DNA甲基化激活磷酸肌醇代谢在口腔白斑癌变中的意义

doi:10.12016/j.issn.2096-1456.2021.10.005

摘要/Abstract

摘要：

目的探讨DNA甲基化修饰在口腔白斑癌变中的作用。方法选取40例口腔鳞状细胞癌（oral squamous cell carcinoma,OSCC）组织和28例口腔白斑（oral leukoplakia,OLK）组织以及40例健康口腔黏膜组织进行DNA甲基化检测。下载GEO（Gene Expression Omnibus）数据库中OSCC、OLK和健康口腔黏膜的表达谱数据。分别对DNA甲基化数据和表达谱数据进行重复性比对,对DNA甲基化数据进行差异分析以及对相应表达谱数据进行IPA分析（Ingenuity Pathway Analysis）。结果数据分析结果显示DNA甲基化具有更大的灵活性和不稳定性。IPA通路分析表明,OLK差异甲基化位点相关基因主要富集在细胞运动和分化过程。OSCC差异甲基化位点相关基因主要富集在细胞增殖、迁移、氧化调节、抗凋亡等过程。OLK和OSCC差异甲基化位点相关基因共同富集于磷酸肌醇代谢过程和磷脂酶C信号通路。结论 DNA甲基化改变参与口腔鳞癌形成,磷酸肌醇代谢激活可能促使口腔白斑癌变。

关键词: 口腔白斑, 口腔鳞状细胞癌, 表观遗传学, DNA甲基化, 肌醇, 磷酸肌醇, 代谢, 磷脂酶C, 炎症, 钙信号

Abstract:

Objective To study the role of DNA methylation in oral leukoplakia carcinogenesis. Methods DNA methylation was detected in forty cases of oral squamous cell carcinoma (OSCC), twenty-eight cases of oral leukoplakia (OLK) and forty cases of healthy oral mucosa. Download the expression profile data of OSCC, OLK and healthy oral mucosa from Gene Expression Omnibus (GEO) database. DNA methylation data and expression profile data were compared for repeatability, DNA methylation data for difference analysis and corresponding expression profile data for IPA pathway analysis. Results The data analysis showed that DNA methylation had greater flexibility and instability. Ingenuity Pathway Analysis (IPA) analysis showed that genes related to OLK differential methylation sites were mainly concentrated in the process of cell movement and differentiation. Genes related to differential methylation sites of OSCC are mainly enriched in cell proliferation, migration, oxidation regulation, and anti-apoptosis processes. The genes associated with OLK and OSCC differential methylation sites are co-enriched in phosphoinositol metabolism and phospholipase C signaling pathway. Conclusion DNA methylation is involved in the formation of oral squamous cell carcinoma, and the activation of phosphoinositol metabolism may promote oral leukinoma.

Key words: oral leukoplakia, oral squamous cell carcinoma, epigenetics, DNA methylation, inositol, phosphoinositol, metabolism, phospholipase C, inflammation, calcium signal

中图分类号:

王广超,刘丽骏,蒋伟文. DNA甲基化激活磷酸肌醇代谢在口腔白斑癌变中的意义[J]. 口腔疾病防治, 2021, 29(10): 677-683.

WANG Guangchao,LIU Lijun,JIANG Weiwen. Significance of phosphoinositol metabolism by DNA methylation may contribute in oral leukoplakia carcinogenesis[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2021, 29(10): 677-683.

