mechanisms underlying mucinous adenocarcinoma of the rectum[J]. Dis Colon Rectum, 2021, 64(6): 677-688. doi: 10.1097/DCR.0000000000001825 [20] Liu X, Liu Y, Du K, et al. EPB41L3 as a prognostic biomarker suppressing cellular malignancy and correlating with the immune microenvironment in glioma[J]. Biochem Biophys Res Commun, 2025, 778: 152305. doi: 10.1016/j.bbrc.2025.152305 [21] Zeng R, Liu Y, Jiang Z J, et al. EPB41L3 is a potential tumor suppressor gene and prognostic indicator in esophageal squamous cell carcinoma[J]. Int J Oncol, 2018, 52(5): 1443-1454. doi: 10.3892/ijo.2018.4316 [22] 王佳, 薛兰辉, 宋炜. 候选肿瘤抑制基因EPB41L3在胃癌和结肠直肠癌中的表达及临床意义[J]. 中国现代普通外科进展, 2021, 24(11): 914-916, 919. [23] Cai M, Guo H, Wang D, et al. Expression, DNA methylation pattern and transcription factor EPB41L3 in gastric cancer: A study of 262 cases[J]. Cell Commun Signal, 2024, 22(1): 470. doi: 10.1186/s12964-024-01849-7 [24] Dickey B L, Nedjai B, Preece M D, et al. Methylation of HPV16 and EPB41L3 in oral gargles and the detection of early and late oropharyngeal cancer[J]. Cancer Med, 2022, 11(20): 3735-3742. doi: 10.1002/cam4.4757 [25] Son H J, Choi E J, Yoo N J, et al. Mutation and expression of a candidate tumor suppressor gene EPB41L3 in gastric and colorectal cancers[J]. Pathol Oncol Res, 2020, 26(3): 2003-2005. doi: 10.1007/s12253-019-00787-x [26] Tuerxun G, Abulimiti T, Abudurexiti G, et al. Over-expression of EPB41L3 promotes apoptosis of human cervical carcinoma cells through PI3K/AKT signaling[J]. Acta Biochim Pol, 2022, 69(2): 283-289. doi: 10.18388/abp.2020_5649 [27] Sun Q, Long L. Diagnostic performances of methylated septin9 gene, CEA CA19-9 and platelet-to-lymphocyte ratio in colorectal cancer[J]. BMC Cancer, 2024, 24(1): 906. doi: 10.1186/s12885-024-12670-3 -
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