浸润性乳腺癌中EphA2基因启动子区DNA低甲基化及与细胞焦亡的相关性

The role of DNA hypomethylation in the promoter region of EphA 2 and its relationship with pyroptosis in invasive breast cancer

目的:探讨浸润性乳腺癌中肝配蛋白A型受体2(ephrin A receptor 2,EphA2)表达及其启动子区DNA低甲基化与细胞焦亡发生的相关性。方法:应用免疫荧光技术和蛋白质印迹法检测42例乳腺癌患者正常乳腺组织、癌旁组织和癌组织中EphA2的表达情况;采用免疫荧光技术和蛋白质印迹法检验细胞焦亡相关蛋白核苷酸结合寡聚结构域样受体家族蛋白3(nucleotide-binding oligomerization domain-like receptor protein 3,NLRP3)的表达水平;采用蛋白质印迹法检测凋亡相关蛋白Caspase 1和炎性细胞因子白细胞介素1β(interleukin-1β,IL-1β)的表达水平;应用巢氏甲基化特异性PCR(nested methylation-specific PCR,nMS-PCR)检验EphA 2基因启动子区DNA甲基化水平;采用蛋白质印迹法测验DNA甲基转移酶1(DNA methyltransferase 1,DNMT1)和DNA甲基转移酶3a(DNA methyltransferase 3a,DNMT3a)蛋白表达水平;应用Pearson相关系数分别对患者癌组织中EphA2的蛋白表达和甲基化水平与NLRP3、Caspase 1、IL-1β、DNMT1和DNMT3a进行相关性分析。结果:与正常乳腺组织及癌旁组织相比,乳腺癌组中EphA2蛋白表达水平明显升高(P均<0.01),NLRP3、Caspase 1和IL-1β的蛋白表达水平明显降低(P均<0.05);同时与正常乳腺组织及癌旁组织相比,乳腺癌组织中EphA2基因启动子区DNA甲基化水平明显降低(P均<0.05),DNMT3a蛋白表达明显降低(P<0.01,P<0.05),而DNMT1蛋白表达的差异未有统计学意义;Pearson相关性分析结果显示,EphA2蛋白与NLRP3蛋白(r=-0.6512,P<0.05)、Caspase 1蛋白(r=-0.5712,P<0.05)、IL-1蛋白(r=-0.6546,P<0.05)和DNMT3a蛋白(r=-0.5374,P<0.05)的表达水平均呈负相关性;而EphA 2基因的甲基化水平与NLRP3蛋白(r=0.6341,P=0.0268)、Caspase 1蛋白(r=0.6728,P=0.0165)、IL-1β蛋白(r=0.6940,P=0.0123)和DNMT3a蛋白(r=0.6871,P=0.0136)的表达水平呈正相关性。结论:乳腺癌组织中EphA2蛋白的表达水平上调并与细胞焦亡呈负相关性;DNMT3a可能参与了EphA 2基因启动子区发生DNA低甲基化的过程。

Objective:To investigate the correlation of the expression of Ephrin A receptor 2(EphA2)and its promoter region DNA hypomethylation with the occurrence of pyroptosis in invasive breast cancer.Methods:The expression level of pyroptosis-related protein EphA2 in normal breast tissue,paracancerous tissues and cancer tissues from 42 breast cancer patients was examined by Immunofluorescence staining and Western blotting.The expression level of pyroptosis related protein nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)was analyzed by immunofluorescence staining and Western blotting.The expression levels of apoptosis related proteins Caspase 1 and inflammatory cytokine interleukin-1β(IL-1β)were studied by Western blotting.The DNA methylation level in the promoter region of EphA 2 was investigated by nested methylation-specific PCR(nMS-PCR).The expression levels of DNA methyltransferase 1(DNMT1)and DNA methyltransferase 3a(DNMT3a)were examined by Western blotting.The correlation of the protein expression and methylation level of EphA2 in cancer tissues with the expression NLRP3,Caspase 1,IL-1β,DNMT1 and DNMT3a was analyzed by Pearson correlation coefficient.Results:Compared with normal breast tissues and paracancerous tissues,the expression level of EphA2 protein was significantly increased(P<0.01),while that of NLRP3,Caspase1 and IL-1βwas significantly decreased(P<0.05)in breast cancer tissues.Meanwhile,compared with normal breast tissues and paracancerous tissues,the DNA methylation level of EphA 2 promoter in breast cancer tissues was significantly decreased(P<0.05),the expression level DNMT3a protein was significantly decreased(P<0.01,P<0.05),and the difference in the expression level of DNMT1 protein was not statistically significant.Pearson correlation analysis showed that the expression level of EphA2 protein is negatively correlated with that of NLRP3(r=-0.6512,P<0.05),Caspase1(r=-0.5712,P<0.05),IL-1β(r=-0.6546,P<0.05)or DNMT3a(r=-0.5374,P<0.05),while the methylation level of EphA 2 was positively correlated with the protein expression level of NLRP3(r=0.6341,P=0.0268),Caspase1(r=0.6728,P=0.0165),IL-1β(r=0.6940,P=0.0123)and DNMT3a(r=0.6871,P=0.0136).Conclusion:The expression of EphA2 protein is upregulated in breast cancer tissues is negatively correlated with pyroptosis.DNMT3a may be involved in the process of DNA hypomethylation in the promoter region of EphA 2.