口腔白斑癌变DNA损伤修复相关基因的芯片检测及表达验证

doi:10.3969/j.issn.1674-8115.2021.03.003

上海交通大学学报(医学版) ›› 2021, Vol. 41 ›› Issue (3): 302-307.doi: 10.3969/j.issn.1674-8115.2021.03.003

口腔白斑癌变DNA损伤修复相关基因的芯片检测及表达验证

刘伟¹(), 朱敏闻², 吴岚³()

^1.上海交通大学医学院附属第九人民医院·口腔医学院口腔颌面头颈肿瘤科，国家口腔疾病临床研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
^2.上海市徐汇区牙病防治所口腔外科，上海 200032
^3.上海交通大学医学院附属第九人民医院·口腔医学院口腔黏膜科，国家口腔疾病临床研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011

收稿日期:2020-03-30 出版日期:2021-03-28 发布日期:2021-04-06
通讯作者: 吴岚 E-mail:liuweb@hotmail.com;teana_wu@sina.com
作者简介:刘伟（1983—），男，主治医师，博士；电子信箱：liuweb@hotmail.com。
基金资助:
国家自然科学基金(82074502);上海交通大学医学院高水平地方高校创新团队(SSMU-ZDCX20180901)

Array detection and expression verification of DNA damage repair related genes in oral leukoplakia cancerization

Wei LIU¹(), Min-wen ZHU², Lan WU³()

^1.Department of Oral and Maxillofacial-Head and Neck Oncology, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China
^2.Shanghai Xuhui District Dental Center, Shanghai 200032, China
^3.Department of Oral Mucosal Diseases, Shanghai Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University School of Medicine, National Clinical Research Center for Oral Diseases, Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China

Received:2020-03-30 Online:2021-03-28 Published:2021-04-06
Contact: Lan WU E-mail:liuweb@hotmail.com;teana_wu@sina.com
Supported by:
National Nature Science Foundation of China(82074502);Innovative Research Team of High-Level Local Universities in Shanghai(SSMU-ZDCX20180901)

摘要/Abstract

摘要：

目的·研究口腔白斑（oral leukoplakia，OL）患者病损发生发展中的DNA损伤修复基因表达谱，并验证关键基因共济失调毛细血管扩张症突变基因（ataxia-telangiectasia mutated，ATM）和组蛋白2A变异体家族成员X（histone variant H2A family member X，H2AX）的mRNA和蛋白表达。方法·利用Affymetrix HTA 2.0芯片对口腔正常黏膜（n=3）、低危白斑（n=4）、高危白斑（n=4）、早期鳞状细胞癌（鳞癌，n=6）进行转录组学的高通量检测，利用基因本体数据库（Gene Ontology，GO）富集分析筛选出生物学功能为DNA损伤修复的相关基因［差异倍数（fold change，FC）对数的绝对值（|log₂FC|）≥2.0且P<0.05］，利用实时定量PCR（real-time quantitative PCR，qPCR）和蛋白质印迹法分别对人正常口腔黏膜角化细胞（HOK）、白斑细胞系（Leuk1）和鳞癌细胞系（HN13）中的关键基因ATM和H2AX进行mRNA和蛋白表达验证。结果·DNA损伤修复基因在白斑发生发展中存在异常表达，从正常黏膜发展到低危白斑有7个|log₂FC|≥2.0的基因，从低危白斑发展到高危白斑有7个|log₂FC|≥2.0的基因，从高危白斑发展到鳞癌有52个|log₂FC|≥2.0的基因。qPCR对关键基因验证结果显示，ATM和H2AX基因在HOK细胞、Leuk1细胞和HN13细胞中的表达量均呈阶梯式升高（P<0.05）。蛋白质印迹法结果显示，磷酸化H2AX（γ-H2AX）蛋白在HOK细胞、Leuk1细胞和HN13细胞中亦逐步上调（P<0.05）。ATM蛋白在Leuk1细胞中的表达比在HOK细胞中表达明显增强（P<0.05），但在HN13细胞中的表达明显降低（P<0.05）。结论·DNA损伤修复基因ATM和H2AX参与OL的发生发展。

