摘要
目的目前关于PCDHA13启动子甲基化在乳腺癌中的作用机制尚未阐明,文中探讨PCDHA13基因启动子甲基化在乳腺癌发生发展中的作用。方法应用MassARRAY质谱甲基化测序检测人乳腺癌组织PCDHA13基因启动子甲基化状态,用培养基配置100μmol/L的5-氮杂胞苷储存液,取融合度60%的ZR-75-1细胞分别加入终浓度为5μmol/L(低浓度组)和10μmol/L(高浓度组)的5-氮杂胞苷储存液,对照组仅加入未经处理的培养基,重亚硫酸盐测序法和甲基化特异性PCR法检测人乳腺癌细胞中PCDHA13基因启动子甲基化状态,并结合半定量RT-PCR的方法分析PCDHA13基因甲基化状态与其mRNA表达的关系。Western blot、MTT以及DAPI染色检测5-Aza处理对乳腺癌ZR-75-1细胞增殖和凋亡的影响。结果乳腺癌组织中PCDHA13基因启动子在第1,4-6,9,10,11个Cp G位点甲基化程度明显高于正常乳腺组织[(0.263 9±0.157 5) vs (0.161 2±0.170 6)、(0.250 9±0.137 7) vs (0.168 8±0.099 2)、(0.420 4±0.208 7) vs (0.262 1±0.173 1)、(0. 376 1±0.140 7) vs (0.282 4±0.148 6)、(0.392 2±0.129 4) vs (0.307 2±0.149 6)],差异有统计学意义(P <0.05),MDA-MB-231细胞和Bcap-37细胞中PCDHA13基因启动子CG位点甲基化率在40%~100%; MCF-7细胞中PCDHA13基因启动子甲基化率在10%~50%之间,呈现低甲基化状态,ZR-75-1细胞中PCDHA13基因启动子表现为高甲基化状态,第4个CG位点甲基化率为60%,第1、8、12个CG位点甲基化率为90%,其余CG位点甲基化率全部为100%,PCDHA13基因在MDA-MB-231和Bcap-37细胞系中低表达,MCF-7细胞系中高表达,而在ZR-75-1中表达缺失;对照组ZR-75-1细胞仅扩增出甲基化PCR产物,而经5-Aza处理的ZR-75-1细胞甲基化和非甲基化引物均扩增出特异性条带,并且高浓度组明显高于低浓度组(P>0.05)。对照组ZR-75-1细胞PCHDA13基因mRNA表达缺失,经5-Aza处理后,ZR-75-1细胞PCDHA13基因mRNA重新恢复表达,高浓度组PCDHA13基因mRNA表达明显高于低浓度组(P>0.05)。对照组ZR-75-1细胞PCHDA13蛋白表达缺失,经5-Aza处理后,ZR-75-1细胞PCDHA13蛋白重新恢复表达,而且高浓度组PCDHA13蛋白表达水平明显高于低浓度组(P>0.05)。经5-Aza处理后24、48和72 h后,低浓度组细胞生长抑制率均较高浓度组降低(P<0.05)。未经药物处理的ZR-75-1细胞核形态基本正常,未出现细胞凋亡。经5-Aza处理后,部分ZR-75-1细胞出现核固缩、染色质凝集、着色较重的现象。结论乳腺癌中PCDHA13启动子高甲基化状态与其mRNA低表达或缺失有关,ZR-75-1细胞PCDHA13表达可被5-Aza逆转,PCHAD13基因重新表达后不仅抑制细胞增殖,而且促进细胞凋亡。PCDHA13基因异常甲基化有望成为乳腺癌潜在的肿瘤生物标志物。
Objective The mechanisms of PCDHA13 promoter methylation in breast cancer have not yet been elucidated at present.This study was to investigate the role of PCDHA13 gene promoter methylation in the development of breast cancer. Methods The methylation state of PCDHA13 gene promoter in human breast cancer tissues was detected by MassARRAY mass spectrum methylation sequencing.100μmol/L 5-Aza was prepared with culture medium.The ZR-75-1 cells with 60%cell confluence were added to the final concentration of 5μmol/L(low concentration group)and 10μmol/L(high concentration group)5-Aza,and the control group was only added culture medium.Detection of methylation status of PCDHA13 gene promoter in human breast cancer cells by bisulfite sequencing and methylation-specific PCR,and analysis of methylation status and mRNA expression of PCDHA13 gene by semi-quantitative RT-PCR.Western blot,MTT and DAPI staining were used to detect the effect of 5-Aza treatment on proliferation and apoptosis of breast cancer ZR-75-1 cells. Results The methylation degree of PCDHA13 gene promoter in the 1,4-6,9,10 and 11 CpG loci in breast cancer tissues was significantly higher than that in normal breast group[(0.263 9±0.157 5)vs(0.161 2±0.170 6),(0.250 