Changes of Genebody Methylation in Lingual Carcinogenesis
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摘要:
目的 了解舌粘膜癌变中基因体甲基化的差异表达基因。 方法 用浓度50 mg/L的4-硝基喹啉-1-氧化物（4NQO）诱导C57BL/6小鼠舌癌。基因芯片和甲基化DNA免疫沉淀测序（MeDIP-Seq）检测0周、12周、28周（代表正常、癌前、癌变）的舌粘膜的基因表达和基因组DNA甲基化。在人正常舌粘膜和癌前病变组织中用qRT-PCR和飞行质谱检测泛素羧基水解酶CYLD的mRNA表达和基因体甲基化。 结果 正常、癌前和舌癌3组间基因体MeDIP-score（mean±s）分别是（5.58±14.80）、（5.79±13.35）、（5.83±17.25），差异无统计学意义（H = 100.75，P > 0.05）。3组间145个差异表达基因伴基因体甲基化改变。CYLD在人癌前病变组织中表达降低伴有基因体甲基化降低（P < 0.05）。 结论 舌粘膜癌变有多个基因体甲基化异常的差异表达基因，CYLD在人癌前病变中低表达可能与基因体甲基化有关。 Abstract:Objective To evaluate differentially expressed genes with correlation of gene body methylation . Methods The lingual carcinogenesis was induced through the delivery of drinking water of 4-nitroquinoline-1-oxide（4NQO） with a density of 50 mg/L. The lingual mucosae specimens were harvested at 0, 12, 28 weeks, which represented normal, pre-cancer, and cancer individually. The microarray and methylated DNA immunoprecipitation sequencing （MeDIP-Seq） were used for the evaluation of gene expression and global DNA methylation in these specimens. The mRNA expression of Ubiquitin carboxyl-terminal hydrolase CYLD was tested with qRT-PCR in human normal and dysplasia mucosa. Results There were no statistical differences in gene body methylation among normal（C）, pre-cancer（M）, and cancer（E）（mean±sofMeDIP-score were 5.58±14.80, 5.79±13.35, 5.83±17.25 individually, H = 100.75, P > 0.05）There presented 145 differentially expressed genes with positive correlation to gene body methylation changes.The expression of CYLD mRNA was decreased in human dysplasia compared to normal mucosa with downregulation of gene body methylation. Conclusions There are many differentially expressed genes with a positive correlation to gene body methylations. The expression of CYLD is downregulated with the downregulation of gene body methylation in earlier stage during tongue carcinogenesis. -
Key words:
- Tongue cancer /
- Animal model /
- Methylation /
- Gene body /
- Differentially expressed gene
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图 1 4NQO诱导C57BL/6舌粘膜癌变（HE，×200）
A：0周，正常粘膜；B：12周，中度不典型增生；C：28周，鳞癌。
Figure 1. The lingual carcinogenesis in C57BL/6 mice induced by 4NQO （HE，×200）
图 2 人正常舌黏膜和异常增生（×200）
A：正常舌黏膜；B：舌黏膜异常增生。
Figure 2. Human normal tongue mucosa and moderate dysplasia. magnification of （×200）
图 3 舌黏膜癌变基因体甲基化构成
MeDIP-score < 4.19定义为未甲基化，介于4.19～41.01定义为部分甲基化，> 41.01为甲基化。
Figure 3. Numbers of methylated，partially methylated and unmethylated gene bodies
图 4 人异常增生舌组织中CYLD的mRNA表达较正常粘膜降低
Figure 4. Expression of CYLD mRNA downregulated in human dysplasia mucosa compared to normal mucosa
图 5 人舌粘膜、异常增生CYLD基因体甲基化飞行质谱检测结果
甲基化圆点图标尺颜色越浅代表甲基化程度越低。基因的序列标尺上方是碱基位置，下方是CpG的标识及大致位置。L12～16为异常增生舌黏膜，L17～21为人正常舌黏膜。
Figure 5. The MassaARRAY results of CYLD gene body methylation
表 1 基因体甲基化异常的差异表达基因
Table 1. Differentially expressed genes with positive correlation to gene body methylation
序号 癌前vs正常 癌变vs正常 癌变vs癌前 上调 下调 上调 下调 上调 下调 1 Gm12657 Ddx25 Hps3 Irf3 Odf2 Msln 2 Rnf14 Cyld Mreg Zfp939 Uba1 Slc35a5 3 Rsbn1l 2610507B11Rik Gm12657 Tesk1 2010012O05Rik Csprs 4 Celf2 5330434G04Rik Edaradd Gab3 Wdyhv1 Ywhaz 5 Mapk3 Dclk2 Gm7120 Ntng1 Cdca5 Ada 6 Rdh9 Ecsit Pemt Sema3b Fbxo24 Bpifb6 7 Ache Il20 Plekhb2 Ptges3l Tmem37 A730008H23Rik 8 Stard10 AI846148 Nop14 Bace2 Spp1 Stard10 9 Klhl7 Iars2 Hivep1 Gm3086 Plekhg4 Casc4 10 Mfsd1 Gm3086 Fam98b Kcnn4 Fam81a Igsf5 11 Arf4 Zfp763 Oas2 a Fubp1 12 Zbtb18 Pptc7 Tbx22 Ewsr1 Cdkl5 13 Kcnj2 Cdc42se2 Mfsd2a Fut2 14 Ap1s2 Slfn4 Shc4 H2-T9 15 Setd1b Npl Tspan32 16 Cyth3 Exoc6 Car4 17 Nek1 Son Mapkapk2 18 Cpn1 Rsbn1l Slc25a39 19 Aqr Nacad Eif5a 20 Abca5 Ddx20 Bace2 21 Pomgnt1 Atf1 Hira 22 Nfkb1 Vps37c Azgp1 23 Kalrn Nfkbiz Hmgcll1 24 Dsg1b Prkaa1 Cfl1 25 Sec62 Pik3c2a Mamstr 26 Camk2a Ankfn1 Celf2 27 Ube2q2 Mfsd1 Gfra4 28 Ppp4r2 Cdk1 Papss2 29 Lrrc58 Bcas2 Smurf2 30 Tnpo1 Rad21 Cat 31 Zfp763 Zfand1 Myoz3 32 Il2rg Mmgt1 Tmed3 33 Slc25a39 Trip13 Prb1 34 Nfe2l1 Dyrk1b 35 Tob1 Prrt2 36 H2-T10 37 Gyk 38 Tmem55b 39 Zfp386 40 Akr1b3 41 Tnfaip6 
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