关键词: 口腔白斑, 鳞状细胞癌, DNA损伤修复, 共济失调毛细血管扩张症突变基因, 组蛋白2A变异体家族成员X

Abstract:

Objective·To study the gene expression profile of DNA damage repair in the occurrence and development of oral leukoplakia (OL) and to verify the expression of mRNA and protein of key genes, ataxia-telangiectasia mutated (ATM) and histone variant H2A family member X (H2AX).

Methods·Affymetrix HTA 2.0 array was used to detect the high-throughput transcriptome of normal mucosa (n=3), low-risk leukoplakia (n=4), high-risk leukoplakia (n=4) and early squamous cell carcinoma (n=6). Gene Ontology function analysis was used to screen out the genes related to DNA damage repair [|log₂(chang fold, FC)|≥2.0 and P<0.05]. Real-time quantitative PCR (qPCR) and Western blotting were used to verify the mRNA and protein expression of key genes (ATM and H2AX) in human normal oral mucosal keratinocytes (HOK), OL (Leuk1) and squamous cell carcinoma cell lines (HN13), respectively.

Results·DNA damage repair genes abnormally expressed in the occurrence and development of leukoplakia. There were 7 genes with |log₂FC|≥2.0 from normal mucosa to low-risk leukoplakia, 7 genes with |log₂FC|≥2.0 from low-risk leukoplakia to high-risk leukoplakia, and 52 genes with |log₂FC|≥2.0 from high-risk leukoplakia to squamous cell carcinoma. qPCR revealed that the expression of ATM and H2AX in HOK cells, Leuk1 cells and HN13 cells increased stepwise (P<0.05). Western blotting revealed that the expression of γ-H2AX protein was also up-regulated in the HOK cells, Leuk1 cells and HN13 cells (P<0.05). ATM protein was up-regulated in Leuk1 cells compared with HOK cells, but down-regulated in HN13 cells (P<0.05).

Conclusion·DNA damage repair genes, ATM and H2AX, are involved in the occurrence and development of OL.

Key words: oral leukoplakia (OL), squamous cell carcinoma, DNA damage repair, ataxia-telangiectasia mutated (ATM), histone variant H2A family member X (H2AX)

中图分类号:

R781.5⁺9

刘伟, 朱敏闻, 吴岚. 口腔白斑癌变DNA损伤修复相关基因的芯片检测及表达验证[J]. 上海交通大学学报(医学版), 2021, 41(3): 302-307.

Wei LIU, Min-wen ZHU, Lan WU. Array detection and expression verification of DNA damage repair related genes in oral leukoplakia cancerization[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(3): 302-307.

图/表 6

表1 qPCR验证ATM和H2AX基因的引物序列

Tab 1 Primer sequence for ATM and H2AX genes by qPCR

Gene	Sequence
ATM	F: 5'-CGCCGCGGTTGATACTACT-3'
	R: 5'-ACGGCAGCAGATAAGCAGAT-3'
H2AX	F: 5'-CAACAAGAAGACGCGAATCA-3'
	R: 5'-CTGGATGTTGGGCAGGAC-3'
β-actin	F: 5'-CCAACCGCGAGAAGATGAC-3'
	R: 5'-GAGGCGTACAGGGATAGCACA-3'

表2 3组研究对象人口学资料比较

Tab 2 Comparison of demographic data between the three groups

Item	Normal mucosa group (N=3)	Leukoplakia group (N=8)	Early squamous cell carcinoma group (N=6)	F/χ² value	P value
Age/year	52.7±8.7	54.5±10.1	55.1±9.8	0.691	0.911
Gender/n				0.032	0.984
Male	2	5	4
Female	1	3	2
Smoking/n	1	3	3	0.314	0.855
Alcohol intake/n	1	3	3	0.314	0.855

