Study on promoter methylation of PCDHA13 gene and breast cancer development
Wen-bin ZHU
;
Xian-chun WEN
;
Hai-tao YU
;
Bin GE
;
Jun LIU
;
Wei-hai CAO
;
Lei LIU
;
Li-ling YUE
Journal of Medical Postgraduates.2018;31(10):1026-1032. doi:10.16571/j.cnki.1008-8199.2018.10.004
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 5 μmol/L(low concentration group) and 10 μmol/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 \[(0.2639±0.1575) vs (0.1612±0.1706), (0.2509±0.1377) vs (0.1688±0.0992), (0.4204±0.2087) vs (0.2621±0.1731), (0.3761±0.1407) vs (0.2824±0.1486), (0.3922±0.1294) vs (0.3072±0.1496)\], 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.
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