受体型蛋白酪氨酸磷酸酶O基因甲基化与乳腺癌细胞株化疗敏感性的关系

Correlation of methylation of protein tyrosine phosphatase receptor-type O gene with the chemosensitivity in breast cancer cells

目的探讨受体型蛋白酪氨酸磷酸酶O(PTPRO)基因不同甲基化状态的乳腺癌细胞株对紫杉醇的敏感性。方法应用甲基化特异性PCR,检测MCF-7、MDA-MB-231和Hs578t乳腺癌细胞株中PTPRO基因启动子Cp G岛甲基化状态,并用RT-PCR法检测PTPRO mRNA表达。采用MTT法检测MCF-7、MDA-MB-231和Hs578t乳腺癌细胞株对0.000 6、0.003 0、0.015 0、0.075 0、0.375 0、1.875 0、9.350 0、46.750 0、234.000 0 mmol/L紫杉醇的敏感性,以及对细胞株进行去甲基化实验后紫杉醇抑制率的改变。两组间均数比较采用配对t检验;在检验方差齐性的前提下,多组间均数比较用单因素方差分析,组间两两比较采用LSD检验。结果乳腺癌MCF-7、MDA-MB-231细胞株中PTPRO基因甲基化,而乳腺癌Hs578t细胞株中PTPRO基因无甲基化。用紫杉醇处理细胞株后再检测PTPRO启动子甲基化情况,结果显示MCF-7、MDA-MB-231细胞株PTPRO基因甲基化率明显降低[(0.861±0.109)比(0.037±0.019),t=23.326,P=0.000;(0.758±0.114)比(0.086±0.010),t=16.109,P=0.000]。并且,MCF-7、MDA-MB-231细胞株经5-杂氮脱氧胞嘧啶(DAC)处理后,PTPRO mRNA表达水平明显高于DAC处理前[(0.033±0.006)比(0.166±0.016),t=28.338,P=0.000;(0.052±0.006)比(0.587±0.087),t=17.257,P=0.000],其表达水平与启动子Cp G岛甲基化状态有关。紫杉醇对MDA-MB-231、MCF-7及Hs578t细胞株的半数抑制浓度(IC50)分别为8.52、5.87和4.52 mmol/L。不同浓度紫杉醇对Hs578t细胞株的抑制率均比MCF-7和MDA-MB-231细胞株高(F=129.93、182.40、46.26、49.26、33.60、80.31、160.05、69.96、79.98,P均=0.000)。去甲基化实验显示:DAC处理MCF-7和MDA-MB-231乳腺癌细胞株后,不同浓度紫杉醇对细胞株的抑制率与处理前相比,差异均有统计学意义(MCF-7:t=14.195、8.328、7.042、6.385、5.450、8.557、4.788、13.351、11.887,P均<0.050;MDA-MB-231:t=16.324、30.443、9.177、12.694、9.528、11.912、10.260、18.109、3.754,P均<0.050)。结论 PTPRO基因甲基化是乳腺癌发生、发展过程中的常见现象;紫杉醇可影响PTPRO基因启动子Cp G岛的甲基化状态,并且对无PTPRO基因甲基化的乳腺癌细胞株杀伤率更高;PTPRO基因甲基化状态的改变影响乳腺癌细胞株对紫杉醇的敏感性。

Objective To determine the role of protein tyrosine phosphatase receptor-type O（PTPRO） gene methylation as a potential molecular biomarker in the treatment of breast cancer and its association with the sensitivity to paclitaxel in the breast cancer cell lines. Methods Methylation-specific PCR was used to detect the methylation status of PTPRO gene promoter CpG island in three breast cancer cell lines MCF-7, MDA-MB- 231 and Hs578t. RT-PCR was used to measure PTPRO mRNA expression. MTT assay was applied to evaluate the sensitivity of MCF-7, MDA-MB-231 and Hs578 cells to paclitaxel under different concentrations, i. e. , 0. 000 6,0. 003 0,0. 015 0,0. 075 0,0. 375 0,1. 875 0,9. 350 0,46.750 0, 234.000 0 mmol/L. The changes of inhibition rate of paclitaxel in cell lines after demethylation were analyzed as well. The mean comparison between two groups was performed using paired t test; after determining the homogeneity of variance, mean comparison among multiple groups were performed using One-Way ANOVA; pairwise comparison using LSD test. Results PTPRO gene was methylated in MCF-7 and MDA-MB-231 cell lines, but unmethylated in Hs578t cell lines. After treated with paclitaxel, the methylation rate of PTPRO in MCF-7 and MDA-MB-231 cell lines was significantly decreased [ （ 0. 861 ±0. 109 ） vs （ 0. 037 ±0. 019 ）, t = 23. 326, P = 0. 000 ; （ 0. 75± 0. 114）vs（0. 086±0. 010） ,t= 16. 109,P=0. 000]. Moreover, PTPRO mRNA expression level in MCF-7 and MDA-MB-231 cell lines was significantly higher after DAC treatment [ （0. 033±0. 006） vs （0. 166±0. 016）, t=28. 338, P=0. 000; （0. 052±0. 006） vs （0. 587±0. 087）, t= 17. 257, P=0. 000], which was correlated with the methylation status of CpG islands. IC50 of paclitaxel on MCF-7, MDA-MB-231 and Hs578 breast cell lines was 8.52, 5.87 and 4. 52 mmol/L, respectively. Hs578t cell line was significantly more sensitive to paclitaxel at different concentrations compared with the other two cell lines （F = 129.93, 182.40, 46. 26, 49.26, 33.60, 80. 31, 160. 05, 69. 96, 79.98, all P=0. 000）. The demethylation experiment showed that after DAC treatment, the inhibition rate of paclitaxel at different concentrations presented a significant difference in MCF-7 and MDA-MB-231 cells （ MCF-7： t = 14. 195, 8. 328, 7. 042, 6. 385, 5.450, 8. 557, 4. 788, 13. 351, 11. 887, all P 〈 O. 050; MDA-MB-231 ： t = 16. 324, 30. 443, 9. 177, 12. 694, 9. 528, 11. 912, 10. 260, 18. 109, 3.754, all P〈0. 050）. Conclusions PTPRO methylation is a common phenomenon in the development of breast cancer. Promoter CpG island methylation status of PTPRO gene could be influenced by paclitaxel and breast cancer cell lines with unmethylated PTPRO are more sensitive to paclitaxel. The changes of methylation status of PTPRO gene significantly affect the sensitivity of breast cancer cells to paclitaxel.