胃癌患者FHIT、hMLH1、p16、RAR-beta、Reprimo和TIMP3基因的甲基化研究
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摘要
目的探究人脆性组氨酸三联体(FHIT)基因、人mut1同源物(h MLH1)基因、p16基因、维甲酸受体(RAR)-beta(RAR-beta)基因、Reprimo基因和基质金属蛋白酶抑制因子3(TIMP3)基因在胃癌及相应的癌旁对照组织中甲基化状态。方法采用亚硫酸氢钠测序法检测42例临床手术切除的胃癌标本及42例相应癌旁组织标本中FHIT基因、h MLH1基因、p16基因、RAR-beta基因、Reprimo基因和TIMP3基因甲基化水平。结果胃癌组织及相应癌旁组织之间各基因的平均甲基化率分别为:FHIT基因(1. 50%,1. 36%)、h MLH1基因(4. 77%,0. 48%)、p16基因(9. 63%,10. 36%)、RAR-beta基因(4. 75%,4. 17%)、Reprimo基因(9. 71%,3. 76%)与TIMP3基因(18. 34%,14. 06%)。癌旁对照组织与胃癌组织的Reprimo基因的平均甲基化率具有统计学差异(P <0. 05)。组织分化程度不同的胃癌患者Reprimo基因启动子甲基化率存在统计学差异(P <0. 05)。结论胃癌中Reprimo基因启动子区胞嘧啶鸟嘌呤二核苷酸岛中存在甲基化现象。Reprimo基因的高甲基化率可以作为胃癌的潜在生物标记物,以便早期发现胃癌。
Objective The aim of this study was to investigate the methylation status of fragile histidine triad( FHIT) gene,human mutl homolog 1( h MLH1) gene,p16 gene,retinoic acid receptor beta( RAR-beta) gene,Reprimo gene and tissue inhibitor of metalloproteinase 3( Timp3) gene in gastric cancer and corresponding paracancerous tissues. Methods The methylation levels of FHIT,h MLH1,p16,RAR-beta,Reprimo and TIMP3 genes in 42 clinically resected gastric cancer specimens and 42 corresponding paracancerous tissues were detected by sodium bisulfite sequencing. Results The average methylation rates of the genes in gastric cancer and corresponding paracancerous tissues were: FHIT( 1. 50%,1. 36%),h MLH1( 4. 77%,0. 48%),p16( 9. 63%,10. 36%),RAR-beta( 4. 75%,4. 17%),Reprimo( 9. 71%,3. 76%) and TIMP3 genes( 18. 34%,14. 06%). Compared with the paracancerous control group,the average methylation rate of Reprimo gene was only statistically different in gastric cancer patients( P = 0. 00787).The difference in methylation rate of Reprimo gene promoter in gastric cancer patients with the degree of tissue differentiation was statistically significant( P < 0. 05). Conclusion There has methylation in the cytosine guanidine dinucleotide island of the Reprimo gene promoter region in gastric cancer. The high methylation rate of the Reprimo gene can be used as a potential biomarker for gastric cancer to detect the early stage of gastric cancer.
Objective The aim of this study was to investigate the methylation status of fragile histidine triad( FHIT) gene,human mutl homolog 1( h MLH1) gene,p16 gene,retinoic acid receptor beta( RAR-beta) gene,Reprimo gene and tissue inhibitor of metalloproteinase 3( Timp3) gene in gastric cancer and corresponding paracancerous tissues. Methods The methylation levels of FHIT,h MLH1,p16,RAR-beta,Reprimo and TIMP3 genes in 42 clinically resected gastric cancer specimens and 42 corresponding paracancerous tissues were detected by sodium bisulfite sequencing. Results The average methylation rates of the genes in gastric cancer and corresponding paracancerous tissues were: FHIT( 1. 50%,1. 36%),h MLH1( 4. 77%,0. 48%),p16( 9. 63%,10. 36%),RAR-beta( 4. 75%,4. 17%),Reprimo( 9. 71%,3. 76%) and TIMP3 genes( 18. 34%,14. 06%). Compared with the paracancerous control group,the average methylation rate of Reprimo gene was only statistically different in gastric cancer patients( P = 0. 00787).The difference in methylation rate of Reprimo gene promoter in gastric cancer patients with the degree of tissue differentiation was statistically significant( P < 0. 05). Conclusion There has methylation in the cytosine guanidine dinucleotide island of the Reprimo gene promoter region in gastric cancer. The high methylation rate of the Reprimo gene can be used as a potential biomarker for gastric cancer to detect the early stage of gastric cancer.
引文
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8 Herman JG,Graff JR,Baylin SB,et al.Methylation-specific PCR:a novel PCR assay for methylation status of Cp Gislands[J].Proc Natl Acad Sci U S A,1996,93(18):9821-9826.
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11 Wojdacz TK,Dobrovic A.Methylation-sensitive high resolution melting(MS-HRM):a new approach for sensitive and high-through put assessment of methylation[J].Nucleic Acids Res,2007,35(6):e41.
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14 Cecener G,Tunca B,Egeli U,et al.The promoter hypermethylation status of GATA6,MGMT,and FHIT in glioblastoma[J].Cell Mol Neurobiol,2012,32(2):237-244.
15 Oue N,Kuraoka K,Kuniyasu H,et al.DNA methylation status of h MLH1,p16(INK4a),and CDH1 is not associated with mRNA expression levels of DNA methyltransferase and DNA demethylase in gastric carcinomas[J].Oncol Rep,2001,8(5):1085-1089.
