MTH1蛋白与结肠癌进展和预后的相关性研究
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摘要
目的探讨MutT同源蛋白1(MutT homolog protein 1, MTH1)的表达对结肠癌(colorectal cancer, CRC)进展和预后的影响。方法笔者首先使用免疫组织化学方法检测了87例结肠癌组织和配对的癌旁正常组织中MTH1蛋白的表达,应用χ~2检验分析MTH1蛋白表达量与结肠癌临床病理特征之间的相关性,应用COX比例回归风险模型分析MTH1蛋白表达对结肠癌根治术后患者的总体生存预后判断价值。笔者使用实时荧光定量PCR和Western blot法分别检测了6株结肠癌细胞HCT116、SW480、SW620、LoVo、COLO320、T84及人胚胎肠黏膜细胞CCC-HIE-2中MTH1基因mRNA和蛋白的表达水平,应用独立t检验比较了肿瘤细胞和正常肠黏膜上皮细胞中MTH1基因mRNA和蛋白相对表达量的均值,并且应用Spearman等级相关系数检测MTH1基因mRNA和蛋白表达的相关性。笔者使用本实验室构建成功的siRNA干扰MTH1基因mRNA表达的HCT116和LoVo细胞株,利用CCK-8和Transwell实验检测MTH1蛋白低表达对结肠癌细胞增殖、迁移、侵袭的影响。结果在87例结肠癌组织中,MTH1蛋白高表达患者手术治疗后总的生存率显著低于MTH1低表达患者(P=0.005)。COX多因素分析表明MTH1蛋白高表达是结肠癌预后不良的独立预测因素(HR=2.256,95%CI:1.008～5.049,P=0.048)。结肠癌细胞株中MTH1基因mRNA和蛋白表达量显著高于正常的肠黏膜细胞,而且mRNA与蛋白的表达呈正相关。siMTH1干扰MTH1基因mRNA表达量的下降与HCT116和LoVo细胞增殖、迁移、侵袭能力的下降显著相关(P<0.05)。结论 MTH1蛋白表达影响了结肠癌的进展和预后。
Objective To explore the effects of the expression of MTH1 protein on colorectal cancer progression and prognosis. Methods We firstly tested the expression of MTH1 protein in 87 colorectal cancer specimens and the paired para-cancerous specimens by performing Immunohistochemistry. Pearson′ χ~2 was used to analyze the correlation between MTH1 protein and clinic-pathological factors. COX proportional hazards models showed the value of MTH1 protein expression in predicting the overall survival and prognosis of CRC patients who had accepted radical operation. Then we selected real-time PCR and western blot to detect the levels of MTH1 gene mRNA and protein in six colorectal cancer cells HCT116, SW480, SW620, LoVo, COLO320, T84 and the human normal intestinal mucous cell line CCC-HIE-2; Student′s t test enabled us to compare mean value of the relative expression of MTH1 gene mRNA and protein in colorectal cancer cells and the human normal intestinal mucous cell line. And by applying Spearman′s rank correlation coefficient, we studied the correlation between the expression of MTH1 protein and its mRNA in these cell lines. Lastly, we used the siRNA we had designed and synthesized to interfere the expression of MTH1 gene mRNA in HCT116 and LoVo; CCK-8 assay and Transwell illustrated the influence of low MTH1 protein expression on proliferation, migration and invasion. Results The analysis of 87 colorectal cancer samples showed that surgically treated patients with high expression of MTH1 protein had a significantly lower overall survival than those with a low expression(P=0.005). According to statistics, MTH1 was an independent prognostic factor of CRC patients(HR=2.256, 95%Cl:1.008-5.049,P=0.048). The expression of MTH1 mRNA and protein in colorectal cancer cells was significantly higher compared with the human normal intestinal mucous cell line, and there was a positive correlation between mRNA levels and MTH1 protein expression. HCT116 and LoVo transfected with siMTH1 showed a significantly decreased ability in proliferation, migration and invasion(P<0.05). Conclusion The expression of MTH1 protein affects colorectal cancer progression and prognosis.
Objective To explore the effects of the expression of MTH1 protein on colorectal cancer progression and prognosis. Methods We firstly tested the expression of MTH1 protein in 87 colorectal cancer specimens and the paired para-cancerous specimens by performing Immunohistochemistry. Pearson′ χ~2 was used to analyze the correlation between MTH1 protein and clinic-pathological factors. COX proportional hazards models showed the value of MTH1 protein expression in predicting the overall survival and prognosis of CRC patients who had accepted radical operation. Then we selected real-time PCR and western blot to detect the levels of MTH1 gene mRNA and protein in six colorectal cancer cells HCT116, SW480, SW620, LoVo, COLO320, T84 and the human normal intestinal mucous cell line CCC-HIE-2; Student′s t test enabled us to compare mean value of the relative expression of MTH1 gene mRNA and protein in colorectal cancer cells and the human normal intestinal mucous cell line. And by applying Spearman′s rank correlation coefficient, we studied the correlation between the expression of MTH1 protein and its mRNA in these cell lines. Lastly, we used the siRNA we had designed and synthesized to interfere the expression of MTH1 gene mRNA in HCT116 and LoVo; CCK-8 assay and Transwell illustrated the influence of low MTH1 protein expression on proliferation, migration and invasion. Results The analysis of 87 colorectal cancer samples showed that surgically treated patients with high expression of MTH1 protein had a significantly lower overall survival than those with a low expression(P=0.005). According to statistics, MTH1 was an independent prognostic factor of CRC patients(HR=2.256, 95%Cl:1.008-5.049,P=0.048). The expression of MTH1 mRNA and protein in colorectal cancer cells was significantly higher compared with the human normal intestinal mucous cell line, and there was a positive correlation between mRNA levels and MTH1 protein expression. HCT116 and LoVo transfected with siMTH1 showed a significantly decreased ability in proliferation, migration and invasion(P<0.05). Conclusion The expression of MTH1 protein affects colorectal cancer progression and prognosis.