图1 从白斑发生发展各阶段的全部基因中筛选出DNA损伤修复基因

Fig 1 DNA damage repair genes were screened from all genes in the development and progression of leukoplakia

图2 口腔早期鳞癌与高危白斑之间的52个差异基因聚类热图Note: NFIX—nuclear factor Ⅸ; POLB—DNA polymerase β; POLR2I—RNA polymeraseⅡ polypeptide Ⅰ; DTX3L—deltex E3 ubiquitin ligase 3L; CTGF—connective tissue growth factor; CDK2—cyclin dependent kinase 2; HELB—DNA helicase B; LYN—LYN proto-oncogene, Src family tyrosine kinase; CENPF—centromere protein F; PCNA—proliferating cell nuclear antigen; PRIM2—DNA primase subunit 2; RAD51AP1—RAD51 associated protein 1; MCM5—minichromosome maintenance complex component 5; MMS22L—MMS22-like, DNA repair protein; NASP—nuclear autoantigenic sperm protein; SMCHD1—structural maintenance of chromosomes flexible hinge domain containing 1; BRIP1—BRCA1 BRCA1 interacting helicase 1; CHEK1—checkpoint kinase 1; CLSPN—claspin; ATAD5—ATPase family AAA domain 5; CDC6—cell division cycle 6; MCM4—minichromosome maintenance complex component 4; TK1—thymidine kinase 1; FANCD2—FA complementation group D2; SUPT16H—SPT16 homolog, facilitates chromatin remodeling subunit; TMX1—thioredoxin related transmembrane protein 1; POLQ—DNA polymerase θ; EXO1—exonuclease 1; ATR—ATR serine/threonine kinase; TOPBP1—DNA topoisomerase Ⅱ binding protein 1; PLK1—polo like kinase 1; TOP2A—DNA topoisomerase Ⅱα; MCM8—minichromosome maintenance 8 homologous recombination repair factor; PRKDC—protein kinase, DNA-activated, catalytic subunit; ESCO2—establishment of sister chromatid cohesion N-acetyltransferase 2; C15orf42—chromosome 15 open reading frame 42; RFWD3—ring finger and WD repeat domain 3; BLM—BLM RecQ like helicase; DTL—denticleless E3 ubiquitin protein ligase homolog; FANCI—FA complementation group Ⅰ; CASP3—caspase 3; DNA2—DNA replication helicase/nuclease 2; SMC3—structural maintenance of chromosomes 3; SMC5—structural maintenance of chromosomes 5; DBF4—DBF4 zinc finger; MCM7—minichromosome maintenance complex component 7; BRCA2—BRCA2 DNA repair associated; RFC3—replication factor C subunit 3; XRCC2—X-ray repair cross complementing 2.

Fig 2 Clustering heat map of 52 different genes between oral early squamous cell carcinoma and high-risk leukoplakia

图3 qPCR验证ATM和H2AX在人正常黏膜、白斑和鳞癌细胞系中的差异表达Note: ①P=0.000， ②P=0.004， ③P=0.008.

Fig 3 qPCR confirmed the differential expression of ATM and H2AX in normal mucosa, leukoplakia and squamous cell carcinoma cell lines

图4 Western blotting检测ATM和γ-H2AX在HOK细胞、Leuk1细胞和HN13细胞中的表达Note:A. Western blotting. B. Gray analysis. ①P=0.006， ②P=0.002， ③P=0.000， ④P=0.009.

Fig 4 Western blotting examined the protein expression of ATM and H2AX in HOK cells, Leuk1 cells and NH13 cells

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口腔白斑癌变DNA损伤修复相关基因的芯片检测及表达验证

Array detection and expression verification of DNA damage repair related genes in oral leukoplakia cancerization

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