16 Bernal C,Aguayo F,Villarroel C,et al.Reprimo as a potential biomarker for early detection in gastric cancer[J].Clin Cancer Res,2008,14(19):6264-6269.
17 Sozzi G,Huebner K,Croce CM.FHIT in human cancer[J].Adv Cancer Res,1998,74:141-166.
18 Croce CM,Sozzi G,Huebner K.Role of FHIT in human cancer[J].J Clin Oncol,1999,17(5):1618-1624.
19 Bronner CE,Baker SM,Morrison PT,et al.Mutation in the DNA mismatch repair gene homologue h MLH1 is associated with hereditary non-polyposis colon cancer[J].Nature,1994,368(6468):258-261.
20 Ohki R,Nemoto J,Murasawa H,et al.Reprimo,a new candidate mediator of the p53-mediated cell cycle arrest at the G2 phase[J].J Biol Chem,2000,275(30):22627-22630.
21 Takahashi T,Suzuki M,Shigematsu H,et al.Aberrant methylation of Reprimo in human malignancies[J].Int JCancer,2005,115(4):503-510.
22 Bernal C,Aguayo F,Villarroel C,et al.Reprimo as a potential biomarker for early detection in gastric cancer[J].Clin Cancer Res,2008,14(19):6264-6269.
23贾德馨,谢蕊,李燕京,等.Reprimo基因在消化道肿瘤中的研究进展[J].现代肿瘤医学,2014,22(11):2749-2751.
2 Choi SJ,Jung SW,Huh S,et al.Alteration of DNA methylation in gastric cancer with chemotherapy[J].J Microbiol Biotechnol,2017,27(8):1367-1378.
3 Zeng XQ,Wang J,Chen SY.Methylation modification in gastric cancer and approaches to targeted epigenetic therapy(Review)[J].Int J Oncol,2017,50(6):1921-1933.
4 Kulis M,Esteller M.DNA methylation and cancer[J].Adv Genet,2010,70(5):27-56.
5 Gopalakrishnan S,Van Emburgh BO,Robertson KD.DNAmethylation in development and human disease[J].Mutat Res,2008,647(1/2):30-38.
6 Shiraishi M,Hayatsu H.High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNAmethylation[J].DNA Research,2004,11(6):409-415.
7 Wajed SA,Laird PW,De Meester R.DNA methylation:an alternative pathway to cancer[J].Ann Surg,2001,234(1):10-20.
8 Herman JG,Graff JR,Baylin SB,et al.Methylation-specific PCR:a novel PCR assay for methylation status of Cp Gislands[J].Proc Natl Acad Sci U S A,1996,93(18):9821-9826.
9 Xiong Z,Laird PW.COBRA:a sensitive and quantitative DNA methylation assay[J].Nucleic Acids Res,1997,25(12):2532-2534.
10 Tost J,Gut IG.DNA methylation analysis by pyrosequencing[J].Nat Protoc,2007,2(9):2265-2275.
11 Wojdacz TK,Dobrovic A.Methylation-sensitive high resolution melting(MS-HRM):a new approach for sensitive and high-through put assessment of methylation[J].Nucleic Acids Res,2007,35(6):e41.
12 Li Y,Tollefsbol TO.DNA methylation detection:bisulfite genomic sequencing analysis[J].Methods Mol Biol,2011,791(3):11-21.
13 Parrish RR,Day JJ,Lubin FD.Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotideresolution from brain tissues[J].Curr Protoc Neurosci,2012,7:24.
14 Cecener G,Tunca B,Egeli U,et al.The promoter hypermethylation status of GATA6,MGMT,and FHIT in glioblastoma[J].Cell Mol Neurobiol,2012,32(2):237-244.
15 Oue N,Kuraoka K,Kuniyasu H,et al.DNA methylation status of h MLH1,p16(INK4a),and CDH1 is not associated with mRNA expression levels of DNA methyltransferase and DNA demethylase in gastric carcinomas[J].Oncol Rep,2001,8(5):1085-1089.
16 Bernal C,Aguayo F,Villarroel C,et al.Reprimo as a potential biomarker for early detection in gastric cancer[J].Clin Cancer Res,2008,14(19):6264-6269.
17 Sozzi G,Huebner K,Croce CM.FHIT in human cancer[J].Adv Cancer Res,1998,74:141-166.
18 Croce CM,Sozzi G,Huebner K.Role of FHIT in human cancer[J].J Clin Oncol,1999,17(5):1618-1624.
19 Bronner CE,Baker SM,Morrison PT,et al.Mutation in the DNA mismatch repair gene homologue h MLH1 is associated with hereditary non-polyposis colon cancer[J].Nature,1994,368(6468):258-261.
20 Ohki R,Nemoto J,Murasawa H,et al.Reprimo,a new candidate mediator of the p53-mediated cell cycle arrest at the G2 phase[J].J Biol Chem,2000,275(30):22627-22630.
21 Takahashi T,Suzuki M,Shigematsu H,et al.Aberrant methylation of Reprimo in human malignancies[J].Int JCancer,2005,115(4):503-510.
22 Bernal C,Aguayo F,Villarroel C,et al.Reprimo as a potential biomarker for early detection in gastric cancer[J].Clin Cancer Res,2008,14(19):6264-6269.
23贾德馨,谢蕊,李燕京,等.Reprimo基因在消化道肿瘤中的研究进展[J].现代肿瘤医学,2014,22(11):2749-2751.