引文
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11 Morak M,Massdorf T,Sykora H,et al.First evidence for digenic inheritance in hereditary colorectal cancer by mutations in the base excision repair genes[J].Eur J Cancer,2011,47(7):1046-1055
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13 Takama F,Kanuma T,Wang D,et al.Mutation analysis of the hMTH1 gene in sporadic human ovarian cancer[J].Int J Oncol,2000,17(3):467-471
14 Hori M,Satou K,Harashima H,et al.Suppression of mutagenesis by 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (7,8-dihydro-8-oxo-2′-deoxyguanosine 5′-triphosphate) by human MTH1,MTH2,and NUDT5[J].Free Radical Biol Med,2010,48(9):1197-1201
15 Gad H,Koolmeister T,Jemth AS,et al.MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool[J].Nature,2014,508(7495):215-221
16 Rajalingam K,Schreck R,Rapp UR,et al.Ras oncogenes and their downstream targets[J].Biochim Biophys Acta,2007,1773(8):1177-1195
17 Patel A,Burton DG,Halvorsen K,et al.MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways[J].Oncogene,2015,34(20):2586-2596
2 Radak Z,Boldogh I.8-oxo-7,8-dihydroguanine:links to gene expression,aging,and defense against oxidative stress[J].Free Radical Biol Med,2010,49(4):587-596
3 LPC.Reaction of HO* with guanine derivatives in aqueous solution:formation of two different redox-active OH-adduct radicals and their unimolecular transformation reactions.Properties of G(-H)* [J].Chemistry (Weinheim An Der Bergstrasse,Germany),2000,3(6):475-484
4 Shibutani S,Takeshita M,Grollman A.Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG[J].Nature,1991,349(6308):431-434
5 KCC.8-Hydroxyguanine,an abundant form of oxidative DNA damage,causes G-T and A-C substitutions[J].J Biol Chem,1992,1(267):166-172
6 Maki H,Sekiguchi M.MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis[J].Nature,1992,355(6357):273-275
7 Taddei F,Hayakawa H,Bouton M,et al.Counteraction by MutT protein of transcriptional errors caused by oxidative damage[J].Science,1997,278(5335):128-130
8 Borrego S,Vazquez A,Dasi F,et al.Oxidative stress and DNA damage in human gastric carcinoma:8-Oxo-7′8-dihydro-2′-deoxyguanosine (8-oxo-dG) as a possible tumor marker[J].Int J Mol Sci,2013,14(2):3467-3486
9 CHK.Overexpression of hMTH1 mRNA:a molecular marker of oxidative stress in lung cancer cells[J].FEBS Letters,1998,1(429):17-20
10 Akiyama S,Saeki H,Nakashima Y,et al.Prognostic impact of MutT homolog-1 expression on esophageal squamous cell carcinoma[J].Cancer Medicine,2017,6(1):258-266
11 Morak M,Massdorf T,Sykora H,et al.First evidence for digenic inheritance in hereditary colorectal cancer by mutations in the base excision repair genes[J].Eur J Cancer,2011,47(7):1046-1055
12 Coskun E,Jaruga P,Jemth AS,et al.Addiction to MTH1 protein results in intense expression in human breast cancer tissue as measured by liquid chromatography-isotope-dilution tandem mass spectrometry[J].DNA Repair,2015,33:101-110
13 Takama F,Kanuma T,Wang D,et al.Mutation analysis of the hMTH1 gene in sporadic human ovarian cancer[J].Int J Oncol,2000,17(3):467-471
14 Hori M,Satou K,Harashima H,et al.Suppression of mutagenesis by 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (7,8-dihydro-8-oxo-2′-deoxyguanosine 5′-triphosphate) by human MTH1,MTH2,and NUDT5[J].Free Radical Biol Med,2010,48(9):1197-1201
15 Gad H,Koolmeister T,Jemth AS,et al.MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool[J].Nature,2014,508(7495):215-221
16 Rajalingam K,Schreck R,Rapp UR,et al.Ras oncogenes and their downstream targets[J].Biochim Biophys Acta,2007,1773(8):1177-1195
17 Patel A,Burton DG,Halvorsen K,et al.MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways[J].Oncogene,2015,34(20):2586-2596