9±0.137 7)vs(0.168 8±0.099 2),(0.420 4±0.208 7)vs(0.262 1±0.173 1),(0.376 1±0.140 7)vs(0.282 4±0.148 6),(0.392 2±0.129 4)vs(0.307 2±0.149 6)],and the difference was statistically significant(P<0.05).The methylation rates of PCDHA13 gene promoter were reached 40%~100%in MDA-MB-231 cells and Bcap-37 cells.The methylation rates of PCDHA13 gene promoter were reached 10%~50%in MCF-7,rendered hypomethylation.The PCDHA13 gene promoter was hypermethylated in ZR-75-1 cells,and themethylation rate were 60%in forth CG site,90%in first,eighth and twelfth CG site,100%in the other CG sites.PCDHA13 gene was low expressed in MDA-MB-231 cells and Bcap-37 cells,high expressed in MCF-7 cells,but absent in ZR-75-1.There were only amplified methylated PCR products from ZR-75-1 cells in control group.While there were amplified methylated and non-methylated PCR products from ZR-75-1 cells treated with 5-Aza,and the high concentration group was significantly higher than the low concentration group(P>0.05).The expression of PCDHA13 mRNA of ZR-75-1 cells was loss in control group,but the expression of PCDHA13 mRNA was reversed after treated with 5-Aza,and the expression of PCDHA13 mRNA was significantly higher in high concentration group than that in low concentration group(P>0.05).After treated with 5-Aza for 24,48 and 72 hours,the growth inhibition rates were lower in low concentration group than that in high concentration group(P>0.05).The morphology of the nuclei was basically normal and there was no apoptosis occurred in ZR-75-1 cells.But after treated with 5-Aza,some ZR-75-1 cells showed nuclear condensation,chromatin agglutination and heavy coloration. Conclusion This study showed that the low expression or loss of mRNA is associated with hypermethylation of the PCDHA13 gene promoter in breast carcinoma.The PCHDA13 gene expression can be reversed by 5-Aza in ZR-75-1 cells.The re-expression of PCHAD13 not only inhibit the proliferation of cells,but also promote apoptosis.Abnormal methylation of PCDHA13 may become a potential tumor marker for breast cancer.
作者
朱文斌
温宪春
于海涛
葛斌
刘军
曹维海
刘雷
岳丽玲
ZHU Wen-bin;WEN Xian-chun;YU Hai-tao;GE Bin;LIU Jun;CAO Wei-hai;LIU Lei;YUE Li-ling(Research Institute of Medical Science and Pharmacy,Qiqihar Medical University,Qiqihar 161006,Heilongjiang,China;Department of Basic Medical Science,Qiqihar Medical University,Qiqihar 161006,Heilongjiang,China;Department of Breast Surgery,First Hospital of Qiqihar,Qiqihar 161006,Heilongjiang,China;Department of Pathology,First Hospital of Qiqihar,Qiqihar 161006,Heilongjiang,China)
出处
《医学研究生学报》
CAS
北大核心
2018年第10期1026-1032,共7页
Journal of Medical Postgraduates
基金
黑龙江省自然科学基金(H2014100)