子宫颈癌患者MSX1基因甲基化与临床病理特征及预后的关联性分析

doi:10.13481/j.1671-587X.20250319

摘要/Abstract

摘要：

目的探讨Msh同源框1（MSX1）基因甲基化与子宫颈癌（CC）患者临床病理特征及预后的关系，分析MSX1基因甲基化在CC诊治过程的应用前景。方法收集2019年1月—2020年6月期间行CC手术120例患者的临床资料和癌组织及癌旁正常组织，并对全部CC患者进行为期3年的随访。采用甲基化特异性PCR（MSP）法检测CC患者癌组织中MSX1基因甲基化状态，分为MSX1基因低甲基化组（n=50）和MSX1基因高甲基化组（n=70），MSP法检测CC患者癌组织中MSX1基因甲基化水平，分析MSX1基因甲基化状态与CC患者临床病理特征和预后的关系，Western blotting法检测CC患者癌组织和癌旁正常组织中MSX1蛋白表达水平。设计靶向MSX1 mRNA的小干扰RNA（siRNA），将CC细胞分为对照组（转染空白载体）和siMSX1组（转染MSX1 siRNA），采用实时荧光定量PCR（RT-qPCR）法验证细胞转染效率，细胞划痕实验检测2组CC细胞迁移活性，Logistic回归分析CC患者预后的影响因素。结果 CC患者癌组织中甲基化发生率为58.33%，明显高于癌旁正常组织甲基化发生率（11.67%，χ2=42.725，P<0.01）。CC患者癌组织中MSX1基因高甲基化状态与患者高危型人类乳头状瘤病毒（HR-HPV）DNA、淋巴结转移和TNM分期有关联（P<0.05），与患者年龄、病理类型和肿瘤大小无关联（P>0.05）。Western blotting法，与癌旁正常组织比较，CC患者癌组织中MSX1蛋白表达水平明显降低（P<0.01）。细胞划痕实验，与对照组比较，siMSX1组细胞MSX1 mRNA表达水平明显降低（P<0.01），提示MSX1基因成功敲除；与对照组比较，siMSX1组细胞迁移活性明显升高（P<0.01）。MSX1基因甲基化者3年累积生存率为54.29%，明显低于MSX1基因未甲基化者（80.00%，χ2=9.717，P=0.002）。Logistic回归分析，阳性HR-HPV DNA、淋巴结转移、TNM分期Ⅲ-Ⅳ期和MSX1基因高甲基化是CC预后的独立危险因素（P<0.05）。结论 MSX1基因甲基化与CC患者较差的临床病理特征及不良预后具有一定关联性，提示其可作为宫颈癌潜在生物标志物。

关键词: 子宫颈癌, MSX1基因, 甲基化, 预后, 小干扰RNA

Abstract:

Objective To discuss the relationship between Msh homeobox 1 （MSX1） gene methylation and the clinicopathological characteristics and prognosis of the patients with cervical cancer （CC）， and to analyze the application prospect of MSX1 gene methylation in the diagnosis and treatment of CC. Methods The clinical data， cancer tissues， and adjacent normal tissues were collected from 120 CC patients who underwent surgery between January 2019 and June 2020， and all CC patients were followed up for 3 years； methylation-specific PCR （MSP） was used to detect the methylation status of the MSX1 gene in cancer tissues of the CC patients， who were divided into MSX1 gene hypo-methylation group （n=50） and MSX1 gene hyper-methylation group （n=70）； MSP was used to detect the methylation level of the MSX1 gene in cancer tissues of the CC patients， and the relationship between MSX1 gene methylation status and clinicopathological characteristics and prognosis of the CC patients was analyzed； Western blotting method was used to detect the expression level of MSX1 protein in cancer tissues and adjacent normal tissues of the CC patients； small interfering RNA （siRNA） targeting MSX1 mRNA was designed； the CC cells were divided into control group （transfected with blank vector） and siMSX1 group （transfected with MSX1 siRNA）； real-time fluorescence quantitative PCR （RT-qPCR） method was used to verify the cell transfection efficiency； cell scratch assay was used to detect the migration activity of the cells in two groups； Logistic regression was used to analyze the influencing factors of prognosis in the CC patients. Results The methylation rate in cancer tissues of the CC patients was 58.33%， which was significantly higher than that in adjacent normal tissues （11.67%， χ2=42.725， P<0.01）； the Western blotting results showed that compared with adjacent normal tissue， the expression level of MSX1 protein in cancer tissue of the CC patients was significantly decreased （P<0.01）； high methylation status of the MSX1 gene in cancer tissue of the CC patients was associated with high-risk human papillomavirus （HR-HPV） DNA， lymph node metastasis， and TNM stage （P<0.05）， but not associated with patient age， pathological type， and tumor size （P>0.05）； the cell scratch assay results showed that compared with control group， the expression level of MSX1 mRNA in the cells in siMSX1 group was significantly decreased （P<0.01）， suggesting successful knockout of the MSX1 gene； the cell scratch assay results showed that compared with control group， the migration activity of the cells in siMSX1 group was significantly increased （P<0.01）； the 3-year cumulative survival rate of patients with MSX1 gene methylation was 54.29%， which was significantly lower than that of patients without MSX1 gene methylation （80.00%， χ2=9.717， P=0.002）； the Logistic regression results showed that positive HR-HPV DNA， lymph node metastasis， TNM stage Ⅲ-Ⅳ and MSX1 gene high methylation were independent risk factors for prognosis in CC patients （P<0.05）. Conclusion MSX1 gene methylation is associated with poorer clinicopathological characteristics and adverse prognosis in the CC patients， suggesting its potential as a biomarker for cervical cancer.