图/表 7

图1 Illumina 450K甲基化芯片部分检测热图

Figure 1 Heatmap of partial detection of Illumina 450K methylation chip Each column represents a sample, and each row represents a DNA methylation site. Only the clustering results of the first twenty loci were shown

表1 本次实验选用的外部表达谱数据集

Table 1 External expression profile data set selected for this experiment

Data set profile	Region	Type	Sample type and sample size
GSE33205	BALTIMORE（USA）	HUEX-1_0-ST	HNSCC（n=50） vs. Normal（n=25）
GSE85195	NAVI MUMBAI（INDIA）	AGILENT-014850	OLK（n=15） vs. OSCC（n=34） vs. Normal（n=1）

表2 DNA甲基化谱在β值最小时的5%重叠率

Table 2 5% overlap of DNA methylation spectrum at the lowest β value

DNA methylation（5%）	OSCC	OLK	Normal
OSCC	0.62	0.61	0.65
OLK	0.61	0.62	0.66
Normal	0.65	0.66	0.67

表3 表达谱在表达量最大时的5%重叠率

Table 3 The 5% overlap rate of the expression profile at the maximum expression level

Expression（5%）	OSCC	OLK	Normal
OSCC	0.80	0.75	0.80
OLK	0.75	0.83	0.82
Normal	0.80	0.82	0.80

图2 口腔白斑差异DNA甲基化位点相关基因主要参与细胞运动和分化

Figure 2 The genes associated with OLK differential DNA methylation sites are mainly involved in cell movement and differentiation This figure shows the major interaction networks of differential DNA methylation sites in OLK tissues. Among them, the changes of DNA methylation status and expression amount of COL18A1, EBF2, IGF1R and KLF4 were supported by data. The activation/suppression of other genes were predicted by IPA internal algorithm. The COL18A1 promoter region is hypopethylated, and the gene host region is hypermethylated, with reduced expression. The EBF2 promoter region is hypomethylated and its expression is elevated. The main body region of IGF1R gene was hypomethylated and its expression was increased. Both KLF4 host region and promoter region were hypopethylated and their expression was increased. COL18A1: collagen type XVIII alpha 1 chain; EBF2: EBF transcription factor 2; IGF1R: insulin-like growth factor 1 receptor; KLF4: Kruppel like factor 4; OLK: oral leukoplakia

图3 口腔鳞状细胞癌差异DNA甲基化相关基因富集网络

Figure 3 OSCC differential DNA methylation related gene enrichment network OSCC: oral squamous cell carcinoma

表4 口腔鳞状细胞癌与口腔白斑差异DNA甲基化位点相关基因显著富集的前6个通路

Table 4 The first six pathways with significant enrichment of genes related to different methylation sites of OSCC and OLK

Canonical pathways	OSCC vs. Normal	OLK vs. Normal
Superpathway of inositol phosphate compounds	2.837	2.324
3-phosphoinositide Biosynthesis	2.828	2.309
D-myo-inositol (1, 4, 5, 6)-tetrakisphosphate biosynthesis	2.673	2.111
D-myo-inositol (3, 4, 5, 6)-tetrakisphosphate biosynthesis	2.673	2.111
D-myo-inositol-5-phosphate metabolism	2.324	2.309
Phospholipase C signaling	-0.943	3.128

