α1-抗胰蛋白酶和组织因子途径抑制物基因多态性的交互作用及其与大肠癌侵袭和转移的关系

Relationship of polymorphism interaction of α1-antitypsin and tissue factor pathway inhibitor genes with invasion and metastasis of colorectal carcinoma

目的:探讨α1-抗胰蛋白酶(α1-antitypsin,α1-AT)基因第5外显子和组织因子途径抑制物(tissue factor pathway inhibitor,TFPI)基因T287C多态性的交互作用及其与大肠癌侵袭和转移的关系。方法:选择河南省新乡医学院第一附属医院2011年7月至2015年7月期间收治的经病理学确诊的大肠癌TNMⅠ、Ⅱ、Ⅲ和Ⅳ期患者各180例,以180例TNM 0期患者作为对照组,用PCR-RFLP技术检测各组患者外周血白细胞两基因的多态性,以Hardy-Weinberg平衡检验分析样本的群体代表性,以Khoury和Wagener模型分析两基因多态性的交互作用。ELISA法检测患者血清α1-AT和TFPI蛋白表达,细胞划痕试验和Transwell侵袭试验分别检测α1-AT和TFPI对人结肠癌SW480细胞迁移和侵袭能力的影响,Western blotting检测SW480细胞胰蛋白酶、组织因子(tissue factor,TF)和蛋白酶激活受体-2(protease-actieated receptor 2,PAR-2)表达水平。结果:α1-AT(MZ)、α1-AT(ZZ)、TFPI(TC)和TFPI(CC)基因型者大肠癌侵袭与转移的风险均显著增加,且在α1-AT(MZ)和TFPI(TC)之间、α1-AT(MZ)和TFPI(CC)之间、α1-AT(ZZ)和TFPI(TC)及α1-AT(ZZ)和TFPI(CC)之间均存在正向交互作用(均γ>1)。Ⅰ、Ⅱ、Ⅲ和Ⅳ期患者血清α1-AT(或TFPI)表达明显低于0期组,且Ⅰ、Ⅱ、Ⅲ和Ⅳ期患者之间血清α1-AT(或TFPI)表达也有明显差异(P<0.01)。同一组携带突变基因型个体的血清α1-AT(或TFPI)表达明显低于携带野生型个体(P<0.01)。体外细胞培养实验显示,α1-AT可明显抑制SW480细胞胰蛋白酶的表达,而TFPI则明显抑制TF表达,而两者均可明显降低细胞PAR-2的表达及细胞迁移和侵袭能力。结论:α1-AT(MZ)、α1-AT(ZZ)、TFPI(TC)和TFPI(CC)基因型均是大肠癌侵袭与转移的高危因素,基因多态性的交互作用增加了大肠癌侵袭与转移的风险。

Objective： To investigate polymorphism interaction of α1-antitypsin（ α1-AT） gene exon 5 and tissue factor pathway inhibitor（ TFPI） gene T287 C and relationship of the gene polymorphism interation with invasion and metastasis of colon carcinoma. Methods： Each of one hundred eighty patients diagnosed as the TNM Ⅰ,Ⅱ,Ⅲ or Ⅳ stage colon carcinoma received and treated by the First Hospital affiliated to Xinxiang Medical University Henan Province during July2011 to July 2015 were selected. One hundred eighty patients with the TNM 0 stage colon carcinoma who without involvement of reginol lymph nodes and distant metastasis were selected as control group. Polymorphisms of the both genes in peripheral blood leukocytes of the patients in each group were tested with PCR-RFLP assay. Hardy-Weinberg equilibrium tests were used to analyze the population representativeness of the samples. Polymorphism interaction of the both genes was examed with Khoury and Wagener model. Expressions of the α1-AT and TFPI proteins in serum were tested by ELISA assay. Scratch test and Transwell invasion assay were used to detect the effect of the α1-AT and TFPI on migration and invation abilities of the human colon carcinoma SW480 line cells respectively. Expression levels of cell trypsin,tissue factor（ TF） and protease-activated receptor-2（ PAR-2） were tested with Western blotting assay. Results： Risks of invasion and metastasis of the colorectal cancer in the patients with genotypes of α1-AT（ MZ）,α1-AT（ ZZ）,TFPI（ TC） and TFPI（ CC） significantly increased,furthermore there was a positive interaction between α1-AT（ MZ） and TFPI（ TC）,α1-AT（ MZ） and TFPI（ CC）,α1-AT（ ZZ） and TFPI（ TC）,and α1-AT（ ZZ） and TFPI（ CC） at all（ all γ 1）. Expressions of the α1-AT and TFPI proteins in the patients with TNM Ⅰ,Ⅱ,Ⅲ,Ⅳ stage were significantly lower than that in the patients with TNM 0 stage,and expressions of serum α1-AT and TFPI proteins among the patients with TNM Ⅰ,Ⅱ,Ⅲ and Ⅳ stage were also obviously different（ P 0. 01）. In the same group,exoressions of serum α1-AT and TFPI proteins in the patients with mutant genotype were significantly lower than those in the patients with wild genotype（ P 0. 01）. Cell culture in vitro experiments shown that α1-AT obviously inhibited exoression of cell trypsin in the SW480 cells,but TFPI inhibited expression of TF evidently. As well as both of them could significantly decrease expression of PAR-2 protein and,migration and invasion abilities of the cells. Conclusion： All genotypes of α1-AT（ MZ）,α1-AT（ ZZ）,TFPI（ TC） and TFPI（ CC） might be risk factors for invasion and metastasis of the colon carcinoma. Polymorphisms and interactions of the gnens could increase invasion and metastasis dangers of the colon cancer.