Key words: Cervical cancer, MSX1 gene, Methylation, Prognosis, Small interfering RNA

中图分类号:

R737.33

黄遐龄,张丹丹,张雪梅. 子宫颈癌患者MSX1基因甲基化与临床病理特征及预后的关联性分析[J]. 吉林大学学报(医学版), 2025, 51(3): 749-756.

Xialing HUANG,Dandan ZHANG,Xuemei ZHANG. Analysis on relationship between methylation of MSX1 gene and clinical pathological features and prognosis of patients with cervical cancer[J]. Journal of Jilin University(Medicine Edition), 2025, 51(3): 749-756.

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Primer sequence	Forward	Reverse
Methylation primer	5'-CGTTTATGGTCGATTATAGGAAGTC-3'	5'-GAAACTCTCGACTTTAACGAACG-3'
Unmethylated primer	5'-GGTGTTTATGGTTGATTATAGGAAGTT-3'	5'-TCAAAACTCTCAACTTTAACAAACAC-3'

Paremeter	Number	MSX1 gene [n（η/%）]		χ2	P
Paremeter	Number	Hypo-methylation（n=50）	Hyper-methylation（n=70）	χ2	P
Age
<50	58	24（41.38）	34（58.62）	0.009	0.974
≥50	62	26（41.94）	36（58.06）	0.009	0.974
Tumor size(cm)
<4	72	32（44.44）	40（55.56）	0.632	0.415
≥4	48	18（37.50）	30（63.50）	0.632	0.415
HR-HPV DNA
Positive	96	34（35.41）	62（64.58）	8.692	0.003
Negative	24	16（66.67）	8（33.33）	8.692	0.003
Histological type
Squamous carcinoma	81	33（40.74）	48（59.26）	0.212	0.612
Adenocarcinoma	39	17（43.59）	22（56.41）	0.212	0.612
Lymphnode metestasis
Yes	33	8（24.24）	25（75.76）	6.912	0.005
No	87	42（48.28）	45（51.72）	6.912	0.005
TNM phase
Ⅰ-Ⅱ	89	42（47.19）	47（52.81）	6.343	0.005
Ⅲ-Ⅳ	31	8（25.81）	23（74.19）	6.343	0.005

Variable	Univariate model					Multivariate model
Variable	β	SE	Z	P	OR(95%CI)	β	SE	Z	P	OR(95%CI)
Age (<50 vs ≥50)	-1.05	0.48	-1.64	0.167	0.816-1.332	-	-	-	-	-
Tumor size (<4 cm vs ≥4 cm)	-0.87	0.82	-0.98	0.213	0.853-1.318	-	-	-	-	-
HR-HPV DNA (negative vs positive)	0.06	0.01	4.40	<0.01	1.322-5.131	0.04	0.03	2.71	<0.01	1.265-4.278
Histological type (adenocarcinoma vssquamous carcinoma)	1.07	0.82	0.62	0.458	0.823-1.278	-	-	-	-	-
Lymphnode metestasis (no vs yes)	0.10	0.02	4.74	<0.01	1.554-4.237	0.06	0.02	2.91	<0.01	1.634-3.928
TNM phase (Ⅰ-Ⅱ vs Ⅲ-Ⅳ)	0.17	0.04	4.32	<0.01	1.721-5.853	0.18	0.05	3.74	<0.01	1.688-5.538
MSX1 gene (unmethylated vs methylated)	0.06	0.02	4.63	<0.01	1.851-4.869	0.04	0.03	3.12	<0.01	1.722-4.639