参考文献 19

[1]	Bewley AF, Farwell DG. Oral leukoplakia and oral cavity squamous cell carcinoma[J]. Clin Dermatol, 2017,35(5):461-467. doi: 10.1016/j.clindermatol.2017.06.008. DOI URL
[2]	Mehanna HM, Rattay T, Smith J, et al. Treatment and follow-up of oral dysplasia-a systematic review and meta-analysis[J]. Head Neck, 2009,31(12):1600-1609. doi: 10.1002/hed.21131. DOI URL
[3]	Hema KN, Smitha T, Sheethal HS, et al. Epigenetics in oral squamous cell carcinoma[J]. J Oral Maxillofac Pathol, 2017,21(2):252-259. doi: 10.4103/jomfp.JOMFP_150_17. DOI URL
[4]	Zhang J, Yang C, Wu C, et al. DNA methyltransferases in cancer: biology, paradox, aberrations, and targeted therapy[J]. Cancers (Basel), 2020,12(8):2123. doi: 10.3390/cancers12082123. DOI URL
[5]	Liu X, Zhang T, Li Y, et al. The role of methylation in the CpG island of the ARHI promoter region in cancers[J]. Adv Exp Med Biol, 2020,1255:123-132. doi: 10.1007/978-981-15-4494-1_10. DOI
[6]	Chatterjee A, Rodger EJ, Morison IM, et al. Tools and strategies for analysis of Genome-Wide and Gene-Specific DNA methylation patterns[J]. Methods Mol Biol, 2017,1537:249-277. doi: 10.1007/978-1-4939-6685-1_15. DOI
[7]	Stansfield JC, Rusay M, Shan R, et al. Toward Signaling-Driven biomarkers immune to normal tissue contamination[J]. Cancer Inform, 2016,15:15-21. doi: 10.4137/CIN.S32468. DOI
[8]	Bhosale PG, Cristea S, Ambatipudi S, et al. Chromosomal alterations and gene expression changes associated with the progression of leukoplakia to advanced gingivobuccal cancer[J]. Transl Oncol, 2017,10(3):396-409. doi: 10.1016/j.tranon.2017.03.008. DOI URL
[9]	Du P, Zhang X, Huang CC, et al. Comparison of beta-value and m-value methods for quantifying methylation levels by microarray analysis[J]. BMC Bioinformatics, 2010,11(1):587. doi: 10.1186/1471-2105-11-587. DOI URL
[10]	Ritchie ME, Phipson B, Wu D, et al. Limma powers differential expression analyses for RNA-sequencing and microarray studies[J]. Nucleic Acids Res, 2015,43(7):e47. doi: 10.1093/nar/gkv007. DOI URL
[11]	Eckersley-Maslin M. Keeping your options open: insights from Dppa2/4 into how epigenetic priming factors promote cell plasticity[J]. Biochem Soc Trans, 2020,48(6):2891-2902. doi: 10.1042/BST20200873. DOI URL
[12]	Oleksiewicz U, Machnik M. Causes, effects, and clinical implications of perturbed patterns within the cancer epigenome[J]. Semin Cancer Biol, 2020:S1044-579X(20)30274. doi: 10.1016/j.semcancer.2020.12.014. DOI
[13]	Wang J, Yang J, Li D, et al. Technologies for targeting DNA methylation modifications: basic mechanism and potential application in cancer[J]. Biochim Biophys Acta Rev Cancer, 2021,1875(1):188454. doi: 10.1016/j.bbcan.2020.188454. DOI URL
[14]	Romanowska K, Sobecka A, Rawłuszko-Wieczorek AA, et al. Head and neck squamous cell carcinoma: epigenetic landscape[J]. Diagnostics (Basel), 2020,11(1):34. doi: 10.3390/diagnostics11010034. DOI
[15]	Arnold L, Enders J, Thomas SM. Activated HGF-c-Met axis in head and neck cancer[J]. Cancers (Basel), 2017,9(12):169. doi: 10.3390/cancers9120169. DOI URL
[16]	Abe M, Yamashita S, Mori Y, et al. High-risk oral leukoplakia is associated with aberrant promoter methylation of multiple genes[J]. BMC Cancer, 2016,16(1):350. doi: 10.1186/s12885-016-2371-5. DOI URL
[17]	Zhang XY, Li M, Sun K, et al. Decreased expression of GRIM-19 by DNA hypermethylation promotes aerobic glycolysis and cell proliferation in head and neck squamous cell carcinoma[J]. Oncotarget, 2015,6(1):101-115. doi: 10.18632/oncotarget.2684. DOI URL
[18]	Schmitt K, Molfenter B, Laureano NK, et al. Somatic mutations and promotor methylation of the ryanodine receptor 2 is a common event in the pathogenesis of head and neck cancer[J]. Int J Cancer, 2019,145(12):3299-3310. doi: 10.1002/ijc.32481. DOI PMID
[19]	Owusu OE, Rusciano I, Marvi MV, et al. Phosphoinositide-Dependent signaling in cancer: a focus on phospholipase C isozymes[J]. Int J Mol Sci, 2020,21(7):2581. doi: 10.3390/ijms21072581. DOI URL